WorldWideScience

Sample records for heating-induced conformational change

  1. Changes in chemical interactions and protein conformation during heat-induced wheat gluten gel formation.

    Science.gov (United States)

    Wang, Kai-Qiang; Luo, Shui-Zhong; Zhong, Xi-Yang; Cai, Jing; Jiang, Shao-Tong; Zheng, Zhi

    2017-01-01

    In order to elucidate the heat-induced wheat gluten gel formation mechanism, changes in chemical interactions and protein conformation were investigated during gelation. The contribution of ionic and hydrogen bonds were found to decrease from 0.746 and 4.133g/L to 0.397 and 2.733g/L, respectively, as the temperature increased from 25 to 90°C. Moreover, the free SH content remarkably decreased from 37.91 to 19.79μmol/g during gelation. Ultraviolet absorption spectra and intrinsic fluorescence spectra suggested that wheat gluten unfolded during the heating process. In addition, wheat gluten gels treated at 80 and 90°C exhibited a "steric hindrance" effect, which can be attributed to the formation of aggregates. Fourier transform infrared spectra suggested that the random coil content increased at low temperatures (40 and 50°C), whereas the content of intermolecular β-sheets due to protein aggregation increased from 38.10% to 44.28% when the gelation temperature was 90°C.

  2. Advances in structure-function relationships of tyrosinase from Agaricus bisporus - investigation on heat-induced conformational changes.

    Science.gov (United States)

    Ioniţă, Elena; Aprodu, Iuliana; Stănciuc, Nicoleta; Râpeanu, Gabriela; Bahrim, Gabriela

    2014-08-01

    A combination of fluorescence spectroscopic measurements, inactivation kinetics and in silico prediction was used in the present study to investigate the heat induced behaviour of tyrosinase from Agaricus bisporus. The phase diagram indicated the existence of at least two distinct species induced by the temperature increase up to 75°C. Regardless of calcium ion presence, the fluorescence intensity results suggest that tyrosinase tends to form aggregates after 10min at 75°C. The quenching experiments using acrylamide and iodide demonstrate a more flexible conformation of tyrosinase at higher temperature. Detailed insights into tyrosinase structure after performing molecular dynamics simulations, suggest important structural rearrangements of the protein with the temperature increase. The copper coordinating His(94) residue was predicted to be involved in salt bridge formation with Glu(98), therefore causing significant alteration of the substrate binding site with increasing temperature. These significant changes in tyrosinase structure at temperatures over 60°C might lead to enzyme inactivation.

  3. Exploring the process-structure-function relationship of horseradish peroxidase through investigation of pH- and heat induced conformational changes

    Science.gov (United States)

    Stănciuc, Nicoleta; Aprodu, Iuliana; Ioniță, Elena; Bahrim, Gabriela; Râpeanu, Gabriela

    2015-08-01

    Given the importance of peroxidase as an indicator for the preservation of vegetables by heat treatment, the present study is focused on enzyme behavior under different pH and temperature conditions, in terms of process-structure-function relationships. Thus, the process-structure-function relationship of peroxidase was investigated by combining fluorescence spectroscopy, in silico prediction methods and inactivation kinetic studies. The fluorescence spectra indicated that at optimum pH value, the Trp117 residue is not located in the hydrophobic core of the protein. Significant blue- and red-shifts were obtained at different pH values, whereas the heat-treatment did not cause significant changes in Trp and Tyr environment. The ANS and quenching experiments demonstrated a more flexible conformation at lower pH and respectively at higher temperature. On the other hand molecular dynamics simulations at different temperatures highlighted that the secondary structure appeared better preserved against temperature, whereas the tertiary structure around the heme was more affected. Temperature dependent changes in the hydrogen bonding and ion paring involving amino acids from the heme-binding region (His170 and Asp247) might trigger miss-coordination of the heme iron atom by His170 residue and further enzyme activity loss.

  4. Conformational changes in biopolymers

    Science.gov (United States)

    Ivanov, Vassili

    2005-12-01

    Biopolymer conformational changes are involved in many biological processes. This thesis summarizes some theoretical and experimental approaches which I have taken at UCLA to explore conformational changes in biopolymers. The reversible thermal denaturation of the DNA double helix is, perhaps, the simplest example of biopolymer conformational change. I have developed a statistical mechanics model of DNA melting with reduced degrees of freedom, which allows base stacking interaction to be taken into account and treat base pairing and stacking separately. Unlike previous models, this model describes both the unpairing and unstacking parts of the experimental melting curves and explains the observed temperature dependence of the effective thermodynamic parameters used in models of the nearest neighbor type. I developed a basic kinetic model for irreversible thermal denaturation of F-actin, which incorporates depolymerization of F-actin from the ends and breaking of F-actin fiber in the middle. The model explains the cooperativity of F-actin thermal denaturation observed by D. Pavlov et al. in differential calorimetry measurements. CG-rich DNA sequences form left-handed Z-DNA at high ionic strength or upon binding of polyvalent ions and some proteins. I studied experimentally the B-to-Z transition of the (CG)6 dodecamer. Improvement of the locally linearized model used to interpret the data gives evidence for an intermediate state in the B-to-Z transition of DNA, contrary to previous research on this subject. In the past 15 years it has become possible to study the conformational changes of biomolecules using single-molecule techniques. In collaboration with other lab members I performed a single-molecule experiment, where we monitored the displacement of a micrometer-size bead tethered to a surface by a DNA probe undergoing the conformational change. This technique allows probing of conformational changes with subnanometer accuracy. We applied the method to detect

  5. New insights into heat induced structural changes of pectin methylesterase on fluorescence spectroscopy and molecular modeling basis

    Science.gov (United States)

    Nistor, Oana Viorela; Stănciuc, Nicoleta; Aprodu, Iuliana; Botez, Elisabeta

    2014-07-01

    Heat-induced structural changes of Aspergillus oryzae pectin methylesterase (PME) were studied by means of fluorescence spectroscopy and molecular modeling, whereas the functional enzyme stability was monitored by inactivation studies. The fluorescence spectroscopy experiments were performed at two pH value (4.5 and 7.0). At both pH values, the phase diagrams were linear, indicating the presence of two molecular species induced by thermal treatment. A red shift of 7 nm was observed at neutral pH by increasing temperature up to 60 °C, followed by a blue shift of 4 nm at 70 °C, suggesting significant conformational rearrangements. The quenching experiments using acrylamide and iodide demonstrate a more flexible conformation of enzyme with increasing temperature, especially at neutral pH. The experimental results were complemented with atomic level observations on PME model behavior after performing molecular dynamics simulations at different temperatures. The inactivation kinetics of PME in buffer solutions was fitted using a first-order kinetics model, resulting in activation energy of 241.4 ± 7.51 kJ mol-1.

  6. Imaging of conformational changes

    Energy Technology Data Exchange (ETDEWEB)

    Michl, Josef [Univ. of Colorado, Boulder, CO (United States)

    2016-03-13

    Control of intramolecular conformational change in a small number of molecules or even a single one by an application of an outside electric field defined by potentials on nearby metal or dielectric surfaces has potential applications in both 3-D and 2-D nanotechnology. Specifically, the synthesis, characterization, and understanding of designed solids with controlled built-in internal rotational motion of a dipole promises a new class of materials with intrinsic dielectric, ferroelectric, optical and optoelectronic properties not found in nature. Controlled rotational motion is of great interest due to its expected utility in phenomena as diverse as transport, current flow in molecular junctions, diffusion in microfluidic channels, and rotary motion in molecular machines. A direct time-resolved observation of the dynamics of motion on ps or ns time scale in a single molecule would be highly interesting but is also very difficult and has yet to be accomplished. Much can be learned from an easier but still challenging comparison of directly observed initial and final orientational states of a single molecule, which is the basis of this project. The project also impacts the understanding of surface-enhanced Raman spectroscopy (SERS) and single-molecule spectroscopic detection, as well as the synthesis of solid-state materials with tailored properties from designed precursors.

  7. Effects of konjac glucomannan on heat-induced changes of wheat gluten structure.

    Science.gov (United States)

    Wang, Yu; Chen, Yiheng; Zhou, Yun; Nirasawa, Satoru; Tatsumi, Eizo; Li, Xiuting; Cheng, Yongqiang

    2017-08-15

    Effects of konjac glucomannan on the structure of wheat gluten were investigated at variable temperatures in this study. Dynamic oscillatory rheology study showed that konjac glucomannan conferred stiffness on gluten with a higher tan δ data at 25°C and 55°C, but this parameter was lower at 75°C and 95°C. Konjac glucomannan decreased the content of thiol equivalent groups and increased the α-helix/β-sheet content ratio, respectively. The thicker layer of gluten protein with 5% konjac glucomannan was observed by scanning electron microscopy. This study revealed that konjac glucomannan could alter the conformations of gluten proteins upon heating via non-covalent interactions and physical entanglements. It is likely that konjac glucomannan promotes protein aggregation by strengthening hydrophobic interaction at 25°C and 55°C, and alleviates heat-induced denaturation by decreasing the flexibility of polypeptide chain at higher 75°C and 95°C. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Application of X-ray phase-contrast tomography in quantative studies of heat induced structural changes in meat

    DEFF Research Database (Denmark)

    Miklos, R.; Nielsen, M. S.; Einarsdottir, Hildur;

    2013-01-01

    X-ray computed tomography is increasingly used in the studies of food structure. This paper describes the perspectives of use of phase contrast computed tomography in studies of heat induced structural changes in meat. From the data it was possible to obtain reconstructed images of the sample str...... structure for visualization and qualitative studies of the sample structure. Further data segmentation allowed structural changes to be quantified.......X-ray computed tomography is increasingly used in the studies of food structure. This paper describes the perspectives of use of phase contrast computed tomography in studies of heat induced structural changes in meat. From the data it was possible to obtain reconstructed images of the sample...

  9. Novel X-ray phase-contrast tomography method for quantitative studies of heat induced structural changes in meat

    DEFF Research Database (Denmark)

    Miklos, Rikke; Nielsen, Mikkel Schou; Einarsdottir, Hildur;

    2014-01-01

    The objective of this study was to evaluate the use of X-ray phase-contrast tomography combined with 3D image segmentation to investigate the heat induced structural changes in meat. The measurements were performed at the Swiss synchrotron radiation light source using a grating interferometric se...... in a qualitative and quantitative manner without prior sample preparation as isolation of single muscle components, calibration or histology....

  10. Heat-induced conformational changes of patatin, the major potato tuber protein

    NARCIS (Netherlands)

    Pots, A.M.; Jongh, H.H.J. de; Gruppen, H.; Hamer, R.J.; Voragen, A.G.J.

    1998-01-01

    This paper presents a first structural characterization of isolated patatin, the major potato tuber protein, at ambient and elevated temperatures. Isolated patatin at room temperature is a highly structured molecule at both secondary and tertiary levels. It is estimated from far-ultraviolet circular

  11. Heat-induced chemical and color changes of extractive-free Black Locust (Rosinia Pseudoacacia) wood

    Science.gov (United States)

    Yao Chen; Jianmin Gao; Yongming Fan; Mandla A. Tshabalala; Nicole M. Stark

    2012-01-01

    To investigate chemical and color changes of the polymeric constituents of black locust (Robinia pseudoacacia) wood during heat treatment, extractive-free wood flour was conditioned to 30% initial moisture content (MC) and heated for 24 h at 120 °C in either an oxygen or nitrogen atmosphere. The color change was measured using the CIELAB color system. Chemical changes...

  12. Effect of calcium chelators on heat coagulation and heat-induced changes of concentrated micellar casein solutions: The role of calcium-ion activity and micellar integrity

    NARCIS (Netherlands)

    Kort, de E.J.P.; Minor, M.; Snoeren, T.A.L.; Hooijdonk, van A.C.M.; Linden, van der E.

    2012-01-01

    There is general consensus that calcium chelators enhance heat stability in milk. However, they increase the heat stability to considerably different extents. For this reason, the effect of various calcium chelators on heat coagulation and heat-induced changes of concentrated micellar casein

  13. Effect of calcium chelators on heat coagulation and heat-induced changes of concentrated micellar casein solutions: The role of calcium-ion activity and micellar integrity

    NARCIS (Netherlands)

    Kort, de E.J.P.; Minor, M.; Snoeren, T.A.L.; Hooijdonk, van A.C.M.; Linden, van der E.

    2012-01-01

    There is general consensus that calcium chelators enhance heat stability in milk. However, they increase the heat stability to considerably different extents. For this reason, the effect of various calcium chelators on heat coagulation and heat-induced changes of concentrated micellar casein solutio

  14. HEAT-INDUCED CHEMICAL AND COLOR CHANGES OF EXTRACTIVE-FREE BLACK LOCUST (ROBINIA PSEUDOACACIA WOOD

    Directory of Open Access Journals (Sweden)

    Yao Chen,

    2012-02-01

    Full Text Available To investigate chemical and color changes of the polymeric constituents of black locust (Robinia pseudoacacia wood during heat treatment, extractive-free wood flour was conditioned to 30% initial moisture content (MC and heated for 24 h at 120 °C in either an oxygen or nitrogen atmosphere. The color change was measured using the CIELAB color system. Chemical changes of the wood components were determined by means of solid state cross-polarization/magic angle spinning 13C-nuclear magnetic resonance (CPMAS-13C-NMR, Fourier transform infrared (FTIR, diffuse reflectance UV-Vis (DRUV spectroscopy, and elemental (CHN analysis. The results showed that lightness (L* decreased, while chromaticity indexes (a* and b* and chroma (C* increased after heat treatment. There was greater color difference (ΔE* in the samples heated in the presence of oxygen compared to nitrogen. CHN analysis showed an increase in hydrogen and oxygen and a decrease in carbon content. NMR spectra confirmed the cleavage of the β-O-4 structure in the lignin, resulting in a decrease in etherified lignin units and an increase in phenolic structures. DRUV and FTIR spectra confirmed the formation of extensive conjugated structures, such as unsaturated ketones and quinones due to the cleavage of the lignin units. Formation of quinones can be attributed to heat treatment in the presence of oxygen.

  15. Monitoring heat-induced changes in soft tissues with 1D transient elastography

    Energy Technology Data Exchange (ETDEWEB)

    Benech, Nicolas; Negreira, Carlos A [Laboratorio de Acustica Ultrasonora, Facultad de Ciencias, Igua 4225, 11400, Montevideo (Uruguay)

    2010-03-21

    In this paper 1D transient elastography was employed in fresh bovine skeletal muscle samples to assess the shear elastic modulus {mu} while the tissue was locally heated by means of an electrical resistance. The investigation is based on the study of the time shift of the shear wave propagation produced by the local temperature variation. The experiments show that the thermal expansion contribution to the time shift is negligible when compared with the shear wave speed variation. In such a case, the quantification of {mu} as a function of temperature becomes possible. Repeated experiments in different samples lead to a reproducible behavior of {mu} as a function of temperature. Irreversible elasticity changes are produced when the temperature exceeds a certain critical value T{sub c}. The proposed method allows estimating this value as well as the spatial extension of the resulting thermal lesion. This point is important when considering applications in monitoring focused ultrasound surgery (FUS) because the surrounding normal tissue should remain unaffected.

  16. Conformational changes in hemoglobin triggered by changing the iron charge

    Energy Technology Data Exchange (ETDEWEB)

    Croci, S., E-mail: simo@unipr.it [University of Parma, Departments of Public Health, INBB Parma (Italy); Achterhold, K. [Physik-Department E17 (Germany); Ortalli, I. [University of Parma, Departments of Public Health, INBB Parma (Italy); Parak, F. G. [Physik-Department E17 (Germany)

    2008-07-15

    In this work the hemoglobin conformational changes induced by changing the iron charge have been studied and compared with Myoglobin. Moessbauer spectroscopy was used to follow the change of the iron conformation. In order to compare the conformational relaxation of hemoglobin and myoglobin, and to study a possible influence of the quaternary structure, an intermediate metastable state of hemoglobin has been created by low temperature X-ray irradiation of methemoglobin. The irradiation reduces the Fe(III) of the heme groups to Fe(II) Low Spin, where the water is still bound on the sixth coordination. Heating cycles performed at temperatures from 140 K to 200 K allow the molecules to overcome an activation energy barrier and to relax into a stable conformation such as deoxy-hemoglobin or carboxy-hemoglobin, if CO is present. Slightly different structures (conformational substates) reveal themselves as a distribution of energy barriers ({Delta}G). The distribution of the activation energy, for the decay of the Fe(II) Low Spin intermediate, has been fitted with a Gaussian. For comparison, published myoglobin data were re-analysed in the same way. The average energy value at characteristic temperature is very similar in case of myoglobin and hemoglobin. The larger Gaussian energy distribution for myoglobin with respect to hemoglobin shows that more conformational substates are available. This may be caused by a larger area exposed to water. In hemoglobin, part of the surface of the chains is not water accessible due to the quaternary structure.

  17. Salmon calcitonin: conformational changes and stabilizer effects

    Directory of Open Access Journals (Sweden)

    Shan-Yang Lin

    2015-11-01

    Full Text Available The therapeutic activity of peptides or protein drugs is highly dependent on their conformational structure. The protein structure is flexible and responds to external conditions, which may compromise the protein's native conformation and influence its physical and chemical stability. The physical and chemical stability of peptides or protein drugs are important characteristics of biopharmaceutical products. Calcitonin (CT is a polypeptide hormone that participates in diverse physiological functions in humans; therefore, it is a potentially useful protein for investigations of different aspects of pharmacology and drug delivery systems. Of the different types of CT available for clinical use, salmon CT (sCT is one of the most potent. In this review article, the commercially available sCT was selected as a suitable peptide candidate for the discussion of its stability and conformational changes in the aqueous and solid states using Fourier transform infrared (FTIR spectroscopic analysis under different external conditions, including pH, temperature, drying method, and added excipients. Particularly, excipients that have been optimized as stabilizers of sCT in aqueous solution and as lyophilized and spray-dried drug formulations are also discussed.

  18. Light and Heat Induced Denaturation of Photosystem Ⅱ Core Antenna Complex CP47

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    Light and heat induced denaturation of CP47, the core antenna complex of photosystem Ⅱ purified from spinach, were investigated using absorption and circular dichroism spectra.Light caused the destruction of chlorophyll a and excitonic interaction of chlorophyll a in CP47, while the protein secondary structure was not apparently changed.Heat induced the destruction of protein secondary structure and excitonic interaction of chlorophyll a, but the chlorophyll a molecule was not damaged.The results suggest that both the chlorophyll a molecular structure and the protein native conformation are necessary for excitonic interaction of chlorophyll a and the energy transfer function of the chlorophyll a binding protein.

  19. Low Temperature Induced Conformation Changes of Aminoacylase

    Institute of Scientific and Technical Information of China (English)

    谢强; 孟凡国; 周海梦

    2004-01-01

    Control of aggregation, by lowering temperature and protein concentrations, can enhance the extent of successful refolding. The low temperature has been used in protein folding studies, as undesired aggregations often occur at higher temperatures. Therefore, it is very important to study the effects of low temperature on the native enzyme to help understand the factors that affect the structure of the proteins. In this paper, aminoacylase was studied at different temperatures by measuring enzyme activity, fluorescence emission spectra, and ultraviolet difference spectra. The results show that aminoacylase conformation changes as the temperature changes, becoming more compact at low temperatures, and having more secondary structural content. However, the activity is very low at low temperature, and totally diminishes at 4℃. Aminoacylase tends therefore to be more condense, with less residues exposed and low enzyme activities at low temperature. This observation might explain the self-protection of organisms under conditions of extreme temperature.

  20. Conformational changes in glycine tri- and hexapeptide

    DEFF Research Database (Denmark)

    Yakubovich, Alexander V.; Solov'yov, Ilia; Solov'yov, Andrey V.

    2006-01-01

    conformations and calculated the energy barriers for transitions between them. Using a thermodynamic approach, we have estimated the times of the characteristic transitions between these conformations. The results of our calculations have been compared with those obtained by other theoretical methods...... also investigated the influence of the secondary structure of polypeptide chains on the formation of the potential energy landscape. This analysis has been performed for the sheet and the helix conformations of chains of six amino acids....

  1. Conformational proofreading: the impact of conformational changes on the specificity of molecular recognition.

    Directory of Open Access Journals (Sweden)

    Yonatan Savir

    Full Text Available To perform recognition, molecules must locate and specifically bind their targets within a noisy biochemical environment with many look-alikes. Molecular recognition processes, especially the induced-fit mechanism, are known to involve conformational changes. This raises a basic question: Does molecular recognition gain any advantage by such conformational changes? By introducing a simple statistical-mechanics approach, we study the effect of conformation and flexibility on the quality of recognition processes. Our model relates specificity to the conformation of the participant molecules and thus suggests a possible answer: Optimal specificity is achieved when the ligand is slightly off target; that is, a conformational mismatch between the ligand and its main target improves the selectivity of the process. This indicates that deformations upon binding serve as a conformational proofreading mechanism, which may be selected for via evolution.

  2. Mapping temperature-induced conformational changes in the Escherichia coli heat shock transcription factor sigma 32 by amide hydrogen exchange

    DEFF Research Database (Denmark)

    Rist, Wolfgang; Jørgensen, Thomas J D; Roepstorff, Peter;

    2003-01-01

    gene transcription. To investigate possible heat-induced conformational changes in sigma 32 we performed amide hydrogen (H/D) exchange experiments under optimal growth and heat shock conditions combined with mass spectrometry. We found a rapid exchange of around 220 of the 294 amide hydrogens at 37...... degrees C, indicating that sigma 32 adopts a highly flexible structure. At 42 degrees C we observed a slow correlated exchange of 30 additional amide hydrogens and localized it to a helix-loop-helix motif within domain sigma 2 that is responsible for the recognition of the -10 region in heat shock...

  3. Heat-induced phytohormone changes are associated with disrupted early reproductive development and reduced yield in rice

    Science.gov (United States)

    Wu, Chao; Cui, Kehui; Wang, Wencheng; Li, Qian; Fahad, Shah; Hu, Qiuqian; Huang, Jianliang; Nie, Lixiao; Peng, Shaobing

    2016-01-01

    Heat stress causes morphological and physiological changes and reduces crop yield in rice (Oryza sativa). To investigate changes in phytohormones and their relationships with yield and other attributes under heat stress, four rice varieties (Nagina22, Huanghuazhan, Liangyoupeijiu, and Shanyou 63) were grown in pots and subjected to three high temperature treatments plus control in temperature-controlled greenhouses for 15 d during the early reproductive phase. Yield reductions in Nagina22, Huanghuazhan, and Liangyoupeijiu were attributed to reductions in spikelet fertility, spikelets per panicle, and grain weight. The adverse effects of high temperature were alleviated by application of exogenous 6-benzylaminopurine (6-BA) in the heat-susceptible Liangyoupeijiu. High temperature stress reduced active cytokinins, gibberellin A1 (GA1), and indole-3-acetic acid (IAA), but increased abscisic acid (ABA) and bound cytokinins in young panicles. Correlation analyses and application of exogenous 6-BA revealed that high temperature-induced cytokinin changes may regulate yield components by modulating the differentiation and degradation of branches and spikelets, panicle exsertion, pollen vigor, anther dehiscence, and grain size. Heat-tolerant Shanyou 63 displayed minor changes in phytohormones, panicle formation, and grain yield under high temperature compared with those of the other three varieties. These results suggest that phytohormone changes are closely associated with yield formation, and a small reduction or stability in phytohormone content is required to avoid large yield losses under heat stress. PMID:27713528

  4. Determination of the season-acclimation of photosynthetic apparatus by heat-induced changes in chlorophyll fluorescence

    Science.gov (United States)

    Nicolay Alexandrovich Gaevsky

    2002-01-01

    Resistance to damage from freezing temperatures is one of the most adaptive properties of "evergreen" (shoot bark, felloderma, needles) woody and bush plant tissues. Species-specific processes accompanying transition of chloroplasts to a frost resistant state (reduction of photochemical activity, the change of chemical structure and structural organization of...

  5. EFFECTS OF METAL IONS ON THE CONFORMATIONAL CHANGES OF DNA

    Institute of Scientific and Technical Information of China (English)

    G. Q. Liu; Y.Y. Meng; S.H. Liu; Y.H. Hu

    2005-01-01

    DNA takes on multi-different conformations such as A-, B-, C-, D- and Z-form. These conformations can transit to one another when DNA deposited in some metal ions solutions or when changing the concentrations of the same metal ions. Here, several major conformational transitions of DNA induced by metal ions under different environment were introduced and the mechanism of the interaction of metal ions with DNA was discuss in detail.

  6. A global overview of drought and heat-induced tree mortality reveals emerging climate change risks for forests

    Science.gov (United States)

    Allen, C.D.; Macalady, A.K.; Chenchouni, H.; Bachelet, D.; McDowell, N.; Vennetier, M.; Kitzberger, T.; Rigling, A.; Breshears, D.D.; Hogg, E.H.(T.); Gonzalez, P.; Fensham, R.; Zhang, Z.; Castro, J.; Demidova, N.; Lim, J.-H.; Allard, G.; Running, S.W.; Semerci, A.; Cobb, N.

    2010-01-01

    Greenhouse gas emissions have significantly altered global climate, and will continue to do so in the future. Increases in the frequency, duration, and/or severity of drought and heat stress associated with climate change could fundamentally alter the composition, structure, and biogeography of forests in many regions. Of particular concern are potential increases in tree mortality associated with climate-induced physiological stress and interactions with other climate-mediated processes such as insect outbreaks and wildfire. Despite this risk, existing projections of tree mortality are based on models that lack functionally realistic mortality mechanisms, and there has been no attempt to track observations of climate-driven tree mortality globally. Here we present the first global assessment of recent tree mortality attributed to drought and heat stress. Although episodic mortality occurs in the absence of climate change, studies compiled here suggest that at least some of the world's forested ecosystems already may be responding to climate change and raise concern that forests may become increasingly vulnerable to higher background tree mortality rates and die-off in response to future warming and drought, even in environments that are not normally considered water-limited. This further suggests risks to ecosystem services, including the loss of sequestered forest carbon and associated atmospheric feedbacks. Our review also identifies key information gaps and scientific uncertainties that currently hinder our ability to predict tree mortality in response to climate change and emphasizes the need for a globally coordinated observation system. Overall, our review reveals the potential for amplified tree mortality due to drought and heat in forests worldwide.

  7. Conformational changes in glycine tri- and hexapeptide

    CERN Document Server

    Yakubovitch, A V; Solovyov, A V; Solovyov, I A; Greiner, Walter; Solov'yov, Andrey V.; Solov'yov, Ilia A.; Yakubovitch, Alexander V.

    2005-01-01

    We have investigated the potential energy surfaces for glycine chains consisting of three and six amino acids. For these molecules we have calculated potential energy surfaces as a function of the Ramachandran angles phi and psi, which are widely used for the characterization of the polypeptide chains. These particular degrees of freedom are essential for the characterization of proteins folding process. Calculations have been carried out within ab initio theoretical framework based on the density functional theory and accounting for all the electrons in the system. We have determined stable conformations and calculated the energy barriers for transitions between them. Using a thermodynamic approach, we have estimated the times of the characteristic transitions between these conformations. The results of our calculations have been compared with those obtained by other theoretical methods and with the available experimental data extracted from the Protein Data Base. This comparison demonstrates a reasonable co...

  8. Ligand-induced conformational changes: Improved predictions of ligand binding conformations and affinities

    DEFF Research Database (Denmark)

    Frimurer, T.M.; Peters, Günther H.J.; Iversen, L.F.

    2003-01-01

    A computational docking strategy using multiple conformations of the target protein is discussed and evaluated. A series of low molecular weight, competitive, nonpeptide protein tyrosine phosphatase inhibitors are considered for which the x-ray crystallographic structures in complex with protein...... tyrosine phosphatase 1 B (PTP1B) are known. To obtain a quantitative measure of the impact of conformational changes induced by the inhibitors, these were docked to the active site region of various structures of PTP1B using the docking program FlexX. Firstly, the inhibitors were docked to a PTP1B crystal...... predicted binding energy and a correct docking mode. Thirdly, to improve the predictability of the docking procedure in the general case, where only a single target protein structure is known, we evaluate an approach which takes possible protein side-chain conformational changes into account. Here, side...

  9. Conformational diversity of wild-type Tau fibrils specified by templated conformation change.

    Science.gov (United States)

    Frost, Bess; Ollesch, Julian; Wille, Holger; Diamond, Marc I

    2009-02-06

    Tauopathies are sporadic and genetic neurodegenerative diseases characterized by aggregation of the microtubule-associated protein Tau. Tau pathology occurs in over 20 phenotypically distinct neurodegenerative diseases, including Alzheimer disease and frontotemporal dementia. The molecular basis of this diversity among sporadic tauopathies is unknown, but distinct fibrillar wild-type (WT) Tau conformations could play a role. Using Fourier transform infrared spectroscopy, circular dichroism, and electron microscopy, we show that WT Tau fibrils and P301L/V337M Tau fibrils have distinct secondary structures, fragilities, and morphologies. Furthermore, P301L/V337M fibrillar seeds induce WT Tau monomer to form a novel fibrillar conformation, termed WT*, that is maintained over multiple seeding reactions. WT* has secondary structure, fragility, and morphology that are similar to P301L/V337M fibrils and distinct from WT fibrils. WT Tau is thus capable of conformational diversity that arises via templated conformation change, as has been described for amyloid beta, beta2-microglobulin, and prion proteins.

  10. Interfacial adsorption of insulin - Conformational changes and reversibility of adsorption

    NARCIS (Netherlands)

    Mollmann, SH; Jorgensen, L; Bukrinsky, JT; Elofsson, U; Norde, W; Frokjaer, S

    2006-01-01

    The adsorption of human insulin to Teflon particles was studied with respect to conformational changes and the reversibility of adsorption was examined by total internal reflection fluorescence (TIRF). Adsorption isotherms for the adsorption of human insulin indicated high affinity adsorption, even

  11. Interfacial adsorption of insulin. Conformational changes and reversibility of adsorption

    NARCIS (Netherlands)

    Mollmann, S.H.; Bukrinsky, J.T.; Elofsson, U.; Norde, W.; Frokjaer, S.

    2006-01-01

    The adsorption of human insulin to Teflon particles was studied with respect to conformational changes and the reversibility of adsorption was examined by total internal reflection fluorescence (TIRF). Adsorption isotherms for the adsorption of human insulin indicated high affinity adsorption, even

  12. A Unifying Hypothesis for the Conformational Change of Tubulin

    CERN Document Server

    Fygenson, D K

    2001-01-01

    Microtubule dynamic instability arises from the hydrolysis of GTP bound to the beta-monomer of the tubulin dimer. The conformational change induced by hydrolysis is unknown, but microtubules disassemble into protofilaments of GDP-bound tubulin that curve away from the microtubule axis. This paper presents the unfolding of a portion of the tubulin molecule into the microtubule interior as a plausible, unifying explanation for diverse structural and kinetic features of microtubules. This is the first specific structural hypothesis for the hydrolysis induced conformational change of tubulin that simultaneously explains weakening of lateral bonds, bending about longitudinal bonds, changes in protofilament supertwist associated with GTP hydrolysis, structural features of GDP-tubulin double rings, faster disassembly at higher temperatures and slower disassembly in the presence of glycerol and deuterium oxide. The hypothesis suggests further theoretical investigations and direct experimental tests.

  13. A chemical chaperone induces inhomogeneous conformational changes in flexible proteins.

    Science.gov (United States)

    Hamdane, Djemel; Velours, Christophe; Cornu, David; Nicaise, Magali; Lombard, Murielle; Fontecave, Marc

    2016-07-27

    Organic osmolytes also known as chemical chaperones are major cellular compounds that favor, by an unclear mechanism, protein's compaction and stabilization of the native state. Here, we have examined the chaperone effect of the naturally occurring trimethylamine N-oxide (TMAO) osmolyte on a loosely packed protein (LPP), known to be a highly flexible form, using an apoprotein mutant of the flavin-dependent RNA methyltransferase as a model. Thermal and chemical denaturation experiments showed that TMAO stabilizes the structural integrity of the apoprotein dramatically. The denaturation reaction is irreversible indicating that the stability of the apoprotein is under kinetic control. This result implies that the stabilization is due to a TMAO-induced reconfiguration of the flexible LPP state, which leads to conformational limitations of the apoprotein likely driven by favorable entropic contribution. Evidence for the conformational perturbation of the apoprotein had been obtained through several biophysical approaches notably analytical ultracentrifugation, circular dichroism, fluorescence spectroscopy, labelling experiments and proteolysis coupled to mass spectrometry. Unexpectedly, TMAO promotes an overall elongation or asymmetrical changes of the hydrodynamic shape of the apoprotein without alteration of the secondary structure. The modulation of the hydrodynamic properties of the protein is associated with diverse inhomogenous conformational changes: loss of the solvent accessible cavities resulting in a dried protein matrix; some side-chain residues initially buried become solvent exposed while some others become hidden. Consequently, the TMAO-induced protein state exhibits impaired capability in the flavin binding process. Our study suggests that the nature of protein conformational changes induced by the chemical chaperones may be specific to protein packing and plasticity. This could be an efficient mechanism by which the cell controls and finely tunes the

  14. Conformational changes of fibrinogen in dispersed carbon nanotubes

    Directory of Open Access Journals (Sweden)

    Park SJ

    2012-08-01

    Full Text Available Sung Jean Park,1 Dongwoo Khang21College of Pharmacy, Gachon University, Yeonsu-gu, Incheon, South Korea; 2School of Nano and Advanced Materials Science Engineering and Center for PRC and RIGET, Gyeongsang National University, Jinju, South KoreaAbstract: The conformational changes of plasma protein structures in response to carbon nanotubes are critical for determining the nanotoxicity and blood coagulation effects of carbon nanotubes. In this study, we identified that the functional intensity of carboxyl groups on carbon nanotubes, which correspond to the water dispersity or hydrophilicity of carbon nanotubes, can induce conformational changes in the fibrinogen domains. Also, elevation of carbon nanotube density can alter the secondary structures (ie, helices and beta sheets of fibrinogen. Furthermore, fibrinogen that had been in contact with the nanoparticle material demonstrated a different pattern of heat denaturation compared with free fibrinogen as a result of a variation in hydrophilicity and concentration of carbon nanotubes. Considering the importance of interactions between carbon nanotubes and plasma proteins in the drug delivery system, this study elucidated the correlation between nanoscale physiochemical material properties of carbon nanotubes and associated structural changes in fibrinogen.Keywords: carbon nanotubes, fibrinogen, nanotoxicity, conformational change, denaturation

  15. A phenomenological relationship between molecular geometry change and conformational energy change

    Science.gov (United States)

    Bodi, Andras; Bjornsson, Ragnar; Arnason, Ingvar

    2010-08-01

    A linear correlation is established between the change in the axial/equatorial conformational energy difference and the change in the molecular geometry transformation during conformational inversion in substituted six-membered ring systems, namely in the 1-substituted cyclohexane/silacyclohexane, cyclohexane/ N-substituted piperidine and 1-substituted silacyclohexane/ P-substituted phosphorinane compound families, and for the analogous gauche/anti conformational isomerism in 1-substituted propanes/1-silapropanes. The nuclear repulsion energy parameterizes the molecular geometry, and changes in the conformational energy between the related compound families are linearly correlated with the changes in the nuclear repulsion energy difference based on DFT (B3LYP, M06-2X), G3B3, and CBS-QB3 calculations. This correlation reproduces the sometimes remarkable contrast between the conformational behavior of analogous compounds, e.g., the lack of a general equatorial preference in silacyclohexanes.

  16. Structure fluctuations and conformational changes in protein binding

    CERN Document Server

    Ruvinsky, Anatoly M; Tuzikov, Alexander V; Vakser, Ilya A

    2011-01-01

    Structure fluctuations and conformational changes accompany all biological processes involving macromolecules. The paper presents a classification of protein residues based on the normalized equilibrium fluctuations of the residue centers of mass in proteins and a statistical analysis of conformation changes in the side-chains upon binding. Normal mode analysis and an elastic network model were applied to a set of protein complexes to calculate the residue fluctuations and develop the residue classification. Comparison with a classification based on normalized B-factors suggests that the B-factors may underestimate protein flexibility in solvent. Our classification shows that protein loops and disordered fragments are enriched with highly fluctuating residues and depleted with weakly fluctuating residues. To calculate the dihedral angles distribution functions, the configuration space was divided into cells by a cubic grid. The effect of protein association on the distribution functions depends on the amino a...

  17. Chiral switching by spontaneous conformational change in adsorbed organic molecules.

    Science.gov (United States)

    Weigelt, Sigrid; Busse, Carsten; Petersen, Lars; Rauls, Eva; Hammer, Bjørk; Gothelf, Kurt V; Besenbacher, Flemming; Linderoth, Trolle R

    2006-02-01

    Self-assembly of adsorbed organic molecules is a promising route towards functional surface nano-architectures, and our understanding of associated dynamic processes has been significantly advanced by several scanning tunnelling microscopy (STM) investigations. Intramolecular degrees of freedom are widely accepted to influence ordering of complex adsorbates, but although molecular conformation has been identified and even manipulated by STM, the detailed dynamics of spontaneous conformational change in adsorbed molecules has hitherto not been addressed. Molecular surface structures often show important stereochemical effects as, aside from truly chiral molecules, a large class of so-called prochiral molecules become chiral once confined on a surface with an associated loss of symmetry. Here, we investigate a model system in which adsorbed molecules surprisingly switch between enantiomeric forms as they undergo thermally induced conformational changes. The associated kinetic parameters are quantified from time-resolved STM data whereas mechanistic insight is obtained from theoretical modelling. The chiral switching is demonstrated to enable an efficient channel towards formation of extended homochiral surface domains. Our results imply that appropriate prochiral molecules may be induced (for example, by seeding) to assume only one enantiomeric form in surface assemblies, which is of relevance for chiral amplification and asymmetric heterogenous catalysis.

  18. Exciton Coupling of Phenylalanine Reveals Conformational Changes of Cationic Peptides

    DEFF Research Database (Denmark)

    Bortolini, Christian; Liu, Lei; Hoffmann, Soren V.

    2017-01-01

    Circular dichroism (CD) is a versatile tool to investigate the secondary structure of proteins. Conventionally, CD signals in the far-UV region are primarily attributed to peptide bond absorption; likewise aromatic residue analysis has typically only focussed on the near-UV absorption characteris...... of overcoming antimicrobial resistance......., we strive towards a quantitative interpretation of CD spectra by detailing the contributions of aromatic chromophores in the far-UV and accurately describing unfolded states of charged amino acid side chains. To this end, we probe conformational changes of cationic peptides, which impact...

  19. Water Evaporation and Conformational Changes from Partially Solvated Ubiquitin

    Directory of Open Access Journals (Sweden)

    Saravana Prakash Thirumuruganandham

    2010-01-01

    Full Text Available Using molecular dynamics simulation, we study the evaporation of water molecules off partially solvated ubiquitin. The evaporation and cooling rates are determined for a molecule at the initial temperature of 300 K. The cooling rate is found to be around 3 K/ns, and decreases with water temperature in the course of the evaporation. The conformation changes are monitored by studying a variety of intermediate partially solvated ubiquitin structures. We find that ubiquitin shrinks with decreasing hydration shell and exposes more of its hydrophilic surface area to the surrounding.

  20. Enthalpy-Entropy Compensation upon Molecular Conformational Changes.

    Science.gov (United States)

    Ahmad, Mazen; Helms, Volkhard; Lengauer, Thomas; Kalinina, Olga V

    2015-04-14

    The change in free energy is the dominant factor in all chemical processes; it usually encompasses enthalpy-entropy compensation (EEC). Here, we use the free energy perturbation formalism to show that EEC is influenced by the molecular conformational changes (CCs) of the entire system comprising the solute and by the already known solvent reorganization. The internal changes of enthalpy and the entropy due to CCs upon modifying the interactions (perturbation) cancel each other exactly. The CCs influence the dissipation of the modified interactions and their contributions to the free energy. Using molecular simulations, we show that, for solvation of six different HIV-1 protease inhibitors, CCs in the solute cause EEC as large as 10-30 kcal/mol. Moreover, the EEC due to CCs in HIV-1 protease is shown to vary significantly upon modifying its bound ligand. These findings have important implications for understanding of EEC phenomena and for interpretation of thermodynamic measurements.

  1. A spin label study of conformational changes in cytochrome c.

    Science.gov (United States)

    Postnikova, G B; Gorbunova, N P; Volkenstein, M V

    1983-04-01

    Spin-labeled pig heart cytochromes c singly modified at Met-65, Tyr-74 and at one of the lysine residues, Lys-72 or Lys-73, were investigated by the ESR method under conditions of different ligand and redox states of the heme and at various pH values. Replacement of Met-80 by the external ligand, cyanide, was shown to produce a sharp increase in the mobility of all the three bound labels while reduction of the spin-labeled ferricytochromes c did not cause any marked changes in their ESR spectra. In the pH range 6-13, two conformational transitions in ferricytochrome c were observed which preceded its alkaline denaturation: the first with pK 9.3 registered by the spin label at the Met-65 position, and the second with pK 11.1 registered by the labels bound to Tyr-74 and Lys-72(73). The conformational changes in the 'left-hand part' of ferricytochrome c are most probably induced in both cases by the exchange of internal protein ligands at the sixth coordination site of the heme.

  2. S-nitrosylation-induced conformational change in blackfin tuna myoglobin.

    Science.gov (United States)

    Schreiter, Eric R; Rodríguez, María M; Weichsel, Andrzej; Montfort, William R; Bonaventura, Joseph

    2007-07-06

    S-nitrosylation is a post-translational protein modification that can alter the function of a variety of proteins. Despite the growing wealth of information that this modification may have important functional consequences, little is known about the structure of the moiety or its effect on protein tertiary structure. Here we report high-resolution x-ray crystal structures of S-nitrosylated and unmodified blackfin tuna myoglobin, which demonstrate that in vitro S-nitrosylation of this protein at the surface-exposed Cys-10 directly causes a reversible conformational change by "wedging" apart a helix and loop. Furthermore, we have demonstrated in solution and in a single crystal that reduction of the S-nitrosylated myoglobin with dithionite results in NO cleavage from the sulfur of Cys-10 and rebinding to the reduced heme iron, showing the reversibility of both the modification and the conformational changes. Finally, we report the 0.95-A structure of ferrous nitrosyl myoglobin, which provides an accurate structural view of the NO coordination geometry in the context of a globin heme pocket.

  3. Exploring ribozyme conformational changes with X-ray crystallography.

    Science.gov (United States)

    Spitale, Robert C; Wedekind, Joseph E

    2009-10-01

    Relating three-dimensional fold to function is a central challenge in RNA structural biology. Toward this goal, X-ray crystallography has long been considered the "gold standard" for structure determinations at atomic resolution, although NMR spectroscopy has become a powerhouse in this arena as well. In the area of dynamics, NMR remains the dominant technique to probe the magnitude and timescales of molecular motion. Although the latter area remains largely unassailable by conventional crystallographic methods, inroads have been made on proteins using Laue radiation on timescales of ms to ns. Proposed 'fourth generation' radiation sources, such as free-electron X-ray lasers, promise ps- to fs-timescale resolution, and credible evidence is emerging that supports the feasibility of single molecule imaging. At present however, the preponderance of RNA structural information has been derived from timescale and motion insensitive crystallographic techniques. Importantly, developments in computing, automation and high-flux synchrotron sources have propelled the rapidity of 'conventional' RNA crystal structure determinations to timeframes of hours once a suitable set of phases is obtained. With a sufficient number of crystal structures, it is possible to create a structural ensemble that can provide insight into global and local molecular motion characteristics that are relevant to biological function. Here we describe techniques to explore conformational changes in the hairpin ribozyme, a representative non-protein-coding RNA catalyst. The approaches discussed include: (i) construct choice and design using prior knowledge to improve X-ray diffraction; (ii) recognition of long-range conformational changes and (iii) use of single-base or single-atom changes to create ensembles. The methods are broadly applicable to other RNA systems.

  4. Large nucleotide-dependent conformational change in Rab28

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Sung Haeng; Baek, Kyuwon; Dominguez, Roberto (UPENN-MED)

    2008-12-01

    Rab GTPases are essential regulators of membrane trafficking. We report crystal structures of Rab28 in the active (GppNHp-bound) and inactive (GDP-3'P-bound) forms at 1.5 and 1.1 {angstrom} resolution. Rab28 is a distant member of the Rab family. While the overall fold of Rab28 resembles that of other Rab GTPases, it undergoes a larger nucleotide-dependent conformational change than other members of this family. Added flexibility resulting from a double-glycine motif at the beginning of switch 2 might partially account for this observation. The double-glycine motif, which is conserved in the Arf family, only occurs in Rab28 and Rab7B of the Rab family, and may have a profound effect on their catalytic activities.

  5. The concerted conformational changes during human rhinovirus 2 uncoating.

    Science.gov (United States)

    Hewat, Elizabeth A; Neumann, Emmanuelle; Blaas, Dieter

    2002-08-01

    Delivery of the rhinovirus genome into the cytoplasm involves a cooperative structural modification of the viral capsid. We have studied this phenomenon for human rhinovirus serotype 2 (HRV2). The structure of the empty capsid has been determined to a resolution of better than 15 A by cryo-electron microscopy, and the atomic structure of native HRV2 was used to examine conformational changes of the capsid. The two proteins around the 5-fold axes make an iris type of movement to open a 10 A diameter channel which allows the RNA genome to exit, and the N terminus of VP1 exits the capsid at the pseudo 3-fold axis. A remarkable modification occurs at the 2-fold axes where the N-terminal loop of VP2 bends inward, probably to detach the RNA.

  6. Conformation change of enzyme molecules in laser radiation field

    Science.gov (United States)

    Leshenyuk, N. S.; Prigun, M. V.; Apanasevitsh, E. E.; Kruglik, G. S.

    2007-06-01

    As a result of an analysis of macromolecules properties in the coherent optical radiation field and with allowance for the experimentally obtained unique data on the interaction of lazer radiation with biomolecules (dependence of the interaction efficiency on the coherence length, presence of the effect in the spectra region far from the absorption band), a mechanism of wave interaction is developed. Using this mathematical model, the calculations of a change in the macromolecules oscillatory energy in the coherent radiation field are performed. It is shown that the increase of macromolecules oscillatory energy depends strongly on the coherence length of radiation. On exposure to noncoherent radiation, the biomolecules oscillatory energy practically does not change, whereas on exposure to laser radiation (coherence length ~3 cm), energy of oscillations of atoms increases by an order of 2÷4, which results in a change in the conformation of biomolecules and activity of enzymes. Recently a lot of data are received concerning the change of lysosomal enzymes activity in blood plasma under action of laser radiation.

  7. Probing conformation and conformational change in proteins is optimally undertaken in relative mode.

    Science.gov (United States)

    Errington, Neil; Rowe, Arthur J

    2003-08-01

    Hydrodynamic bead modelling has been widely used in attempts to assess the 3D conformation of proteins in solution. Initially, simple models employing only a small number of beads were used, with a considerable degree of success. Latterly, high-resolution bead models based upon atomic coordinates have been developed, and much more sophisticated questions can in principle be addressed. A detailed analysis is presented of the errors involved in the generation of such models and associated prediction of (translational friction) parameters, and in the practical measurement of these parameters for comparison. It is shown that in most cases, for a particle of only moderate asymmetry, the errors are such that it is not feasible to determine, on an absolute basis, which of a range of candidate conformers is the "correct" one. However, when the properties of the candidate conformers can be compared in relation to those of a "paradigm conformer", whose structure in solution, on the basis of external evidence, can be accepted as correct, then errors cancel and very precise comparisons become possible. The generation of 3D bead models (and hence 3D data files) for a range of candidate conformers is a simple matter, using the existing program MacBEADS, further facilitated by a 3D display module (pro Fit).

  8. Ligand-Induced Conformational Changes and Conformational Dynamics in the Solution Structure of the Lactose Repressor Protein

    Science.gov (United States)

    Taraban, Marc; Zhan, Hongli; Whitten, Andrew E.; Langley, David B.; Matthews, Kathleen S.; Swint-Kruse, Liskin; Trewhella, Jill

    2008-01-01

    SUMMARY We present here the results of a series of small-angle X-ray scattering studies aimed at understanding the role of conformational changes and structural flexibility in DNA binding and allosteric signaling in a bacterial transcription regulator, Lactose repressor protein (LacI). Experiments were designed to detect possible conformational changes that occur when LacI binds either DNA or the inducer IPTG, or both. Our studies included the native LacI dimer of homodimers and a dimeric variant (R3), enabling us to probe conformational changes within the homodimers and distinguish them from those involving changes in the homodimer-homodimer relationships. The scattering data indicate that removal of operator DNA (oDNA) from R3 results in an unfolding and extension of the hinge-helix that connects the LacI regulatory and DNA-binding domains. In contrast, only very subtle conformational changes occur in the R3 dimer-oDNA complex upon IPTG binding, indicative of small adjustments in the orientations of domains and/or sub-domains within the structure. The binding of IPTG to native (tetrameric) LacI-oDNA complexes also appears to facilitate a modest change in the average homodimer-homodimer disposition. Notably, the crystal structure of the native LacI-oDNA complex differs significantly from the average solution conformation. The solution scattering data are best-fit by an ensemble of structures that includes (1) ~60% of the V-shaped dimer-of-homodimers observed in the crystal structure, and (2) ~40% of molecules with more “open” forms, such as those generated when the homodimers move with respect to each other about the tetramerization domain. In gene regulation, such a flexible LacI would be beneficial for the interaction of its two DNA binding domains, positioned at the tips of the V, with the required two of three LacI operators needed for full repression. PMID:18164724

  9. Probing structural evolution in heat induced protein gelation by scattering techniques

    Science.gov (United States)

    Kumar, Sugam; Piplani, Pulkit; Mehan, S.; Aswal, V. K.

    2017-05-01

    The heating of a globular protein is known to elicit conformational changes in the protein molecules, resulting in the formation of a gel depending on the solution conditions. We have used dynamic light scattering (DLS) and small-angle neutron scattering (SANS) to investigate the phase behavior and structure evolution in heat induced gelation of Bovine Serum Albumin (BSA) protein as a function of pH and ionic strength. The gelation temperature is found to be increasing with increase in pH and decrease in ionic strength. The structure of the protein molecule remains intact very close to the gelation temperature. However, on further increase in temperature, the protein molecules form small aggregates which eventually lead to a network gel at gelation temperature. The gel structure is characterized by a mass fractal having a fractal dimension about 2.

  10. Ligand-driven conformational changes of MurD visualized by paramagnetic NMR.

    Science.gov (United States)

    Saio, Tomohide; Ogura, Kenji; Kumeta, Hiroyuki; Kobashigawa, Yoshihiro; Shimizu, Kazumi; Yokochi, Masashi; Kodama, Kota; Yamaguchi, Hiroto; Tsujishita, Hideki; Inagaki, Fuyuhiko

    2015-11-19

    Proteins, especially multi-domain proteins, often undergo drastic conformational changes upon binding to ligands or by post-translational modifications, which is a key step to regulate their function. However, the detailed mechanisms of such dynamic regulation of the functional processes are poorly understood because of the lack of an efficient tool. We here demonstrate detailed characterization of conformational changes of MurD, a 47 kDa protein enzyme consisting of three domains, by the use of solution NMR equipped with paramagnetic lanthanide probe. Quantitative analysis of pseudocontact shifts has identified a novel conformational state of MurD, named semi-closed conformation, which is found to be the key to understand how MurD regulates the binding of the ligands. The modulation of the affinity coupled with conformational changes accentuates the importance of conformational state to be evaluated in drug design.

  11. Conformational changes in DNA gyrase revealed by limited proteolysis

    DEFF Research Database (Denmark)

    Kampranis, S C; Maxwell, A

    1998-01-01

    -induced dimerization of the B subunits is a key step of the gyrase mechanism. The proteolytic fingerprint of this conformation (stabilized by the non-hydrolyzable ATP analog 5'-adenylyl-beta, gamma-imidodiphosphate (ADPNP) shows a protection of the 43-kDa N-terminal domain of the B subunit. The presence of quinolones...

  12. Effects of lipid environment on the conformational changes of an ABC importer.

    Science.gov (United States)

    Rice, Austin J; Alvarez, Frances J D; Davidson, Amy L; Pinkett, Heather W

    2014-01-01

    In order to shuttle substrates across the lipid bilayer, membrane proteins undergo a series of conformation changes that are influenced by protein structure, ligands, and the lipid environment. To test the effect of lipid on conformation change of the ABC transporter MolBC, EPR studies were conducted in lipids and detergents of variable composition. In both a detergent and lipid environment, MolBC underwent the same general conformation changes as detected by site-directed EPR spectroscopy. However, differences in activity and the details of the EPR analysis indicate conformational rigidity that is dependent on the lipid environment. From these observations, we conclude that native-like lipid mixtures provide the transporter with greater activity and conformational flexibility as well as technical advantages such as reconstitution efficiency and protein stability.

  13. Heat Induced Damage Detection by Terahertz (THz) Radiation

    Science.gov (United States)

    Rahani, Ehsan Kabiri; Kundu, Tribikram; Wu, Ziran; Xin, Hao

    2011-06-01

    Terahertz (THz) and sub-terahertz imaging and spectroscopy are becoming increasingly popular nondestructive evaluation techniques for damage detection and characterization of materials. THz radiation is being used for inspecting ceramic foam tiles used in TPS (Thermal Protection System), thick polymer composites and polymer tiles that are not good conductors of ultrasonic waves. Capability of THz electromagnetic waves in detecting heat induced damage in porous materials is investigated in this paper. Porous pumice stone blocks are subjected to long time heat exposures to produce heat induced damage in the block. The dielectric properties extracted from THz TDS (Time Domain Spectroscopy) measurements are compared for different levels of heat exposure. Experimental results show noticeable and consistent change in dielectric properties with increasing levels of heat exposure, well before its melting point.

  14. Transglutaminase 2 undergoes a large conformational change upon activation.

    Directory of Open Access Journals (Sweden)

    Daniel M Pinkas

    2007-12-01

    Full Text Available Human transglutaminase 2 (TG2, a member of a large family of enzymes that catalyze protein crosslinking, plays an important role in the extracellular matrix biology of many tissues and is implicated in the gluten-induced pathogenesis of celiac sprue. Although vertebrate transglutaminases have been studied extensively, thus far all structurally characterized members of this family have been crystallized in conformations with inaccessible active sites. We have trapped human TG2 in complex with an inhibitor that mimics inflammatory gluten peptide substrates and have solved, at 2-A resolution, its x-ray crystal structure. The inhibitor stabilizes TG2 in an extended conformation that is dramatically different from earlier transglutaminase structures. The active site is exposed, revealing that catalysis takes place in a tunnel, bridged by two tryptophan residues that separate acyl-donor from acyl-acceptor and stabilize the tetrahedral reaction intermediates. Site-directed mutagenesis was used to investigate the acyl-acceptor side of the tunnel, yielding mutants with a marked increase in preference for hydrolysis over transamidation. By providing the ability to visualize this activated conformer, our results create a foundation for understanding the catalytic as well as the non-catalytic roles of TG2 in biology, and for dissecting the process by which the autoantibody response to TG2 is induced in celiac sprue patients.

  15. Evidence for protein conformational change at a Au(110)/protein interface

    Science.gov (United States)

    Messiha, H. L.; Smith, C. I.; Scrutton, N.S.; Weightman, P.

    2008-01-01

    Evidence is presented that reflection anisotropy spectroscopy (RAS) can provide real-time measurements of conformational change in proteins induced by electron transfer reactions. A bacterial electron transferring flavoprotein (ETF) has been modified so as to adsorb on an Au(110) electrode and enable reversible electron transfer to the protein cofactor in the absence of mediators. Reversible changes are observed in the RAS of this protein that are interpreted as arising from conformational changes accompanying the transfer of electrons. PMID:19325933

  16. Triggered drug release from dynamic microspheres via a protein conformational change.

    Science.gov (United States)

    King, William J; Pytel, Nicholas J; Ng, Kelvin; Murphy, William L

    2010-06-11

    In this study we formed and characterized dynamic hydrogel microspheres in which a protein conformational change was used to control microsphere volume changes and the release of an encapsulated drug. In particular, a specific biochemical ligand, trifluoperazine, induced calmodulin's nanometer scale conformation change, which translated to a 48.7% microsphere volume decrease. This specific, ligand-induced volume change triggered the release of a model drug, vascular endothelial growth factor (VEGF), at pre-determined times. After release from the microspheres, 85.6 +/- 10.5% of VEGF was in its native conformation. Taken together, these results suggest that protein conformational change could serve as a useful mechanism to control drug release from dynamic hydrogels.

  17. Ligand induced conformational changes of the human serotonin transporter revealed by molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Heidi Koldsø

    Full Text Available The competitive inhibitor cocaine and the non-competitive inhibitor ibogaine induce different conformational states of the human serotonin transporter. It has been shown from accessibility experiments that cocaine mainly induces an outward-facing conformation, while the non-competitive inhibitor ibogaine, and its active metabolite noribogaine, have been proposed to induce an inward-facing conformation of the human serotonin transporter similar to what has been observed for the endogenous substrate, serotonin. The ligand induced conformational changes within the human serotonin transporter caused by these three different types of ligands, substrate, non-competitive and competitive inhibitors, are studied from multiple atomistic molecular dynamics simulations initiated from a homology model of the human serotonin transporter. The results reveal that diverse conformations of the human serotonin transporter are captured from the molecular dynamics simulations depending on the type of the ligand bound. The inward-facing conformation of the human serotonin transporter is reached with noribogaine bound, and this state resembles a previously identified inward-facing conformation of the human serotonin transporter obtained from molecular dynamics simulation with bound substrate, but also a recently published inward-facing conformation of a bacterial homolog, the leucine transporter from Aquifex Aoelicus. The differences observed in ligand induced behavior are found to originate from different interaction patterns between the ligands and the protein. Such atomic-level understanding of how an inhibitor can dictate the conformational response of a transporter by ligand binding may be of great importance for future drug design.

  18. Ligand induced conformational changes of the human serotonin transporter revealed by molecular dynamics simulations.

    Science.gov (United States)

    Koldsø, Heidi; Autzen, Henriette Elisabeth; Grouleff, Julie; Schiøtt, Birgit

    2013-01-01

    The competitive inhibitor cocaine and the non-competitive inhibitor ibogaine induce different conformational states of the human serotonin transporter. It has been shown from accessibility experiments that cocaine mainly induces an outward-facing conformation, while the non-competitive inhibitor ibogaine, and its active metabolite noribogaine, have been proposed to induce an inward-facing conformation of the human serotonin transporter similar to what has been observed for the endogenous substrate, serotonin. The ligand induced conformational changes within the human serotonin transporter caused by these three different types of ligands, substrate, non-competitive and competitive inhibitors, are studied from multiple atomistic molecular dynamics simulations initiated from a homology model of the human serotonin transporter. The results reveal that diverse conformations of the human serotonin transporter are captured from the molecular dynamics simulations depending on the type of the ligand bound. The inward-facing conformation of the human serotonin transporter is reached with noribogaine bound, and this state resembles a previously identified inward-facing conformation of the human serotonin transporter obtained from molecular dynamics simulation with bound substrate, but also a recently published inward-facing conformation of a bacterial homolog, the leucine transporter from Aquifex Aoelicus. The differences observed in ligand induced behavior are found to originate from different interaction patterns between the ligands and the protein. Such atomic-level understanding of how an inhibitor can dictate the conformational response of a transporter by ligand binding may be of great importance for future drug design.

  19. Furan: A critical heat induced dietary contaminant

    DEFF Research Database (Denmark)

    Mariotti, María S.; Granby, Kit; Rozowski, Jaime

    2013-01-01

    The presence of furan in a broad range of heat processed foods (0-6000 μg kg-1) has received considerable attention due to the fact that this heat induced contaminant is considered as a "possible carcinogenic compound to humans". Since a genotoxic mode of action could be associated with furan...

  20. 76 FR 4244 - Source Rules Involving U.S. Possessions and Other Conforming Changes; Correction

    Science.gov (United States)

    2011-01-25

    ... Federal Register on Wednesday, April 9, 2008 (73 FR 19350) providing rules under section 937(b) of the... Conforming Changes; Correction AGENCY: Internal Revenue Service (IRS), Treasury. ACTION: Correcting...

  1. Assessing Energy-Dependent Protein Conformational Changes in the TonB System.

    Science.gov (United States)

    Larsen, Ray A

    2017-01-01

    Changes in conformation can alter a protein's vulnerability to proteolysis. Thus, in vivo differential proteinase sensitivity provides a means for identifying conformational changes that mark discrete states in the activity cycle of a protein. The ability to detect a specific conformational state allows for experiments to address specific protein-protein interactions and other physiological components that potentially contribute to the function of the protein. This chapter presents the application of this technique to the TonB-dependent energy transduction system of Gram-negative bacteria, a strategy that has refined our understanding of how the TonB protein is coupled to the ion electrochemical gradient of the cytoplasmic membrane.

  2. 47 CFR 68.348 - Changes in equipment and circuitry subject to a Supplier's Declaration of Conformity.

    Science.gov (United States)

    2010-10-01

    ... Supplier's Declaration of Conformity. 68.348 Section 68.348 Telecommunication FEDERAL COMMUNICATIONS... a Supplier's Declaration of Conformity. (a) No change shall be made in terminal equipment or... Declaration of Conformity Statement furnished to users. (b) Any other changes in terminal equipment...

  3. The Structure of Lombricine Kinase: Implications for Phosphagen Conformational Changes

    Energy Technology Data Exchange (ETDEWEB)

    Bush, D. Jeffrey; Kirillova, Olga; Clark, Shawn A.; Davulcu, Omar; Fabiola, Felcy; Xie, Qing; Somasundaram, Thayumanasamy; Ellington, W. Ross; Chapman, Michael S. (Oregon HSU); (FSU)

    2012-05-29

    Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309-317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His{sup 178}. Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.

  4. Mg(2+)-induced conformational changes in the btuB riboswitch from E. coli.

    Science.gov (United States)

    Choudhary, Pallavi K; Sigel, Roland K O

    2014-01-01

    Mg(2+) has been shown to modulate the function of riboswitches by facilitating the ligand-riboswitch interactions. The btuB riboswitch from Escherichia coli undergoes a conformational change upon binding to its ligand, coenzyme B12 (adenosyl-cobalamine, AdoCbl), and down-regulates the expression of the B12 transporter protein BtuB in order to control the cellular levels of AdoCbl. Here, we discuss the structural folding attained by the btuB riboswitch from E. coli in response to Mg(2+) and how it affects the ligand binding competent conformation of the RNA. The btuB riboswitch notably adopts different conformational states depending upon the concentration of Mg(2+). With the help of in-line probing, we show the existence of at least two specific conformations, one being achieved in the complete absence of Mg(2+) (or low Mg(2+) concentration) and the other appearing above ∼0.5 mM Mg(2+). Distinct regions of the riboswitch exhibit different dissociation constants toward Mg(2+), indicating a stepwise folding of the btuB RNA. Increasing the Mg(2+) concentration drives the transition from one conformation toward the other. The conformational state existing above 0.5 mM Mg(2+) defines the binding competent conformation of the btuB riboswitch which can productively interact with the ligand, coenzyme B12, and switch the RNA conformation. Moreover, raising the Mg(2+) concentration enhances the ratio of switched RNA in the presence of AdoCbl. The lack of a AdoCbl-induced conformational switch experienced by the btuB riboswitch in the absence of Mg(2+) indicates a crucial role played by Mg(2+) for defining an active conformation of the riboswitch.

  5. Predicting protein conformational changes for unbound and homology docking: learning from intrinsic and induced flexibility.

    Science.gov (United States)

    Chen, Haoran; Sun, Yuanfei; Shen, Yang

    2017-03-01

    Predicting protein conformational changes from unbound structures or even homology models to bound structures remains a critical challenge for protein docking. Here we present a study directly addressing the challenge by reducing the dimensionality and narrowing the range of the corresponding conformational space. The study builds on cNMA-our new framework of partner- and contact-specific normal mode analysis that exploits encounter complexes and considers both intrinsic and induced flexibility. First, we established over a CAPRI (Critical Assessment of PRedicted Interactions) target set that the direction of conformational changes from unbound structures and homology models can be reproduced to a great extent by a small set of cNMA modes. In particular, homology-to-bound interface root-mean-square deviation (iRMSD) can be reduced by 40% on average with the slowest 30 modes. Second, we developed novel and interpretable features from cNMA and used various machine learning approaches to predict the extent of conformational changes. The models learned from a set of unbound-to-bound conformational changes could predict the actual extent of iRMSD with errors around 0.6 Å for unbound proteins in a held-out benchmark subset, around 0.8 Å for unbound proteins in the CAPRI set, and around 1 Å even for homology models in the CAPRI set. Our results shed new insights into origins of conformational differences between homology models and bound structures and provide new support for the low-dimensionality of conformational adjustment during protein associations. The results also provide new tools for ensemble generation and conformational sampling in unbound and homology docking. Proteins 2017; 85:544-556. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  6. Two Na+ Sites Control Conformational Change in a Neurotransmitter Transporter Homolog.

    Science.gov (United States)

    Tavoulari, Sotiria; Margheritis, Eleonora; Nagarajan, Anu; DeWitt, David C; Zhang, Yuan-Wei; Rosado, Edwin; Ravera, Silvia; Rhoades, Elizabeth; Forrest, Lucy R; Rudnick, Gary

    2016-01-15

    In LeuT, a prokaryotic homolog of neurotransmitter transporters, Na(+) stabilizes outward-open conformational states. We examined how each of the two LeuT Na(+) binding sites contributes to Na(+)-dependent closure of the cytoplasmic pathway using biochemical and biophysical assays of conformation. Mutating either of two residues that contribute to the Na2 site completely prevented cytoplasmic closure in response to Na(+), suggesting that Na2 is essential for this conformational change, whereas Na1 mutants retained Na(+) responsiveness. However, mutation of Na1 residues also influenced the Na(+)-dependent conformational change in ways that varied depending on the position mutated. Computational analyses suggest those mutants influence the ability of Na1 binding to hydrate the substrate pathway and perturb an interaction network leading to the extracellular gate. Overall, the results demonstrate that occupation of Na2 stabilizes outward-facing conformations presumably through a direct interaction between Na(+) and transmembrane helices 1 and 8, whereas Na(+) binding at Na1 influences conformational change through a network of intermediary interactions. The results also provide evidence that N-terminal release and helix motions represent distinct steps in cytoplasmic pathway opening.

  7. Two Na+ Sites Control Conformational Change in a Neurotransmitter Transporter Homolog*

    Science.gov (United States)

    Tavoulari, Sotiria; Margheritis, Eleonora; Nagarajan, Anu; DeWitt, David C.; Zhang, Yuan-Wei; Rosado, Edwin; Ravera, Silvia; Rhoades, Elizabeth; Forrest, Lucy R.; Rudnick, Gary

    2016-01-01

    In LeuT, a prokaryotic homolog of neurotransmitter transporters, Na+ stabilizes outward-open conformational states. We examined how each of the two LeuT Na+ binding sites contributes to Na+-dependent closure of the cytoplasmic pathway using biochemical and biophysical assays of conformation. Mutating either of two residues that contribute to the Na2 site completely prevented cytoplasmic closure in response to Na+, suggesting that Na2 is essential for this conformational change, whereas Na1 mutants retained Na+ responsiveness. However, mutation of Na1 residues also influenced the Na+-dependent conformational change in ways that varied depending on the position mutated. Computational analyses suggest those mutants influence the ability of Na1 binding to hydrate the substrate pathway and perturb an interaction network leading to the extracellular gate. Overall, the results demonstrate that occupation of Na2 stabilizes outward-facing conformations presumably through a direct interaction between Na+ and transmembrane helices 1 and 8, whereas Na+ binding at Na1 influences conformational change through a network of intermediary interactions. The results also provide evidence that N-terminal release and helix motions represent distinct steps in cytoplasmic pathway opening. PMID:26582198

  8. Low pH-induced conformational changes in 33 kD protein of photosystem Ⅱ

    Institute of Scientific and Technical Information of China (English)

    WENG Jun; TAN Cuiyan; YU Yong; RUAN Kangcheng; XU Chunhe

    2004-01-01

    33 kD protein, located on the lumen side of thylakoid membranes, is one of three extrinsic proteins of photosystemⅡ(PSⅡ). Previous study showed that NBS modification of W241, the only tryptophan in 33 kD protein, is helpful for understanding the function of W241 in maintaining functional conformation of 33 kD protein. In this paper, studies of both circular dichroism and fluorescence spectra showed that upon decreasing pH from 6.2 to 2.5, the conformation of soluble 33 kD protein changed significantly, with an increase or a decrease in percentage of random coil or ?-helix and turns. The changes in secondary structures of this protein are pH reversible. After NBS modification at pH 2.5, the conformational change of 33 kD protein was kept fixed. The CD ellipticity at 200 nm for NBS-modified 33 kD protein is much lower than that for control, indicating that the unfolding degree of 33 kD protein was enhanced after the NBS modification. Moreover, the conformational flexibility is lost in NBS-modified 33 kD protein, and the conformational change becomes pH irreversible, indicating that NBS modification blocked the reversibility of conformational change of 33 kD protein. The specific binding capability of NBS-modi- fied 33 kD protein is much lower than that of low pH-treated control. Furthermore, the rebinding of modified protein on PSⅡ membranes cannot restore the activity of oxygen evolution. We suggest that it is low pH but not NBS modification of W241 that leads to the conformational change of 33 kD protein from one functional to another non-functional state. The significant capability of proton transport of 33 kD protein is discussed.

  9. Coarse-grained Simulations of Conformational Changes in Multidrug Resistance Transporters

    Science.gov (United States)

    Jewel, S. M. Yead; Dutta, Prashanta; Liu, Jin

    2016-11-01

    The overexpression of multidrug resistance (MDR) systems on the gram negative bacteria causes serious problems for treatment of bacterial infectious diseases. The system effectively pumps the antibiotic drugs out of the bacterial cells. During the pumping process one of the MDR components, AcrB undergoes a series of large-scale conformational changes which are responsible for drug recognition, binding and expelling. All-atom simulations are unable to capture those conformational changes because of computational cost. Here, we implement a hybrid coarse-grained force field that couples the united-atom protein models with the coarse-grained MARTINI water/lipid, to investigate the proton-dependent conformational changes of AcrB. The simulation results in early stage ( 100 ns) of proton-dependent conformational changes agree with all-atom simulations, validating the coarse-grained model. The coarse-grained force field allows us to explore the process in microsecond simulations. Starting from the crystal structures of Access(A)/Binding(B)/Extrusion(E) monomers in AcrB, we find that deprotonation of Asp407 and Asp408 in monomer E causes a series of large-scale conformational changes from ABE to AAA in absence of drug molecules, which is consistent with experimental findings. This work is supported by NIH Grant: 1R01GM122081-01.

  10. Sulphate removal induces a major conformational change in Leishmania mexicana pyruvate kinase in the crystalline state.

    Science.gov (United States)

    Tulloch, Lindsay B; Morgan, Hugh P; Hannaert, Véronique; Michels, Paul A M; Fothergill-Gilmore, Linda A; Walkinshaw, Malcolm D

    2008-11-14

    We report X-ray structures of pyruvate kinase from Leishmania mexicana (LmPYK) that are trapped in different conformations. These, together with the previously reported structure of LmPYK in its inactive (T-state) conformation, allow comparisons of three different conformers of the same species of pyruvate kinase (PYK). Four new site point mutants showing the effects of side-chain alteration at subunit interfaces are also enzymatically characterised. The LmPYK tetramer crystals grown with ammonium sulphate as precipitant adopt an active-like conformation, with sulphate ions at the active and effector sites. The sulphates occupy positions similar to those of the phosphates of ligands bound to active (R-state) and constitutively active (nonallosteric) PYKs from several species, and provide insight into the structural roles of the phosphates of the substrates and effectors. Crystal soaking in sulphate-free buffers was found to induce major conformational changes in the tetramer. In particular, the unwinding of the Aalpha6' helix and the inward hinge movement of the B domain are coupled with a significant widening (4 A) of the tetramer caused by lateral movement of the C domains. The two new LmPYK structures and the activity studies of site point mutations described in this article are consistent with a developing picture of allosteric activity in which localised changes in protein flexibility govern the distribution of conformer families adopted by the tetramer in its active and inactive states.

  11. 75 FR 76260 - Conforming Changes to Applicant Submission Requirements; Implementing Federal Financial Report...

    Science.gov (United States)

    2010-12-08

    ... Information Relay Service at 800-877-8339. ] SUPPLEMENTARY INFORMATION: I. Background On July 15, 2010 (75 FR... published at 75 FR 41087 on July 15, 2010, is adopted as final without change. ] Dated: December 1, 2010... URBAN DEVELOPMENT 24 CFR Parts 5, 84, and 85 RIN 2501-AD50 Conforming Changes to Applicant...

  12. Conformational changes in human serum albumin induced by sodium perfluorooctanoate in aqueous solutions.

    Science.gov (United States)

    Messina, Paula V; Prieto, Gerardo; Ruso, Juan M; Sarmiento, Félix

    2005-08-18

    Conformational changes in the bulk solution and at the air-aqueous interface of human serum albumin (HSA) induced by changes in concentration of sodium perfluorooctanoate (C(7)F(15)COO(-)Na(+)) were studied by difference spectroscopy, zeta-potential data, and axisymmetric drop shape analysis. zeta-potential was used to monitor the formation of the HSA-C(7)F(15)COO(-)Na(+) complex and the surface charge of the complex. The conformational transition of HSA in the bulk solution was followed as a function of denaturant concentration by absorbance measurements at 280 nm. The data were analyzed to obtain values for the Gibbs energies of the transition in water (DeltaG(0)(W)) and in a hydrophobic environment (DeltaG(0)(hc)) pertaining to saturated protein-surfactant complexes. The conformational changes that surfactants induce in HSA molecules alter its absorption behavior at the air-water interface. Dynamic surface measurements were used to evaluate this behavior. At low [C(7)F(15)COO(-)Na(+)], proteins present three adsorption regimes: induction time, monolayer saturation, and interfacial gelation. When surfactant concentration increases and conformational changes in the bulk solution occur, the adsorption regimes disappear. HSA molecules in an intermediate conformational state migrate to the air-water interface and form a unique monolayer. At high [C(7)F(15)COO(-)Na(+)], the adsorption of denatured molecules exhibits a behavior analogous to that of dilute solutions.

  13. On the Roles of Substrate Binding and Hinge Unfolding in Conformational Changes of Adenylate Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Brokaw, Jason B.; Chu, Jhih-wei

    2010-11-17

    We characterized the conformational change of adenylate kinase (AK) between open and closed forms by conducting five all-atom molecular-dynamics simulations, each of 100 ns duration. Different initial structures and substrate binding configurations were used to probe the pathways of AK conformational change in explicit solvent, and no bias potential was applied. A complete closed-to-open and a partial open-to-closed transition were observed, demonstrating the direct impact of substrate-mediated interactions on shifting protein conformation. The sampled configurations suggest two possible pathways for connecting the open and closed structures of AK, affirming the prediction made based on available x-ray structures and earlier works of coarse-grained modeling. The trajectories of the all-atom molecular-dynamics simulations revealed the complexity of protein dynamics and the coupling between different domains during conformational change. Calculations of solvent density and density fluctuations surrounding AK did not show prominent variation during the transition between closed and open forms. Finally, we characterized the effects of local unfolding of an important hinge near Pro177 on the closed-to-open transition of AK and identified a novel mechanism by which hinge unfolding modulates protein conformational change. The local unfolding of Pro177 hinge induces alternative tertiary contacts that stabilize the closed structure and prevent the opening transition.

  14. Redox-linked conformation change and electron transfer between monoheme c-type cytochromes and oxides

    Energy Technology Data Exchange (ETDEWEB)

    Khare, Nidhi; Lovelace, David M.; Eggleston, Carrick M.; Swenson, Michael; Magnuson, Timothy S.

    2006-06-15

    Electron transfer between redox active proteins and mineral oxides is important in a variety of natural as well as technological processes, including electron transfer from dissimilatory metal-reducing bacteria to minerals. One of the pathways that could trigger electron transfer between proteins and minerals is redox-linked conformation change. We present electrochemical evidence that mitochondrial cytochrome c (Mcc) undergoes significant conformation change upon interaction with hematite and indium-tin oxide (ITO) surfaces. The apparent adsorption-induced conformation change causes the protein to become more reducing, which makes it able to transfer electrons to the hematite conduction band. Although Mcc is not a protein thought to be involved in interaction with mineral surfaces, it shares (or can be conformed so as to share) some characteristics with multiheme outer-membrane cytochromes thought to be involved in the transfer of electrons from dissimilatory iron-reducing bacteria to ferric minerals during respiration. We present evidence that a 10.1 kDa monohoeme cytochrome isolated and purified from Acidiphilium cryptum, with properties similar to those of Mcc, also undergoes conformation change as a result of interaction with hematite surfaces.

  15. Infrared spectroscopic studies on reaction induced conformational changes in the NADH ubiquinone oxidoreductase (complex I).

    Science.gov (United States)

    Hellwig, Petra; Kriegel, Sébastien; Friedrich, Thorsten

    2016-07-01

    Redox-dependent conformational changes are currently discussed to be a crucial part of the reaction mechanism of the respiratory complex I. Specialized difference Fourier transform infrared techniques allow the detection of side-chain movements and minute secondary structure changes. For complex I, (1)H/(2)H exchange kinetics of the amide modes revealed a better accessibility of the backbone in the presence of NADH and quinone. Interestingly, the presence of phospholipids, that is crucial for the catalytic activity of the isolated enzyme complex, changes the overall conformation. When comparing complex I samples from different species, very similar electrochemically induced FTIR difference spectra and very similar rearrangements are reported. Finally, the information obtained with variants and from Zn(2+) inhibited samples for the conformational reorganization of complex I upon electron transfer are discussed in this review. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.

  16. Saxs study of structure and conformational changes of crotamine.

    OpenAIRE

    Beltran, J R; Mascarenhas,Y.P.; Craievich, A F; Laure, C J

    1985-01-01

    The radius of gyration of crotamine is determined by the small angle x-ray scattering technique. Several molecular solutions have been studied to correct for concentration effects. The apparent molecular radius of gyration is also determined as a function of pH. An important change between pH 9.5 and 12.5 is attributed to a dominant effect of molecular aggregation.

  17. Driving Calmodulin Protein towards Conformational Shift by Changing Ionization States of Select Residues

    Science.gov (United States)

    Negi, Sunita; Rana Atilgan, Ali; Atilgan, Canan

    2012-12-01

    Proteins are complex systems made up of many conformational sub-states which are mainly determined by the folded structure. External factors such as solvent type, temperature, pH and ionic strength play a very important role in the conformations sampled by proteins. Here we study the conformational multiplicity of calmodulin (CaM) which is a protein that plays an important role in calcium signaling pathways in the eukaryotic cells. CaM can bind to a variety of other proteins or small organic compounds, and mediates different physiological processes by activating various enzymes. Binding of calcium ions and proteins or small organic molecules to CaM induces large conformational changes that are distinct to each interacting partner. In particular, we discuss the effect of pH variation on the conformations of CaM. By using the pKa values of the charged residues as a basis to assign protonation states, the conformational changes induced in CaM by reducing the pH are studied by molecular dynamics simulations. Our current view suggests that at high pH, barrier crossing to the compact form is prevented by repulsive electrostatic interactions between the two lobes. At reduced pH, not only is barrier crossing facilitated by protonation of residues, but also conformations which are on average more compact are attained. The latter are in accordance with the fluorescence resonance energy transfer experiment results of other workers. The key events leading to the conformational change from the open to the compact conformation are (i) formation of a salt bridge between the N-lobe and the linker, stabilizing their relative motions, (ii) bending of the C-lobe towards the N-lobe, leading to a lowering of the interaction energy between the two-lobes, (iii) formation of a hydrophobic patch between the two lobes, further stabilizing the bent conformation by reducing the entropic cost of the compact form, (iv) sharing of a Ca+2 ion between the two lobes.

  18. Molecular Dynamics Simulations of Insulin: Elucidating the Conformational Changes that Enable Its Binding.

    Directory of Open Access Journals (Sweden)

    Anastasios Papaioannou

    Full Text Available A sequence of complex conformational changes is required for insulin to bind to the insulin receptor. Recent experimental evidence points to the B chain C-terminal (BC-CT as the location of these changes in insulin. Here, we present molecular dynamics simulations of insulin that reveal new insights into the structural changes occurring in the BC-CT. We find three key results: 1 The opening of the BC-CT is inherently stochastic and progresses through an open and then a "wide-open" conformation--the wide-open conformation is essential for receptor binding, but occurs only rarely. 2 The BC-CT opens with a zipper-like mechanism, with a hinge at the Phe24 residue, and is maintained in the dominant closed/inactive state by hydrophobic interactions of the neighboring Tyr26, the critical residue where opening of the BC-CT (activation of insulin is initiated. 3 The mutation Y26N is a potential candidate as a therapeutic insulin analogue. Overall, our results suggest that the binding of insulin to its receptor is a highly dynamic and stochastic process, where initial docking occurs in an open conformation and full binding is facilitated through interactions of insulin receptor residues with insulin in its wide-open conformation.

  19. Substrate-Induced Conformational Changes Occur in All Cleaved Forms of Caspase-6

    Energy Technology Data Exchange (ETDEWEB)

    S Vaidya; E Velazquez-Delgado; G Abbruzzese; J Hardy

    2011-12-31

    Caspase-6 is an apoptotic cysteine protease that also governs disease progression in Huntington's and Alzheimer's diseases. Caspase-6 is of great interest as a target for treatment of these neurodegenerative diseases; however, the molecular basis of caspase-6 function and regulation remains poorly understood. In the recently reported structure of caspase-6, the 60's and 130's helices at the base of the substrate-binding groove extend upward, in a conformation entirely different from that of any other caspase. Presently, the central question about caspase-6 structure and function is whether the extended conformation is the catalytically competent conformation or whether the extended helices must undergo a large conformational rearrangement in order to bind substrate. We have generated a series of caspase-6 cleavage variants, including a novel constitutively two-chain form, and determined crystal structures of caspase-6 with and without the intersubunit linker. This series allows evaluation of the role of the prodomain and intersubunit linker on caspase-6 structure and function before and after substrate binding. Caspase-6 is inherently more stable than closely related caspases. Cleaved caspase-6 with both the prodomain and the linker present is the most stable, indicating that these two regions act in concert to increase stability, but maintain the extended conformation in the unliganded state. Moreover, these data suggest that caspase-6 undergoes a significant conformational change upon substrate binding, adopting a structure that is more like canonical caspases.

  20. Molecular dynamics simulations of conformation changes of HIV-1 regulatory protein on graphene

    Science.gov (United States)

    Zhao, Daohui; Li, Libo; He, Daohang; Zhou, Jian

    2016-07-01

    The fragment of viral protein R (Vpr), Vpr13-33, plays an important role in regulating nuclear importing of HIV genes through channel formation in which it adopts a leucine-zipper-like alpha-helical conformation. A recent experimental study reported that helical Vpr13-33 would transform to β-sheet or random coil structures and aggregate on the surface of graphene or graphene oxide through hydrophobic interactions. Due to experimental limitations, however, there is still a considerable lack of understanding on the adsorption dynamics at the early stage of the conformational transition at water-graphene interface and the underlying driving force at molecular level. In this study, atomistic molecular dynamics simulations were used to explore the conformation transition phenomena. Vpr13-33 kept α-helical structure in solution, but changed to β-sheet structure when strongly adsorbed onto graphene. Preferential adsorption of Vpr13-33 on graphene is dominated by hydrophobic interactions. The cluster analysis identified the most significant populated conformation and the early stage of structure conversion from α-helical to β-sheet was found, but the full β-sheet propagation was not observed. Free energy landscape analysis further complemented the transformation analysis of peptide conformations. These findings are consistent with experimental results, and give a molecular level interpretation for the reduced cytotoxicity of Vpr13-33 to some extent upon graphene exposure. Meanwhile, this study provides some significant insights into the detailed mechanism of graphene-induced protein conformation transition.

  1. Sequential conformation change and activation of chicken liver dihydrofolate reductase in low concentration of guanidine hydrochloride

    Institute of Scientific and Technical Information of China (English)

    范映辛; 朱笠; 周筠梅; 邹承鲁

    1997-01-01

    The conformation changes of dihydrofolate reductase (DHFR) from chicken liver in guanidine hy-drochloride were monitored by protein intrinsic fluorescence, hydrophobic fluorescence probe TNS and limited proteol-ysis by proteinase K. The kinetics of the enzyme denaturation were also studied and compared with its activity changes. It was indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene (TNS) that a subtle conforma-tional change of the enzyme in dilute GuHCl parallels GuHCl-induced activation. At GuHCl concentration higher than 0.75 mol/L, the conformational change can be detected by increased susceptibility of the enzyme to proteinase K, but no significant gross conformational change of the enzyme molecule is observed by intrinsic fluorescence up to a GuHCl concentration of 1.2 mol/L. The results suggest that the denaturation of DHFR by GuHCl does not follow strictly the two-state model. The enzyme seems to open up sequentially with increasing concentrations of denaturants, mainly at th

  2. Conformational changes in dopamine transporter intracellular regions upon cocaine binding and dopamine translocation.

    Science.gov (United States)

    Dehnes, Yvette; Shan, Jufang; Beuming, Thijs; Shi, Lei; Weinstein, Harel; Javitch, Jonathan A

    2014-07-01

    The dopamine transporter (DAT), a member of the neurotransmitter:sodium symporter family, mediates the reuptake of dopamine at the synaptic cleft. DAT is the primary target for psychostimulants such as cocaine and amphetamine. We previously demonstrated that cocaine binding and dopamine transport alter the accessibility of Cys342 in the third intracellular loop (IL3). To study the conformational changes associated with the functional mechanism of the transporter, we made cysteine substitution mutants, one at a time, from Phe332 to Ser351 in IL3 of the background DAT construct, X7C, in which 7 endogenous cysteines were mutated. The accessibility of the 20 engineered cysteines to polar charged sulfhydryl reagents was studied in the absence and presence of cocaine or dopamine. Of the 11 positions that reacted with methanethiosulfonate ethyl ammonium, as evidenced by inhibition of ligand binding, 5 were protected against this inhibition by cocaine and dopamine (S333C, S334C, N336C, M342C and T349C), indicating that reagent accessibility is affected by conformational changes associated with inhibitor and substrate binding. In some of the cysteine mutants, transport activity is disrupted, but can be rescued by the presence of zinc, most likely because the distribution between inward- and outward-facing conformations is restored by zinc binding. The experimental data were interpreted in the context of molecular models of DAT in both the inward- and outward-facing conformations. Differences in the solvent accessible surface area for individual IL3 residues calculated for these states correlate well with the experimental accessibility data, and suggest that protection by ligand binding results from the stabilization of the outward-facing configuration. Changes in the residue interaction networks observed from the molecular dynamics simulations also revealed the critical roles of several positions during the conformational transitions. We conclude that the IL3 region of DAT

  3. Enhancing Architecture-Implementation Conformance with Change Management and Support for Behavioral Mapping

    Science.gov (United States)

    Zheng, Yongjie

    2012-01-01

    Software architecture plays an increasingly important role in complex software development. Its further application, however, is challenged by the fact that software architecture, over time, is often found not conformant to its implementation. This is usually caused by frequent development changes made to both artifacts. Against this background,…

  4. Predicting the reaction coordinates of millisecond light-induced conformational changes in photoactive yellow protein

    NARCIS (Netherlands)

    Vreede, J.; Juraszek, J.; Bolhuis, P.G.

    2010-01-01

    Understanding the dynamics of large-scale conformational changes in proteins still poses a challenge for molecular simulations. We employ transition path sampling of explicit solvent molecular dynamics trajectories to obtain atomistic insight in the reaction network of the millisecond timescale part

  5. Applications of the Local Mode Model to CH Bond Length Changes, Molecular Conformations and Vibrational Dynamics

    OpenAIRE

    Henry, Bryan R.; Gough, Kathleen M.

    1983-01-01

    The theoretical basis for the local mode model is reviewed. The model is applied to gas phase overtone spectra of aromatic molecules to investigate both substituent induced CH bond length changes and conformationally inequivalent hydrogens. The dynamic implications of the local mode model are discussed.

  6. A Large Conformational Change of a Bridged β-Cyclodextrin Dimer in Aqueous Solution

    Institute of Scientific and Technical Information of China (English)

    Xiao Qi ZHENG; Yong Hui WANG; Qing Xiang GUO; Li YANG; You Cheng LIU

    2003-01-01

    A novel bridged β-CD dimer in which two β-cyclodextrins were linked by a naphthalene at positions 2 and 7 has been synthesized. 1H and 13CNMR measurements showed that a large change in the conformation of the dimer occurred in aqueous solution. The dimer interacted with methyl and ethyl orange to form stable inclusion complexes via "induced fit" mechanism.

  7. Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis

    DEFF Research Database (Denmark)

    Villa, Elizabeth; Sengupta, Jayati; Trabuco, Leonard G.

    2009-01-01

    In translation, elongation factor Tu (EF-Tu) molecules deliver aminoacyl-tRNAs to the mRNA-programmed ribosome. The GTPase activity of EF-Tu is triggered by ribosome-induced conformational changes of the factor that play a pivotal role in the selection of the cognate aminoacyl-tRNAs. We present a...

  8. Enhancing Architecture-Implementation Conformance with Change Management and Support for Behavioral Mapping

    Science.gov (United States)

    Zheng, Yongjie

    2012-01-01

    Software architecture plays an increasingly important role in complex software development. Its further application, however, is challenged by the fact that software architecture, over time, is often found not conformant to its implementation. This is usually caused by frequent development changes made to both artifacts. Against this background,…

  9. Vibrational circular dichroism analysis reveals a conformational change of the baccatin III ring of paclitaxel: visualization of conformations using a new code for structure-activity relationships.

    Science.gov (United States)

    Izumi, Hiroshi; Ogata, Atsushi; Nafie, Laurence A; Dukor, Rina K

    2008-03-21

    The comparison between measured and conformer-weighted calculated VCD spectra of the baccatin III ring of paclitaxel and visualization of the conformations using the new code for structure-activity relationships are reported for the first time. The VCD spectrum of paclitaxel closely resembles that of the baccatin III ring. The large characteristic nuCO VCD bands with bisignate signs (1732 cm-1, Deltaepsilon = -1.6 x 10(-1); 1715 cm(-1), Deltaepsilon = 2.4 x 10(-1)) strongly reflect the structural property of the family of conformations bacc-ABC32F defined using the new code. The comparison with the conformation of the baccatin III core in the electron micrograph of the crystal structure of tubulin-paclitaxel (1JFF) suggests a conformational change of paclitaxel corresponding to a switch through the binding with beta-tublin and the intermolecular interactions involving the hydroxyl group (D) and carbonyl of acetoxy group (E). The representation of conformational codes allows complicated conformations to be very easily compared and facilitates future computational analyses such as those for the large-molecule calculations as well as genome analysis.

  10. Intramembrane aspartic acid in SCAP protein governs cholesterol-induced conformational change

    Science.gov (United States)

    Feramisco, Jamison D.; Radhakrishnan, Arun; Ikeda, Yukio; Reitz, Julian; Brown, Michael S.; Goldstein, Joseph L.

    2005-01-01

    The polytopic membrane protein SCAP transports sterol regulatory element-binding proteins (SREBPs) from the endoplasmic reticulum (ER) to the Golgi, thereby activating cholesterol synthesis. Cholesterol accumulation in the ER membranes changes SCAP to an alternate conformation in which it binds ER retention proteins called Insigs, thereby terminating cholesterol synthesis. Here, we show that the conserved Asp-428 in the sixth transmembrane helix of SCAP is essential for SCAP's dissociation from Insigs. In transfected hamster cells, mutant SCAP in which Asp-428 is replaced by alanine (D428A) remained in an Insig-binding conformation when cells were depleted of sterols. As a result, mutant SCAP failed to dissociate from Insigs, and it failed to carry SREBPs to the Golgi. These data identify an important functional residue in SCAP, and they provide genetic evidence that the conformation of SCAP dictates the rate of cholesterol synthesis in animal cells. PMID:15728349

  11. Atomic force microscopy evidence for conformational changes of fibronectin adsorbed on unmodified and sulfonated polystyrene surfaces.

    Science.gov (United States)

    Kowalczyńska, Hanna M; Kołos, Robert; Nowak-Wyrzykowska, Małgorzata; Dobkowski, Jacek; Elbaum, Danek; Szczepankiewicz, Andrzej; Kamiński, Jarosław

    2009-12-15

    The effect of polystyrene surface polarity on the conformation of adsorbed fibronectin (FN) has been studied with atomic force microscopy. We demonstrated that bare sulfonated and nonsulfonated polystyrene surfaces featured similar topographies. After the FN adsorption, direct comparison of both types of substrata revealed drastically different topographies, roughness values, and also cell-adhesive properties. This was interpreted in terms of FN conformational changes induced by the surface polarity. At high-solute FN concentrations the multilayer FN adsorption took place resulting, for the sulfonated substratum, in an increase of surface roughness, whereas for the nonsulfonated one the roughness was approximately stable. Conversely, the FN conformation characteristic for the first saturative layer tended to be conserved in the consecutive layers, as evidenced by height histograms. The height of individual FN molecules indicated, consonantly with the derived thickness of the adsorbed protein layer (the latter value being 1.4 nm and 0.6 nm, respectively, for an unmodified and sulfonated polystyrene surface), that molecules are flattened on polar surfaces and more compact on nonsulfonated ones. It was also demonstrated that the FN adsorption and conformation on polymeric substrata, and hence the resultant cell-adhesive properties, depended on the chemistry of the original surface rather than on its topography. Our results also demonstrated the ability of surface polarity to influence the protein conformation and its associated biological activity.

  12. Conformational changes in the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum detected using phosphorescence polarization.

    Science.gov (United States)

    Restall, C J; Coke, M; Murray, E K; Chapman, D

    1985-02-28

    The technique of time-averaged phosphorescence has been used to study the interaction of calcium ions and ATP with the (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum vesicles. The presence of excess calcium ions was found to cause a 20% decrease in the phosphorescence emission anisotropy. This is interpreted as being due to a conformational change in the protein and is supported by data from time-resolved phosphorescence measurements which also show a lowering of the anisotropy. This change in the decay of the emission anisotropy is associated with only minor changes in the rotational relaxation time of the protein and is again suggestive of a conformational change in the protein. In some cases ATP was also observed to lower the time-averaged phosphorescence anisotropy possibly via an interaction with the low-affinity regulatory site of the protein.

  13. Conformational Transitions and Glycation of Serum Albumin in Patients with Minimal-Change Glomerulopathy

    Science.gov (United States)

    Hong, Sae Yong; Lee, Eun Young; Yang, Jong Oh; Kim, Tae Yeong; Kim, Eun Hee; Cheong, Mi Young; Kim, Soo Hyun; Cheong, Chae Joon

    2004-01-01

    Background There has been a lack of study on the structural changes of serum albumin in patients with minimal change disease (MCD). To determine whether glycation and/or conformational transitions of albumin are involved in the pathogenesis of albuminuria, nine patients with MCD were enrolled in a prospective follow-up study for comparison of these parameters in serum albumin during the remission and relapse of nephrotic syndrome. Methods Circular dichroism measurements were made with purified albumin. Ellipticities at each wavelength were transformed to mean residue ellipticity. Monosaccharide composition was analyzed by high-pH anion-exchange chromatography with pulsed amperometric detection. Results There was no difference in the proportions of α-helix, β-conformation, and β-turn of albumin between the sera of control patients and those with nephrotic syndrome. However, the proportion of the random configuration was slightly higher in the plasma albumin of patients in relapse than in those in remission. The proportion of the random configuration was lower in the albumin of the serum than in the urine of patients with nephrotic syndrome, but there was no difference in the proportions of α-helix, β-conformation, and β-turn of albumin between their plasma and urine. Conclusion Our results suggest that conformational changes in albumin are involved in albuminuria in patients with MCD. PMID:15481604

  14. Binding induced conformational changes of proteins correlate with their intrinsic fluctuations: a case study of antibodies

    Directory of Open Access Journals (Sweden)

    Keskin Ozlem

    2007-05-01

    Full Text Available Abstract Background How antibodies recognize and bind to antigens can not be totally explained by rigid shape and electrostatic complimentarity models. Alternatively, pre-existing equilibrium hypothesis states that the native state of an antibody is not defined by a single rigid conformation but instead with an ensemble of similar conformations that co-exist at equilibrium. Antigens bind to one of the preferred conformations making this conformation more abundant shifting the equilibrium. Results Here, two antibodies, a germline antibody of 36–65 Fab and a monoclonal antibody, SPE7 are studied in detail to elucidate the mechanism of antibody-antigen recognition and to understand how a single antibody recognizes different antigens. An elastic network model, Anisotropic Network Model (ANM is used in the calculations. Pre-existing equilibrium is not restricted to apply to antibodies. Intrinsic fluctuations of eight proteins, from different classes of proteins, such as enzymes, binding and transport proteins are investigated to test the suitability of the method. The intrinsic fluctuations are compared with the experimentally observed ligand induced conformational changes of these proteins. The results show that the intrinsic fluctuations obtained by theoretical methods correlate with structural changes observed when a ligand is bound to the protein. The decomposition of the total fluctuations serves to identify the different individual modes of motion, ranging from the most cooperative ones involving the overall structure, to the most localized ones. Conclusion Results suggest that the pre-equilibrium concept holds for antibodies and the promiscuity of antibodies can also be explained this hypothesis: a limited number of conformational states driven by intrinsic motions of an antibody might be adequate to bind to different antigens.

  15. Tracing the conformational changes in BSA using FRET with environmentally-sensitive squaraine probes

    Science.gov (United States)

    Govor, Iryna V.; Tatarets, Anatoliy L.; Obukhova, Olena M.; Terpetschnig, Ewald A.; Gellerman, Gary; Patsenker, Leonid D.

    2016-06-01

    A new potential method of detecting the conformational changes in hydrophobic proteins such as bovine serum albumin (BSA) is introduced. The method is based on the change in the Förster resonance energy transfer (FRET) efficiency between protein-sensitive fluorescent probes. As compared to conventional FRET based methods, in this new approach the donor and acceptor dyes are not covalently linked to protein molecules. Performance of the new method is demonstrated using the protein-sensitive squaraine probes Square-634 (donor) and Square-685 (acceptor) to detect the urea-induced conformational changes of BSA. The FRET efficiency between these probes can be considered a more sensitive parameter to trace protein unfolding as compared to the changes in fluorescence intensity of each of these probes. Addition of urea followed by BSA unfolding causes a noticeable decrease in the emission intensities of these probes (factor of 5.6 for Square-634 and 3.0 for Square-685), and the FRET efficiency changes by a factor of up to 17. Compared to the conventional method the new approach therefore demonstrates to be a more sensitive way to detect the conformational changes in BSA.

  16. Molecular dynamics simulations of conformation changes of HIV-1 regulatory protein on graphene

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Daohui; Li, Libo; He, Daohang; Zhou, Jian, E-mail: jianzhou@scut.edu.cn

    2016-07-30

    Graphical abstract: Preferential adsorption of Vpr13-33 on graphene accompanied by early conformational change from α-helix to β-sheet structures was observed by molecular simulations. This work presents the molecular mechanism of graphene-induced peptide conformational alteration and sheds light on developing graphene-based materials to inhibit HIV. - Highlights: • Graphene induced early structural transition of Vpr13-33 is studied by MD simulations. • Both π-π stacking and hydrophobic interactions orchestrate the peptide adsorption. • Vpr has an increased propensity of β-sheet content on graphene surface. • To develop graphene-based materials to inhibit HIV is possible. - Abstract: The fragment of viral protein R (Vpr), Vpr13-33, plays an important role in regulating nuclear importing of HIV genes through channel formation in which it adopts a leucine-zipper-like alpha-helical conformation. A recent experimental study reported that helical Vpr13-33 would transform to β-sheet or random coil structures and aggregate on the surface of graphene or graphene oxide through hydrophobic interactions. Due to experimental limitations, however, there is still a considerable lack of understanding on the adsorption dynamics at the early stage of the conformational transition at water-graphene interface and the underlying driving force at molecular level. In this study, atomistic molecular dynamics simulations were used to explore the conformation transition phenomena. Vpr13-33 kept α-helical structure in solution, but changed to β-sheet structure when strongly adsorbed onto graphene. Preferential adsorption of Vpr13-33 on graphene is dominated by hydrophobic interactions. The cluster analysis identified the most significant populated conformation and the early stage of structure conversion from α-helical to β-sheet was found, but the full β-sheet propagation was not observed. Free energy landscape analysis further complemented the transformation analysis of

  17. Real-time monitoring of protein conformational changes using a nano-mechanical sensor.

    Directory of Open Access Journals (Sweden)

    Livan Alonso-Sarduy

    Full Text Available Proteins can switch between different conformations in response to stimuli, such as pH or temperature variations, or to the binding of ligands. Such plasticity and its kinetics can have a crucial functional role, and their characterization has taken center stage in protein research. As an example, Topoisomerases are particularly interesting enzymes capable of managing tangled and supercoiled double-stranded DNA, thus facilitating many physiological processes. In this work, we describe the use of a cantilever-based nanomotion sensor to characterize the dynamics of human topoisomerase II (Topo II enzymes and their response to different kinds of ligands, such as ATP, which enhance the conformational dynamics. The sensitivity and time resolution of this sensor allow determining quantitatively the correlation between the ATP concentration and the rate of Topo II conformational changes. Furthermore, we show how to rationalize the experimental results in a comprehensive model that takes into account both the physics of the cantilever and the dynamics of the ATPase cycle of the enzyme, shedding light on the kinetics of the process. Finally, we study the effect of aclarubicin, an anticancer drug, demonstrating that it affects directly the Topo II molecule inhibiting its conformational changes. These results pave the way to a new way of studying the intrinsic dynamics of proteins and of protein complexes allowing new applications ranging from fundamental proteomics to drug discovery and development and possibly to clinical practice.

  18. Ligand-specific conformational changes in the alpha1 glycine receptor ligand-binding domain

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Lynch, Joseph W

    2009-01-01

    indicate that channel opening is accompanied by conformational rearrangements in both beta-sheets. In an attempt to resolve ligand-dependent movements in the ligand-binding domain, we employed voltage-clamp fluorometry on alpha1 glycine receptors to compare changes mediated by the agonist, glycine......, and by the antagonist, strychnine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. In the inner beta-sheet, we labeled residues in loop 2 and in binding domain loops D and E....... At each position, strychnine and glycine induced distinct maximal fluorescence responses. The pre-M1 domain responded similarly; at each of four labeled positions glycine produced a strong fluorescence signal, whereas strychnine did not. This suggests that glycine induces conformational changes...

  19. The molecular mechanism of toxin-induced conformational changes in a potassium channel : relation to C-type inactivation

    NARCIS (Netherlands)

    Zachariae, U.; Schneider, R.; Velisetty, P.; Lange, A.; Seeliger, D.; Wacker, S.J.; Karimi-Nejad, Y.; Vriend, G.; Becker, S.; Pongs, O.; Baldus, M.; Groot, B.L. de

    2008-01-01

    Recently, a solid-state NMR study revealed that scorpion toxin binding leads to conformational changes in the selectivity filter of potassium channels. The exact nature of the conformational changes, however, remained elusive. We carried out all-atom molecular dynamics simulations that enabled us to

  20. Ligand-induced conformational changes in wild-type and mutant yeast pyruvate kinase.

    Science.gov (United States)

    Collins, R A; Kelly, S M; Price, N C; Fothergill-Gilmore, L A; Muirhead, H

    1996-12-01

    A mutant form of pyruvate kinase in which serine 384 has been mutated to proline has been engineered in the yeast Saccharomyces cerevisiae. Residue 384 is located in a helix in a subunit interface of the tetrameric enzyme, and the mutation was anticipated to alter the conformation of the helix and hence destabilize the interface. Previous results indicate that the mutant favours the T quaternary conformation over the R conformation, and this is confirmed by the results presented here. Addition of phosphoenol-pyruvate (PEP), ADP and fructose-1, 6-bisphosphate (Fru-1.6-P2) singly to the wild-type and mutant enzymes results in a significant quenching of tryptophan fluorescence (12-44%), and for Fru-1,6-P2, a red shift of 15 nm in the emission maximum. Fluorescence titration experiments showed that PEP, ADP and Fru-1,6-P2 induce conformations which have similar ligand-binding properties in the wild-type and mutant enzymes. However, the Fru-1,6-P2 induced conformation is demonstrably different from those induced by either ADP or PEP. The enzymes differ in their susceptibility to trypsin digestion and N-ethylmaleimide inhibition. The thermal stability of the enzyme is unaltered by the mutation. Far-UV CD spectra show that both enzymes adopt a similar overall secondary structure in solution. Taken together, the results suggest that the Ser384-Pro mutation causes the enzyme to adopt a different tertiary and/or quaternary structure from the wild-type enzyme and affects the type and extent of the conformational changes induced in the enzyme upon ligand binding. A simplified minimal reaction mechanism is proposed in which the R and T states differ in both affinity and kcat. Thus, in terms of the models of cooperativity and allosteric interaction, pyruvate kinase is both a K and a V system.

  1. Raman spectroscopic evidence of tissue restructuring in heat-induced tissue fusion.

    Science.gov (United States)

    Su, Lei; Cloyd, Kristy L; Arya, Shobhit; Hedegaard, Martin A B; Steele, Joseph A M; Elson, Daniel S; Stevens, Molly M; Hanna, George B

    2014-09-01

    Heat-induced tissue fusion via radio-frequency (RF) energy has gained wide acceptance clinically and here we present the first optical-Raman-spectroscopy study on tissue fusion samples in vitro. This study provides direct insights into tissue constituent and structural changes on the molecular level, exposing spectroscopic evidence for the loss of distinct collagen fibre rich tissue layers as well as the denaturing and restructuring of collagen crosslinks post RF fusion. These findings open the door for more advanced optical feedback-control methods and characterization during heat-induced tissue fusion, which will lead to new clinical applications of this promising technology.

  2. Conformational changes during nucleotide selection by Sulfolobus solfataricus DNA polymerase Dpo4.

    Science.gov (United States)

    Eoff, Robert L; Sanchez-Ponce, Raymundo; Guengerich, F Peter

    2009-07-31

    The mechanism of nucleotide selection by Y-family DNA polymerases has been the subject of intense study, but significant structural contacts and/or conformational changes that relate to polymerase fidelity have been difficult to identify. Here we report on the conformational dynamics of a model Y-family polymerase Dpo4 from Sulfolobus solfataricus. Hydrogen-deuterium exchange in tandem with mass spectrometry was used to monitor changes in Dpo4 structure as a function of time and the presence or absence of specific substrates and ligands. Analysis of the data revealed previously unrecognized structural changes that accompany steps in the catalytic cycle leading up to phosphoryl transfer. For example, the solvent accessibility of the alphaB-loop-alphaC region in the finger domain decreased in the presence of all four dNTP insertion events, but the rate of deuterium exchange, an indicator of conformational flexibility, only decreased during an accurate insertion event. Of particular note is a change in the region surrounding the H-helix of the thumb domain. Upon binding DNA and Mg2+, the H-helix showed a decrease in solvent accessibility and flexibility that was relaxed only upon addition of dCTP, which forms a Watson-Crick base pair with template dG and not during mispairing events. The current study expands upon a previous report from our group that used a fluorescent probe located near the thumb domain to measure the kinetic properties of Dpo4 conformational changes. We now present a model for nucleotide selection by Dpo4 that arises from a synthesis of both structural and kinetic data.

  3. Volume analysis of heat-induced cracks in human molars: A preliminary study

    Directory of Open Access Journals (Sweden)

    Michael A. Sandholzer

    2014-01-01

    Full Text Available Context: Only a few methods have been published dealing with the visualization of heat-induced cracks inside bones and teeth. Aims : As a novel approach this study used nondestructive X-ray microtomography (micro-CT for volume analysis of heat-induced cracks to observe the reaction of human molars to various levels of thermal stress. Materials and Methods: Eighteen clinically extracted third molars were rehydrated and burned under controlled temperatures (400, 650, and 800°C using an electric furnace adjusted with a 25°C increase/min. The subsequent high-resolution scans (voxel-size 17.7 μm were made with a compact micro-CT scanner (SkyScan 1174. In total, 14 scans were automatically segmented with Definiens XD Developer 1.2 and three-dimensional (3D models were computed with Visage Imaging Amira 5.2.2. The results of the automated segmentation were analyzed with an analysis of variance (ANOVA and uncorrected post hoc least significant difference (LSD tests using Statistical Package for Social Sciences (SPSS 17. A probability level of P < 0.05 was used as an index of statistical significance. Results: A temperature-dependent increase of heat-induced cracks was observed between the three temperature groups (P < 0.05, ANOVA post hoc LSD. In addition, the distributions and shape of the heat-induced changes could be classified using the computed 3D models. Conclusion: The macroscopic heat-induced changes observed in this preliminary study correspond with previous observations of unrestored human teeth, yet the current observations also take into account the entire microscopic 3D expansions of heat-induced cracks within the dental hard tissues. Using the same experimental conditions proposed in the literature, this study confirms previous results, adds new observations, and offers new perspectives in the investigation of forensic evidence.

  4. Single-molecule fluorescence polarization study of conformational change in archaeal group II chaperonin.

    Directory of Open Access Journals (Sweden)

    Ryo Iizuka

    Full Text Available Group II chaperonins found in archaea and in eukaryotic cytosol mediate protein folding without a GroES-like cofactor. The function of the cofactor is substituted by the helical protrusion at the tip of the apical domain, which forms a built-in lid on the central cavity. Although many studies on the change in lid conformation coupled to the binding and hydrolysis of nucleotides have been conducted, the molecular mechanism of lid closure remains poorly understood. Here, we performed a single-molecule polarization modulation to probe the rotation of the helical protrusion of a chaperonin from a hyperthermophilic archaeum, Thermococcus sp. strain KS-1. We detected approximately 35° rotation of the helical protrusion immediately after photorelease of ATP. The result suggests that the conformational change from the open lid to the closed lid state is responsible for the approximately 35° rotation of the helical protrusion.

  5. Conformational change of pseudouridine 55 synthase upon its association with RNA substrate.

    Science.gov (United States)

    Phannachet, Kulwadee; Huang, Raven H

    2004-01-01

    Pseudouridine 55 synthase (Psi55S) catalyzes isomerization of uridine (U) to pseudouridine (Psi) at position 55 in transfer RNA. The crystal structures of Thermotoga maritima Psi55S, and its complex with RNA, have been determined at 2.9 and 3.0 A resolutions, respectively. Structural comparisons with other families of pseudouridine synthases (PsiS) indicate that Psi55S may acquire its ability to recognize a stem-loop RNA substrate by two insertions of polypeptides into the PsiS core. The structure of apo-Psi55S reveals that these two insertions interact with each other. However, association with RNA substrate induces substantial conformational change in one of the insertions, resulting in disruption of interaction between insertions and association of both insertions with the RNA substrate. Specific interactions between two insertions, as well as between the insertions and the RNA substrate, account for the molecular basis of the conformational change.

  6. Protein Conformational Change Based on a Two-dimensional Generalized Langevin Equation

    Institute of Scientific and Technical Information of China (English)

    Ying-xi Wang; Shuang-mu Linguang; Nan-rong Zhao; Yi-jing Yan

    2011-01-01

    A two-dimensional generalized Langevin equation is proposed to describe the protein conformational change,compatible to the electron transfer process governed by atomic packing density model.We assume a fractional Gaussian noise and a white noise through bond and through space coordinates respectively,and introduce the coupling effect coming from both fluctuations and equilibrium variances.The general expressions for autocorrelation functions of distance fluctuation and fluorescence lifetime variation are derived,based on which the exact conformational change dynamics can be evaluated with the aid of numerical Laplace inversion technique.We explicitly elaborate the short time and long time approximations.The relationship between the two-dimensional description and the one-dimensional theory is also discussed.

  7. Continuum molecular simulation of large conformational changes during ion-channel gating.

    Directory of Open Access Journals (Sweden)

    Ali Nekouzadeh

    Full Text Available A modeling framework was developed to simulate large and gradual conformational changes within a macromolecule (protein when its low amplitude high frequency vibrations are not concerned. Governing equations were derived as alternative to Langevin and Smoluchowski equations and used to simulate gating conformational changes of the Kv7.1 ion-channel over the time scale of its gating process (tens of milliseconds. The alternative equations predict the statistical properties of the motion trajectories with good accuracy and do not require the force field to be constant over the diffusion length, as assumed in Langevin equation. The open probability of the ion-channel was determined considering cooperativity of four subunits and solving their concerted transition to the open state analytically. The simulated open probabilities for a series of voltage clamp tests produced current traces that were similar to experimentally recorded currents.

  8. Non-enzymatic Glycation of Almond Cystatin Leads to Conformational Changes and Altered Activity.

    Science.gov (United States)

    Siddiqui, Azad A; Sohail, Aamir; Bhat, Sheraz A; Rehman, Md T; Bano, Bilqees

    2015-01-01

    The non-enzymatic reaction between proteins and reducing sugars, known as glycation, leads to the formation of inter and intramolecular cross-links of proteins. Stable end products called as advanced Maillard products or advanced glycation end products (AGEs) have received tremendous attention since last decades. It was suggested that the formation of AGEs not only modify the conformation of proteins but also induces altered biological activity. In this study, cystatin purified from almond was incubated with three different sugars namely D-ribose, fructose and lactose to monitor the glycation process. Structural changes induced in cystatin on glycation were studied using UV-visible spectroscopy, fluorescence spectroscopy, CD and FTIR techniques. Glycated cystatin was found to migrate slower on electrophoresis as compared to control cystatin. Biological activity data of glycated cystatin showed that D-ribose was most effective in inducing conformational changes with maximum altered activity.

  9. Comparison between conformational change and inactivation rates of aminoacylase during denaturation in urea solutions

    Institute of Scientific and Technical Information of China (English)

    王洪睿; 王希成; 张彤; 周海梦

    1995-01-01

    The kinetic method of the substrate reaction in the presence of mactivator previously described by Tsou has been applied to the determination of inactivation rates of aminoacylase during denaturation in urea solutions. The protective effect of substrate on the inactivation of aminoacylase by urea has been investigated. Simultaneously, the comparison between conformational change and inactivation rates of enzyme in the urea solutions of different concentrations has been studied. Results obtained show that the inactivation rate constants of the enzyme are larger than the rate constants of conformational changes. The present results show that the active site of metal enzyme-aminoacylase is also located in a limited and flexible region of the molecule that is more sensitive to denaturants than the enzyme as a whole.

  10. A user-friendly web portal for analyzing conformational changes in structures of Mycobacterium tuberculosis.

    Science.gov (United States)

    Hassan, Sameer; Thangam, Manonanthini; Vasudevan, Praveen; Kumar, G Ramesh; Unni, Rahul; Devi, P K Gayathri; Hanna, Luke Elizabeth

    2015-10-01

    Initiation of the Tuberculosis Structural Consortium has resulted in the expansion of the Mycobacterium tuberculosis (MTB) protein structural database. Currently, 969 experimentally solved structures are available for 354 MTB proteins. This includes multiple crystal structures for a given protein under different functional conditions, such as the presence of different ligands or mutations. In depth analysis of the multiple structures reveal that subtle differences exist in conformations of a given protein under varied conditions. Therefore, it is immensely important to understand the conformational differences between the multiple structures of a given protein in order to select the most suitable structure for molecular docking and structure-based drug designing. Here, we introduce a web portal ( http://bmi.icmr.org.in/mtbsd/torsion.php ) that we developed to provide comparative data on the ensemble of available structures of MTB proteins, such as Cα root means square deviation (RMSD), sequence identity, presence of mutations and torsion angles. Additionally, torsion angles were used to perform principal component analysis (PCA) to identify the conformational differences between the structures. Additionally, we present a few case studies to demonstrate this database. Graphical Abstract Conformational changes seen in the structures of the enoyl-ACP reductase protein encoded by the Mycobacterial gene inhA.

  11. Reversible Conformational Changes of PsbO Protein Detected by Terahertz Time-Domain Spectroscopy

    Institute of Scientific and Technical Information of China (English)

    CHEN Hua; CHEN Gui-Ying; LI Shu-Qin; WANG Li

    2009-01-01

    We used a terahertz time-domain spectroscope (THz-TDS) to detect the reversible conformations2 changes of PsbO protein induced by N-bromosuccinimide and Guanidine Hydrochloride.The veracity and sensitivity are confirmed by the fluorescence emission spectra.The results demonstrate that THz-TDS has both advantages and disadvantages in monitoring the denaturation process of proteins,which is important in applying THz-TDS technique to studying biomolecules.

  12. Switch region for pathogenic structural change in conformational disease and its prediction.

    Directory of Open Access Journals (Sweden)

    Xin Liu

    Full Text Available Many diseases are believed to be related to abnormal protein folding. In the first step of such pathogenic structural changes, misfolding occurs in regions important for the stability of the native structure. This destabilizes the normal protein conformation, while exposing the previously hidden aggregation-prone regions, leading to subsequent errors in the folding pathway. Sites involved in this first stage can be deemed switch regions of the protein, and can represent perfect binding targets for drugs to block the abnormal folding pathway and prevent pathogenic conformational changes. In this study, a prediction algorithm for the switch regions responsible for the start of pathogenic structural changes is introduced. With an accuracy of 94%, this algorithm can successfully find short segments covering sites significant in triggering conformational diseases (CDs and is the first that can predict switch regions for various CDs. To illustrate its effectiveness in dealing with urgent public health problems, the reason of the increased pathogenicity of H5N1 influenza virus is analyzed; the mechanisms of the pandemic swine-origin 2009 A(H1N1 influenza virus in overcoming species barriers and in infecting large number of potential patients are also suggested. It is shown that the algorithm is a potential tool useful in the study of the pathology of CDs because: (1 it can identify the origin of pathogenic structural conversion with high sensitivity and specificity, and (2 it provides an ideal target for clinical treatment.

  13. Redox-Dependent Conformational Changes in Cytochrome c Oxidase Suggest a Gating Mechanism for Proton Uptake

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Ling; Liu, Jian; Mills, Denise A.; Proshlyakov, Denis A.; Hiser, Carrie; Ferguson-Miller, Shelagh; (MSU)

    2009-08-05

    A role for conformational change in the coupling mechanism of cytochrome c oxidase is the subject of controversy. Relatively small conformational changes have been reported in comparisons of reduced and oxidized crystal structures of bovine oxidase but none in bacterial oxidases. Comparing the X-ray crystal structures of the reduced (at 2.15 {angstrom} resolution) and oxidized forms of cytochrome c oxidase from Rhodobacter sphaeroides, we observe a displacement of heme a3 involving both the porphyrin ring and the hydroxyl farnesyl tail, accompanied by protein movements in nearby regions, including the mid part of helix VIII of subunit I which harbors key residues of the K proton uptake path, K362 and T359. The conformational changes in the reduced form are reversible upon reoxidation. They result in an opening of the top of the K pathway and more ordered waters being resolved in that region, suggesting an access path for protons into the active site. In all high-resolution structures of oxidized R. sphaeroides cytochrome c oxidase, a water molecule is observed in the hydrophobic region above the top of the D path, strategically positioned to facilitate the connection of residue E286 of subunit I to the active site or to the proton pumping exit path. In the reduced and reduced plus cyanide structures, this water molecule disappears, implying disruption of proton conduction from the D path under conditions when the K path is open, thus providing a mechanism for alternating access to the active site.

  14. Conformational Changes of Calmodulin on Calcium and Peptide Binding Monitored by Film Bulk Acoustic Resonators

    Directory of Open Access Journals (Sweden)

    Janos Vörös

    2011-12-01

    Full Text Available Film bulk acoustic resonators (FBAR are mass sensitive, label-free biosensors that allow monitoring of the interaction between biomolecules. In this paper we use the FBAR to measure the binding of calcium and the CaMKII peptide to calmodulin. Because the mass of the calcium is too small to be detected, the conformational change caused by the binding process is measured by monitoring the resonant frequency and the motional resistance of the FBAR. The resonant frequency is a measure for the amount of mass coupled to the sensor while the motional resistance is influenced by the viscoelastic properties of the adsorbent. The measured frequency shift during the calcium adsorptions was found to be strongly dependent on the surface concentration of the immobilized calmodulin, which indicates that the measured signal is significantly influenced by the amount of water inside the calmodulin layer. By plotting the measured motional resistance against the frequency shift, a mass adsorption can be distinguished from processes involving measurable conformational changes. With this method three serial processes were identified during the peptide binding. The results show that the FBAR is a promising technology for the label-free measurement of conformational changes.

  15. Conformational changes in DNA-binding proteins: relationships with precomplex features and contributions to specificity and stability.

    Science.gov (United States)

    Andrabi, Munazah; Mizuguchi, Kenji; Ahmad, Shandar

    2014-05-01

    Both Proteins and DNA undergo conformational changes in order to form functional complexes and also to facilitate interactions with other molecules. These changes have direct implications for the stability and specificity of the complex, as well as the cooperativity of interactions between multiple entities. In this work, we have extensively analyzed conformational changes in DNA-binding proteins by superimposing DNA-bound and unbound pairs of protein structures in a curated database of 90 proteins. We manually examined each of these pairs, unified the authors' annotations, and summarized our observations by classifying conformational changes into six structural categories. We explored a relationship between conformational changes and functional classes, binding motifs, target specificity, biophysical features of unbound proteins, and stability of the complex. In addition, we have also investigated the degree to which the intrinsic flexibility can explain conformational changes in a subset of 52 proteins with high quality coordinate data. Our results indicate that conformational changes in DNA-binding proteins contribute significantly to both the stability of the complex and the specificity of targets recognized by them. We also conclude that most conformational changes occur in proteins interacting with specific DNA targets, even though unbound protein structures may have sufficient information to interact with DNA in a nonspecific manner. Copyright © 2013 Wiley Periodicals, Inc.

  16. Conformational Change and Epimerization of Diketopiperazines Containing Proline Residue in Water.

    Science.gov (United States)

    Ishizu, Takashi; Tsutsumi, Hiroyuki; Yokoyama, Emi; Kawamoto, Haruka; Yokota, Runa

    2017-01-01

    In water, diketopiperazines cyclo(L-Pro-L-Xxx) and cyclo(L-Pro-D-Xxx) (Xxx=Phe, Tyr) formed an intramolecular hydrophobic interaction between the main skeleton part and their benzene ring, and both cyclo(L-Pro-L-Xxx) and cyclo(L-Pro-D-Xxx) took a folded conformation. The conformational changes from folded to extended conformation by addition of several deuterated organic solvents (acetone-d6, metanol-d4, dimethyl sulfoxide-d6 (DMSO-d6)) and the temperature rise were investigated using (1)H-NMR spectra. The results suggested that the intrarmolecular hydrophobic interaction of cyclo(L-Pro-D-Xxx) formed more strongtly than that of cyclo(L-Pro-L-Xxx). Under a basic condition of 1.0×10(-1) mol/L potassium deuteroxide, enolization of O1-C1-C9-H9 moiety of cyclo(L-Pro-L-Xxx) occurred, while that of the O4-C4-C3-H3 moiety did not. Cyclo(L-Pro-L-Xxx) epimerized to cyclo(D-Pro-L-Xxx), while cyclo(L-Pro-D-Xxx) did not change.

  17. Sulfmyoglobin Conformational Change: A Role in the Decrease of Oxy-Myoglobin Functionality

    Science.gov (United States)

    Román-Morales, Elddie; López-Alfonzo, Erika; Pietri, Ruth; López-Garriga, Juan

    2016-01-01

    This work is focused at understanding the interaction of H2S with Myoglobin (Mb), in particular the Sulfmyoglobin (SMb) product, whose physiological role is controversial and not well understood. The scattering curves, Guinier, Kratky, Porod and P(r) plots were analyzed for oxy-Mb and oxy-Hemoglobin I (oxyHbI) in the absence and presence of H2S, using Small and Wide Angle X-ray Scattering (SAXS/WAXS) technique. Three dimensional models were also generated from the SAXS/WAXS data. The results show that SMb formation, produced by oxyMb and H2S interaction, induces a change in the protein conformation where its envelope has a very small cleft and the protein is more flexible, less rigid and compact. Based on the direct relationship between Mb’s structural conformation and its functionality, we suggest that the conformational change observed upon SMb formation plays a contribution to the protein decrease in O2 affinity and, therefore, on its functionality. PMID:28138567

  18. Conformation-changing aggregation in hydroxyacetone: a combined low-temperature FTIR, jet, and crystallographic study.

    Science.gov (United States)

    Sharma, Archna; Reva, Igor; Fausto, Rui; Hesse, Susanne; Xue, Zhifeng; Suhm, Martin A; Nayak, Susanta K; Sathishkumar, Ranganthan; Pal, Rumpa; Row, Tayur N Guru

    2011-12-21

    Aggregation in hydroxyacetone (HA) is studied using low-temperature FTIR, supersonic jet expansion, and X-ray crystallographic (in situ cryocrystallization) techniques. Along with quantum chemical methods (MP2 and DFT), the experiments unravel the conformational preferences of HA upon aggregation to dimers and oligomers. The O-H···O═C intramolecular hydrogen bond present in the gas-phase monomer partially opens upon aggregation in supersonic expansions, giving rise to intermolecular cooperatively enhanced O-H···O-H hydrogen bonds in competition with isolated O-H···O═C hydrogen bonds. On the other hand, low-temperature IR studies on the neat solid and X-ray crystallographic data reveal that HA undergoes profound conformational changes upon crystallization, with the HOCC dihedral angle changing from ~0° in the gas phase to ~180° in the crystalline phase, hence giving rise to a completely new conformation. These conclusions are supported by theoretical calculations performed on the geometry derived from the crystalline phase. © 2011 American Chemical Society

  19. Atomic snapshots of an RNA packaging motor reveal conformational changes linking ATP hydrolysis to RNA translocation.

    Science.gov (United States)

    Mancini, Erika J; Kainov, Denis E; Grimes, Jonathan M; Tuma, Roman; Bamford, Dennis H; Stuart, David I

    2004-09-17

    Many viruses package their genome into preformed capsids using packaging motors powered by the hydrolysis of ATP. The hexameric ATPase P4 of dsRNA bacteriophage phi12, located at the vertices of the icosahedral capsid, is such a packaging motor. We have captured crystallographic structures of P4 for all the key points along the catalytic pathway, including apo, substrate analog bound, and product bound. Substrate and product binding have been observed as both binary complexes and ternary complexes with divalent cations. These structures reveal large movements of the putative RNA binding loop, which are coupled with nucleotide binding and hydrolysis, indicating how ATP hydrolysis drives RNA translocation through cooperative conformational changes. Two distinct conformations of bound nucleotide triphosphate suggest how hydrolysis is activated by RNA binding. This provides a model for chemomechanical coupling for a prototype of the large family of hexameric helicases and oligonucleotide translocating enzymes.

  20. Binding interaction between rice glutelin and amylose: Hydrophobic interaction and conformational changes.

    Science.gov (United States)

    Xu, Xingfeng; Liu, Wei; Zhong, Junzhen; Luo, Liping; Liu, Chengmei; Luo, Shunjing; Chen, Lin

    2015-11-01

    The interaction of rice glutelin (RG) with amylose was characterized by spectroscopic and molecular docking studies. The intrinsic fluorescence of RG increased upon the addition of amylose. The binding sites, binding constant and thermodynamic features indicated that binding process was spontaneous and the main driving force of the interaction was hydrophobic interaction. The surface hydrophobicity of RG decreased with increasing amount of amylose. Furthermore, synchronous fluorescence and circular dichroism (CD) spectra provided data concerning conformational and micro-environmental changes of RG. With the concentration of amylose increasing, the polarity around the tyrosine residues increased while the hydrophobicity decreased. Alteration of protein conformation was observed with increasing of α-helix and reducing of β-sheet. Finally, a visual representation of two binding sites located in the amorphous area of RG was presented by molecular modeling studies.

  1. Aggregation and conformational change of mushroom (Agaricus bisporus) polyphenoloxidase subjected to thermal treatment.

    Science.gov (United States)

    Zhou, Lei; Liu, Wei; Zou, Liqiang; Xiong, Zhiqiang; Hu, Xiuting; Chen, Jun

    2017-01-01

    This study investigated changes in the activity, conformation and microstructure of mushroom polyphenoloxidase (PPO) subjected to thermal treatment. The inactivation of PPO can be achieved by high temperature-short time or mild temperature-long time treatment. Circular dichroism and fluorescence spectra suggested that heating process induced the rearrangement of secondary structure and the disruption of tertiary structure. Red shifts of fluorescence spectra showed positive correlations with the inactivation rate of PPO. There were significant differences in the conformation and molecular microstructure among PPO samples with the same relative activity, which were obtained by treating PPO at 45, 55 and 65°C for different times. In summary, PPO molecules were deformed at mild temperature, while higher temperature induced the formation of large aggregates. PPO with the same relative activity might exist in different forms.

  2. Probing Conformational Change of Bovine Serum Albumin–Dextran Conjugates under Controlled Dry Heating

    Energy Technology Data Exchange (ETDEWEB)

    Xia, Shuqin; Li, Yunqi; Zhao, Qin; Li, Ji; Xia, Qiuyang; Zhang, Xiaoming; Huang, Qingrong (Rutgers); (Chinese Aca. Sci.); (Jiangnan)

    2015-04-29

    The time-dependent conformational change of bovine serum album (BSA) during Maillard reaction with dextran under controlled dry heating has been studied by small-angle X-ray scattering, fluorescence spectroscopy, dynamic light scattering, and circular dichroism analysis. Through the research on the radii of gyration (Rg), intrinsic fluorescence, and secondary structure, conjugates with dextran coating were found to inhibit BSA aggregation and preserve the secondary structure of native BSA against long-time heat treatment during Maillard reaction. The results suggested that the hydrophilic dextran was conjugated to the compact protein surface and enclosed it and more dextran chains were attached to BSA with the increase of the heating time. The study presented here will be beneficial to the understanding of the conformational evolution of BSA molecules during the dry-heating Maillard reaction and to the control of the protein–polysaccharide conjugate structure.

  3. RNA conformational changes in the life cycles of RNA viruses, viroids, and virus-associated RNAs.

    Science.gov (United States)

    Simon, Anne E; Gehrke, Lee

    2009-01-01

    The rugged nature of the RNA structural free energy landscape allows cellular RNAs to respond to environmental conditions or fluctuating levels of effector molecules by undergoing dynamic conformational changes that switch on or off activities such as catalysis, transcription or translation. Infectious RNAs must also temporally control incompatible activities and rapidly complete their life cycle before being targeted by cellular defenses. Viral genomic RNAs must switch between translation and replication, and untranslated subviral RNAs must control other activities such as RNA editing or self-cleavage. Unlike well characterized riboswitches in cellular RNAs, the control of infectious RNA activities by altering the configuration of functional RNA domains has only recently been recognized. In this review, we will present some of these molecular rearrangements found in RNA viruses, viroids and virus-associated RNAs, relating how these dynamic regions were discovered, the activities that might be regulated, and what factors or conditions might cause a switch between conformations.

  4. Crystal structures of β-1,4-galactosyltransferase 7 enzyme reveal conformational changes and substrate binding.

    Science.gov (United States)

    Tsutsui, Yuko; Ramakrishnan, Boopathy; Qasba, Pradman K

    2013-11-01

    The β-1,4-galactosyltransferase 7 (β4GalT7) enzyme is involved in proteoglycan synthesis. In the presence of a manganese ion, it transfers galactose from UDP-galactose to xylose on a proteoglycan acceptor substrate. We present here the crystal structures of human β4GalT7 in open and closed conformations. A comparison of these crystal structures shows that, upon manganese and UDP or UDP-Gal binding, the enzyme undergoes conformational changes involving a small and a long loop. We also present the crystal structures of Drosophila wild-type β4GalT7 and D211N β4GalT7 mutant enzymes in the closed conformation in the presence of the acceptor substrate xylobiose and the donor substrate UDP-Gal, respectively. To understand the catalytic mechanism, we have crystallized the ternary complex of D211N β4GalT7 mutant enzyme in the presence of manganese with the donor and the acceptor substrates together in the same crystal structure. The galactose moiety of the bound UDP-Gal molecule forms seven hydrogen bonds with the protein molecule. The nonreducing end of the xylose moiety of xylobiose binds to the hydrophobic acceptor sugar binding pocket created by the conformational changes, whereas its extended xylose moiety forms hydrophobic interactions with a Tyr residue. In the ternary complex crystal structure, the nucleophile O4 oxygen atom of the xylose molecule is found in close proximity to the C1 and O5 atoms of the galactose moiety. This is the first time that a Michaelis complex of a glycosyltransferase has been described, and it clearly suggests an SN2 type catalytic mechanism for the β4GalT7 enzyme.

  5. Conformational Changes in the Capsid of a Calicivirus upon Interaction with Its Functional Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Ossiboff, Robert J.; Zhou, Yi; Lightfoot, Patrick J.; Prasad, B.V. Venkataram; Parker, John S.L. (Baylor); (Cornell)

    2010-07-19

    Nonenveloped viral capsids are metastable structures that undergo conformational changes during virus entry that lead to interactions of the capsid or capsid fragments with the cell membrane. For members of the Caliciviridae, neither the nature of these structural changes in the capsid nor the factor(s) responsible for inducing these changes is known. Feline functional adhesion molecule A (fJAM-A) mediates the attachment and infectious viral entry of feline calicivirus (FCV). Here, we show that the infectivity of some FCV isolates is neutralized following incubation with the soluble receptor at 37 C. We used this property to select mutants resistant to preincubation with the soluble receptor. We isolated and sequenced 24 soluble receptor-resistant (srr) mutants and characterized the growth properties and receptor-binding activities of eight mutants. The location of the mutations within the capsid structure of FCV was mapped using a new 3.6-{angstrom} structure of native FCV. The srr mutations mapped to the surface of the P2 domain were buried at the protruding domain dimer interface or were present in inaccessible regions of the capsid protein. Coupled with data showing that both the parental FCV and the srr mutants underwent increases in hydrophobicity upon incubation with the soluble receptor at 37 C, these findings indicate that FCV likely undergoes conformational change upon interaction with its receptor. Changes in FCV capsid conformation following its interaction with fJAM-A may be important for subsequent interactions of the capsid with cellular membranes, membrane penetration, and genome delivery.

  6. Single color FRET based measurements of conformational changes of proteins resulting from translocation inside cells.

    Science.gov (United States)

    Gahl, Robert F; Tekle, Ephrem; Tjandra, Nico

    2014-03-15

    Translocation of proteins to different parts of the cell is necessary for many cellular mechanisms as a means for regulation and a variety of other functions. Identifying how these proteins undergo conformational changes or interact with various partners during these events is critical to understanding how these mechanisms are executed. A protocol is presented that identifies conformational changes in a protein that occur during translocation while overcoming challenges in extracting distance information in very different environments of a living cell. Only two samples are required to be prepared and are observed with one optical setup. Live-cell FRET imaging has been applied to identify conformational changes between two native cysteines in Bax, a member of the Bcl-2 family of proteins that regulates apoptosis. Bax exists in the cytosol and translocates to the mitochondria outer membrane upon apoptosis induction. The distance, r, between the two native cysteines in the cytosolic structure of Bax necessitates the use of a FRET donor-accepter pair with R0~r as the most sensitive probe for identifying structural changes at these positions. Alexa Fluor 546 and Dabcyl, a dark acceptor, were used as FRET pairs - resulting in single color intensity variations of Alexa-546 as a measure of FRET efficiency. An internal reference, conjugated to Bax, was employed to normalize changes in fluorescence intensity of Alexa Fluor 546 due to inherent inhomogeneities in the living cell. This correction allowed the true FRET effects to be measured with increased precision during translocation. Normalization of intensities to the internal reference identified a FRET efficiency of 0.45±0.14 in the cytosol and 0.11±0.20 in the mitochondria. The procedure for the conjugation of the internal reference and FRET probes as well as the data analysis is presented. Published by Elsevier Inc.

  7. Thermally induced conformational changes of Ca-arachidate Langmuir-Blodgett Films at different compression

    Science.gov (United States)

    Weber, Jan; Beier, Andre; Hasselbrink, Eckart; Balgar, Thorsten

    2014-07-01

    The conformational order in Ca-arachidate Langmuir-Blodgett films on solid glass supports is investigated by means of vibrational sum-frequency generation spectroscopy (VSFG). The symmetric C-H stretching vibrations of both the terminal methyl and the methylene groups are utilized to monitor the chain conformation at various sample temperatures under ambient conditions. At room temperature the film is well-ordered consisting almost entirely of all-trans configured chains. Between 340 and 430 K we observe a marked increase in gauche-defects before oxidative degeneration starts at sample temperatures above 470 K. The temperature dependence of the data is well represented by apparent enthalpy changes for the formation of gauche-defects, sharply increasing with packing density from 29 to 62 kJ/mol; values, which are an order of magnitude larger than those of the gas phase molecule. These large apparent enthalpies do not prevent the formation of a high degree of conformational disorder at elevated temperatures.

  8. Targeting C-Reactive Protein in Inflammatory Disease by Preventing Conformational Changes

    Directory of Open Access Journals (Sweden)

    J. R. Thiele

    2015-01-01

    Full Text Available C-reactive protein (CRP is a pentraxin that has long been employed as a marker of inflammation in clinical practice. Recent findings brought up the idea of CRP to be not only a systemic marker but also a mediator of inflammation. New studies focused on structural changes of the plasma protein, revealing the existence of two distinct protein conformations associated with opposed inflammatory properties. Native, pentameric CRP (pCRP is considered to be the circulating precursor form of monomeric CRP (mCRP that has been identified to be strongly proinflammatory. Recently, a dissociation mechanism of pCRP has been identified on activated platelets and activated/apoptotic cells associated with the amplification of the proinflammatory potential. Correspondingly, CRP deposits found in inflamed tissues have been identified to exhibit the monomeric conformation by using conformation-specific antibodies. Here we review the current literature on the causal role of the dissociation mechanism of pCRP and the genesis of mCRP for the amplification of the proinflammatory potential in inflammatory reactions such as atherosclerosis and ischemia/reperfusion injury. The chance to prevent the formation of proinflammatory mediators in ubiquitous inflammatory cascades has pushed therapeutic strategies by targeting pCRP dissociation in inflammation. In this respect, the development of clinically applicable derivatives of the palindromic compound 1,6-bis(phosphocholine-hexane (1,6-bis PC should be a major focus of future CRP research.

  9. Raman Spectroscopy of Conformational Changes in Membrane-Bound Sodium Potassium ATPase

    DEFF Research Database (Denmark)

    Helix Nielsen, Claus; Abdali, Salim; Lundbæk, Jens August

    2007-01-01

    In this investigation we assess the potential of Raman spectroscopy as a tool for probing conformational changes in membrane-spanning proteins — in this case, the sodium potassium adenosine triphosphatase (Na+,K+-ATPase). Spectral analysis of protein-lipid complexes is complicated by the presence....... This illustrates the stabilizing role of the N-terminal domain under physiological conditions. More generally, it shows that Raman spectroscopy might be a useful tool in understanding the relationship between functional states and structural changes in membrane-bound proteins.......+,K+-ATPase stabilized by N-terminal truncation differs from that induced by Na+ binding, and that the N-terminal truncation leads to changes in protein structure that affect the average hydrophobic environment of protein Tyr, possibly reflecting changes in the hydrophobic coupling between protein and membrane...

  10. Ligand-induced changes in estrogen receptor conformation as measured by site-directed spin labeling.

    Science.gov (United States)

    Hurth, Kyle M; Nilges, Mark J; Carlson, Kathryn E; Tamrazi, Anobel; Belford, R Linn; Katzenellenbogen, John A

    2004-02-24

    Site-directed spin labeling (SDSL), the site-specific incorporation of nitroxide spin-labels into a protein, has allowed us to investigate ligand-induced conformational changes in the ligand-binding domain of human estrogen receptor alpha (hERalpha-LBD). EPR (electron paramagnetic resonance) spectroscopy of the nitroxide probe attached to ER produces different spectra depending upon the identity of the bound ligand; these differences are indicative of changes in the type and degree of motional character of the spin-label induced by different ligand-induced conformations of labeled ER. Visual inspection of EPR spectra, construction of B versus C cross-correlation plots, and cross-comparison of spectral pairs using a relative squared difference (RSD) calculation allowed receptor-ligand complexes to be profiled according to their conformational character. Plotting B and C parameters allowed us to evaluate the liganded receptor according to the motional characteristics of the attached spin-label, and they were particularly illustrative for the receptor labeled at position 530, which had motion between the fast and intermediate regimes. RSD analysis allowed us to directly compare the similarity or difference between two different spectra, and these comparisons produced groupings that paralleled those seen in B versus C cross-correlation plots, again relating meaningfully with the pharmacological nature of the bound ligand. RSD analysis was also particularly useful for qualifying differences seen with the receptor labeled at position 417, which had motion between the intermediate and slow motional regimes. This work demonstrates that B and C formulas from EPR line shape theory are useful for qualitative analysis of spectra with differences subtler than those that are often analyzed by EPR spectroscopists. This work also provides evidence that the ER can exist in a range of conformations, with specific conformations resulting from preferential stabilization of ER by the

  11. Evidence of Conformational Changes in Adsorbed Lysozyme Molecule on Silver Colloids

    CERN Document Server

    Chandra, Goutam; Dasgupta, Swagata; Roy, Anushree

    2010-01-01

    In this article, we discuss metal-protein interactions in the Ag-lysozyme complex by spectroscopic measurements. The analysis of the variation in relative intensities of SERS bands reveal the orientation and the change in conformation of the protein molecules on the Ag surface with time. The interaction kinetics of metal-protein complexes has been analyzed over a period of three hours via both Raman and absorption measurements. Our analysis indicates that the Ag nanoparticles most likely interact with Trp-123 which is in close proximity to Phe-34 of the lysozyme molecule.

  12. Heat induces gene amplification in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Bin, E-mail: yanbin@mercyhealth.com [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Mercy Cancer Center, Mercy Medical Center-North Iowa, Mason City, IA 50401 (United States); Ouyang, Ruoyun [Department of Respiratory Medicine, The Second Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410011 (China); Huang, Chenghui [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Department of Oncology, The Third Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410013 (China); Liu, Franklin [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Neill, Daniel [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Li, Chuanyuan [Dermatology, Duke University Medical Center, Durham, NC 27710 (United States); Dewhirst, Mark [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. Black-Right-Pointing-Pointer Hyperthermia induces DNA double strand breaks. Black-Right-Pointing-Pointer DNA double strand breaks are considered to be required for the initiation of gene amplification. Black-Right-Pointing-Pointer The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 Degree-Sign C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) {gamma}H2AX immunostaining to detect {gamma}H2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 Degree-Sign C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 Degree-Sign C for 30 min induces DNA double strand breaks in HCT116 cells as shown by {gamma}H2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and

  13. gamma-Ray-mediated oxidative labeling for detecting protein conformational changes by electrospray mass spectrometry.

    Science.gov (United States)

    Tong, Xin; Wren, J Clara; Konermann, Lars

    2008-03-15

    Exposure of proteins to hydroxyl radicals induces the incorporation of oxygen atoms into solvent-exposed side chains. Earlier studies have employed this approach for mapping protein-protein interactions in mass spectrometry-based footprinting experiments. This work explores whether the overall level of gamma-ray mediated oxidative labeling can be used for monitoring large-scale conformational changes. According to a recently developed kinetic model (Tong, X.; Wren, J. C.; Konermann, L. Anal. Chem. 2007, 79, 6376-6382), the apparent first-order rate constant for oxidative labeling can be approximated as k(app) = k(RAD)/([P](tot) + C/k(u)), where k(RAD) is the primary rate of *OH formation, [P](tot) is the protein concentration, C reflects the presence of competing radical deactivation channels, and ku is the rate constant at which hydroxyl radicals react with the protein. The current study introduces conformational effects into this model by proposing that k(u) = [see text for formula] , where N is the number of amino acids, alphai is a measure for the solvent exposure of residue i, and k(ch)(i) is the oxidation rate constant that would apply for a completely solvent-exposed side chain. Using myoglobin and cytochrome c as model systems, it is demonstrated that unfolding by addition of H(3)PO(4) increases k(app) by up to 30% and 70%, respectively. Unfolding by other commonly used denaturants such as organic acids or urea results in dramatically lower oxidation levels than for the native state, a behavior that is due to the radical scavenging activity of these substances (corresponding to an increased value of C). Control experiments on model peptides are suitable for identifying such "secondary" effects, i.e., factors that modify oxidation levels without being related to conformational changes. In conclusion, the overall *OH labeling level represents a viable probe of large-scale protein conformational changes only under conditions where secondary effects are known

  14. Optical properties of gelatin/CdS bionanocomposite and its conformational change**

    Institute of Scientific and Technical Information of China (English)

    Tang Shi-hua; Li You-qun; Wang Jun; Wang Bai-yang

    2013-01-01

    BACKGROUND:The properties, integration mechanism and conformation change of protein are closely related with the biological effects of nanocomposites. OBJECTIVE:To study the optical properties and integration mechanism of gelatin/CdS bionanocomposite, and to analyze the conformation change of gelatin macromolecule at pH 12.0. METHODS:The gelatin/CdS bionanocomposites were synthesized via one-pot chemical route with the materials of gelatin, cadmium acetate and Na 2 S•9H 2 O through adding the Cd2+and S2−solution into the gelatin dilute solution at the temperature of 296, 302 and 308 K, and with the concentration of 8×10−6-1.2×10−3 RESULTS AND CONCLUSION:Transmission electron microscopy images showed that the morphology of gelatin/CdS nanocomposites was mainly chain-shaped. The ultraviolet-visible spectroscopy showed that the gelatin/CdS nanocomposites band gap was decreased with the increasing in both the CdS concentration and temperature, and they showed an obvious quantum size effect. The dynamic light scattering showed that theζpotential was negative and decreased slightly with the increasing in concentration of CdS. The Fourier transform infrared spectroscopy showed that level ofα-helix andβ-sheets in gelatin macromolecular conformation was decreased and theβ-turns level was increased. The gelatin/Cd 2+complex and gelatin/CdS nanocomposites were formed on the basis of various observations, the most plausible mechanism is proposed for the integration of gelatin/CdS nanocomposites, which includes the coordination (Cd2+with the oxygen of carbonyl group in gelatin molecular chain), vulcanization and surface coated. The shape,ζpotential and optical properties of samples were characterized by transmission electron microscope, dynamic light scattering, X-ray diffraction, ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy.

  15. Effect of proteins and their conformation change during brushite transformation to hydroxyapatite

    Science.gov (United States)

    Xie, Jing

    2000-10-01

    Hydroxyapatite (HA, Ca5(PO4)3OH) coatings on metallic orthopedic implant are being used to achieve implant integration. However, HA is stable in physiological solutions, other more reactive calcium phosphate ceramics (CPC) such as brushite (CaHPO4·2H 2O) have been found to release calcium and phosphate ions during their transformation to HA. The release of these ions may induce faster bone growth and enhance implant integration. This work examines the biocompatibility of the CPC phases that form during the transformation process. Since biocompatibility is associated with cellular response, which in turn is initiated by protein adsorption, this work focuses on the mutual effect between protein adsorption and CPC transformation. The first part of the study is focused on the influence of protein adsorption on transformation kinetics and chemistry. Brushite coated samples immersed in protein free and proteinaceous physiological solutions were retrieved after different exposures times. These were examined using XRD, EDS and FTIR/reflectance. Results show that the presence of Bovine Serum Albumin (BSA) in physiological solution retards the transformation, but the presence of Fibronectin (FN) accelerates the transformation to HA. Interestingly, neither BSA nor FN alters the transformation chemistry. Due to the limitations of the techniques used, this part of the work does not monitor the effect of transformation on adsorbed proteins but only the effect of adsorbed protein on the transforming calcium phosphate coating. The second part of the work examines in situ conformational changes of adsorbed proteins during the CPC transformation using FTIR/ATR. Protein adsorbed on different surfaces such as germanium, CPC, zinc selenide and titanium shows different conformation indicated by the Amide I and II absorption bands in the infrared spectra. During the transformation of brushite to HA, both BSA and FN show a continuous change in conformation, which suggests that the

  16. DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction.

    Science.gov (United States)

    Algasaier, Sana I; Exell, Jack C; Bennet, Ian A; Thompson, Mark J; Gotham, Victoria J B; Shaw, Steven J; Craggs, Timothy D; Finger, L David; Grasby, Jane A

    2016-04-08

    Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5'-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5'-terminiin vivo Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5'-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5'-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Tyr(40), Asp(181), and Arg(100)and a reacting duplex 5'-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound and 5'-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage.

  17. Conformational changes opening and closing the CFTR chloride channel: insights from cysteine scanning mutagenesis.

    Science.gov (United States)

    El Hiani, Yassine; Linsdell, Paul

    2014-12-01

    Cystic fibrosis, the most common lethal genetic disease affecting young people in North America, is caused by failure of the chloride ion channel known as CFTR (cystic fibrosis transmembrane conductance regulator). CFTR belongs to the large family of ATP-binding cassette (ABC) membrane transporters. In CFTR, ATP-driven events at the nucleotide-binding domains (NBDs) open and close a gate that controls chloride permeation. However, the conformational changes concomitant with opening and closing of the CFTR gate are unknown. Diverse techniques including substituted cysteine accessibility method, disulfide cross-linking, and patch-clamp recording have been used to explore CFTR channel structure. Here, we consider the architecture of both the open and the closed CFTR channel. We review how CFTR channel structure changes between the closed and the open channel conformations and portray the relative function of both cytoplasmic and vestigial gates during the gating cycle. Understanding how the CFTR channel gates chloride permeation is central for understanding how CFTR defects lead to CF. Such knowledge opens the door for novel ways to maximize CFTR channel activity in a CF setting.

  18. Coarse-grained Simulations of Sugar Transport and Conformational Changes of Lactose Permease

    Science.gov (United States)

    Liu, Jin; Jewel, S. M. Yead; Dutta, Prashanta

    2016-11-01

    Escherichia coli lactose permease (LacY) actively transports lactose and other galactosides across cell membranes through lactose/H+ symport process. Lactose/H+ symport is a highly complex process that involves sugar translocation, H+ transfer, as well as large-scale protein conformational changes. The complete picture of lactose/H+ symport is largely unclear due to the complexity and multiscale nature of the process. In this work, we develop the force field for sugar molecules compatible with PACE, a hybrid and coarse-grained force field that couples the united-atom protein models with the coarse-grained MARTINI water/lipid. After validation, we implement the new force field to investigate the transport of a β-D-galactopyranosyl-1-thio- β-D-galactopyranoside (TDG) molecule across a wild-type LacY during lactose/H+ symport process. Results show that the local interactions between TDG and LacY at the binding pocket are consistent with the X-ray experiment. Protonation of Glu325 stabilizes the TDG and inward-facing conformation of LacY. Protonation of Glu269 induces a dramatic protein structural reorganization and causes the expulsion of TDG from LacY to both sides of the membrane. The structural changes occur primarily in the N-terminal domain of LacY. This work is supported by NSF Grants: CBET-1250107 and CBET -1604211.

  19. Conformational responses to changes in the state of ionization of titrable groups in proteins

    Science.gov (United States)

    Richman, Daniel Eric

    Electrostatic energy links the structural properties of proteins with some of their important biological functions, including catalysis, energy transduction, and binding and recognition. Accurate calculation of electrostatic energy is essential for predicting and for analyzing function from structure. All proteins have many ionizable residues at the protein-water interface. These groups tend to have ionization equilibria (pK a values) shifted slightly relative to their values in water. In contrast, groups buried in the hydrophobic interior usually have highly anomalous p Ka values. These shifts are what structure-based calculations have to reproduce to allow examination of contributions from electrostatics to stability, solubility and interactions of proteins. Electrostatic energies are challenging to calculate accurately because proteins are heterogeneous dielectric materials. Any individual ionizable group can experience very different local environments with different dielectric properties. The studies in this thesis examine the hypothesis that proteins reorganize concomitant with changes in their state of ionization. It appears that the pKa value measured experimentally reflects the average of pKa values experienced in the different electrostatic environments corresponding to different conformational microstates. Current computational models fail to sample conformational reorganization of the backbone correctly. Staphyloccocal nuclease (SNase) was used as a model protein in nuclear magnetic resonance (NMR) spectroscopy studies to characterize the conformational rearrangements of the protein coupled to changes in the ionization state of titrable groups. One set of experiments tests the hypothesis that proton binding to surface Asp and Glu side chains drives local unfolding by stabilizing less-native, more water-solvated conformations in which the side chains have normalized pKa values. Increased backbone flexibility in the ps-ns timescale, hydrogen bond (H

  20. Mechanical Activation of a Multimeric Adhesive Protein through Domain Conformational Change

    Science.gov (United States)

    Wijeratne, Sithara S.; Botello, Eric; Yeh, Hui-Chun; Zhou, Zhou; Bergeron, Angela; Frey, Eric W.; Patel, Jay M.; Moake, Joel; Dong, Jing-fei; Kiang, Ching-Hwa

    2013-01-01

    The mechanical force-induced activation of the adhesive protein von Willebrand Factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWF’s crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (pVWF) multimers to bind platelets. Here we showed that a pathological level of high shear stress exposure of pVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of multimeric VWF. We found that shear-activated pVWF multimers (spVWF) are more resistant to mechanical unfolding than non-sheared pVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of pVWF multimers. PMID:23521301

  1. Konjac glucomannan-induced changes in thiol/disulphide exchange and gluten conformation upon dough mixing.

    Science.gov (United States)

    Zhou, Yun; Zhao, Dan; Foster, Tim J; Liu, Yixin; Wang, Yu; Nirasawa, Satoru; Tatsumi, Eizo; Cheng, Yongqiang

    2014-01-15

    Effects of konjac glucomannan (KGM) on the changes in gluten upon dough mixing were investigated in this study. Wheat flour was blended with KGM and processed into dough. Farinographic analysis showed that KGM caused a significant increase in water absorption and dough development time to reach maximum consistency. Comparison of electrophoretic protein profile from control dough and KGM-dough revealed that protein fractions were similar in molecular size distribution, but the lability of glutenin aggregates slightly differed. Addition of KGM to gluten induced negative effects on exchange between sulfhydryl groups and disulphide bonds. Fourier transform-Raman spectroscopy indicated that secondary structure of gluten proteins was differentially modified related with water absorption of flours before dough formation. This study reveals that when KGM is added to the dough, conformational behaviours of gluten proteins are changed and the hydroxyl groups of KGM might be involved in the interaction by forming strong intermolecular hydrogen bonding system.

  2. Molecular dynamics analysis of conformational change of paramyxovirus F protein during the initial steps of membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Martin-Garcia, Fernando; Mendieta-Moreno, Jesus Ignacio; Mendieta, Jesus [Centro de Biologia Molecular ' Severo Ochoa' (CSIC/UAM), C/ Nicolas Cabrera, 1, Cantoblanco, 28049 Madrid (Spain); Biomol-Informatics SL, Parque Cientifico de Madrid, C/ Faraday, 7, Cantoblanco, 28049 Madrid (Spain); Gomez-Puertas, Paulino, E-mail: pagomez@cbm.uam.es [Centro de Biologia Molecular ' Severo Ochoa' (CSIC/UAM), C/ Nicolas Cabrera, 1, Cantoblanco, 28049 Madrid (Spain)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Initial conformational change of paramyxovirus F protein is caused only by mechanical forces. Black-Right-Pointing-Pointer HRA region undergoes a structural change from a beta + alpha conformation to an extended coil and then to an all-alpha conformation. Black-Right-Pointing-Pointer HRS domains of F protein form three single {alpha}-helices prior to generation of the coiled coil. -- Abstract: The fusion of paramyxovirus to the cell membrane is mediated by fusion protein (F protein) present in the virus envelope, which undergoes a dramatic conformational change during the process. Unlike hemagglutinin in orthomyxovirus, this change is not mediated by an alteration of environmental pH, and its cause remains unknown. Steered molecular dynamics analysis leads us to suggest that the conformational modification is mediated only by stretching mechanical forces once the transmembrane fusion peptide of the protein is anchored to the cell membrane. Such elongating forces will generate major secondary structure rearrangement in the heptad repeat A region of the F protein; from {beta}-sheet conformation to an elongated coil and then spontaneously to an {alpha}-helix. In addition, it is proposed that the heptad repeat A region adopts a final three-helix coiled coil and that this structure appears after the formation of individual helices in each monomer.

  3. Fluorescence analysis of the lipid binding-induced conformational change of apolipoprotein E4.

    Science.gov (United States)

    Mizuguchi, Chiharu; Hata, Mami; Dhanasekaran, Padmaja; Nickel, Margaret; Phillips, Michael C; Lund-Katz, Sissel; Saito, Hiroyuki

    2012-07-17

    Apolipoprotein (apo) E is thought to undergo conformational changes in the N-terminal helix bundle domain upon lipid binding, modulating its receptor binding activity. In this study, site-specific fluorescence labeling of the N-terminal (S94) and C-terminal (W264 or S290) helices in apoE4 by pyrene maleimide or acrylodan was employed to probe the conformational organization and lipid binding behavior of the N- and C-terminal domains. Guanidine denaturation experiments monitored by acrylodan fluorescence demonstrated the less organized, more solvent-exposed structure of the C-terminal helices compared to the N-terminal helix bundle. Pyrene excimer fluorescence together with gel filtration chromatography indicated that there are extensive intermolecular helix-helix contacts through the C-terminal helices of apoE4. Comparison of increases in pyrene fluorescence upon binding of pyrene-labeled apoE4 to egg phosphatidylcholine small unilamellar vesicles suggests a two-step lipid-binding process; apoE4 initially binds to a lipid surface through the C-terminal helices followed by the slower conformational reorganization of the N-terminal helix bundle domain. Consistent with this, fluorescence resonance energy transfer measurements from Trp residues to acrylodan attached at position 94 demonstrated that upon binding to the lipid surface, opening of the N-terminal helix bundle occurs at the same rate as the increase in pyrene fluorescence of the N-terminal domain. Such a two-step mechanism of lipid binding of apoE4 is likely to apply to mostly phospholipid-covered lipoproteins such as VLDL. However, monitoring pyrene fluorescence upon binding to HDL(3) suggests that not only apoE-lipid interactions but also protein-protein interactions are important for apoE4 binding to HDL(3).

  4. Detection of Receptor-Induced Glycoprotein Conformational Changes on Enveloped Virions by Using Confocal Micro-Raman Spectroscopy

    Science.gov (United States)

    Lu, Xiaonan; Liu, Qian; Benavides-Montano, Javier A.; Nicola, Anthony V.; Aston, D. Eric; Rasco, Barbara A.

    2013-01-01

    Conformational changes in the glycoproteins of enveloped viruses are critical for membrane fusion, which enables viral entry into cells and the pathological cell-cell fusion (syncytia) associated with some viral infections. However, technological capabilities for identifying viral glycoproteins and their conformational changes on actual enveloped virus surfaces are generally scarce, challenging, and time-consuming. Our model, Nipah virus (NiV), is a syncytium-forming biosafety level 4 pathogen with a high mortality rate (40 to 75%) in humans. Once the NiV attachment glycoprotein (G) (NiV-G) binds the cell receptor ephrinB2 or -B3, G triggers conformational changes in the fusion glycoprotein (F) that result in membrane fusion and viral entry. We demonstrate that confocal micro-Raman spectroscopy can, within minutes, simultaneously identify specific G and F glycoprotein signals and receptor-induced conformational changes in NiV-F on NiV virus-like particles (VLPs). First, we identified reproducible G- and F-specific Raman spectral features on NiV VLPs containing M (assembly matrix protein), G, and/or F or on NiV/vesicular stomatitis virus (VSV) pseudotyped virions via second-derivative transformations and principal component analysis (PCA). Statistical analyses validated our PCA models. Dynamic temperature-induced conformational changes in F and G or receptor-induced target membrane-dependent conformational changes in F were monitored in NiV pseudovirions in situ in real time by confocal micro-Raman spectroscopy. Advantageously, Raman spectroscopy can identify specific protein signals in relatively impure samples. Thus, this proof-of-principle technological development has implications for the rapid identification and biostability characterization of viruses in medical, veterinary, and food samples and for the analysis of virion glycoprotein conformational changes in situ during viral entry. PMID:23283947

  5. Thermal Performance Study of Composite Phase Change Material with Polyacrylicand Conformal Coating.

    Science.gov (United States)

    Kee, Shin Yiing; Munusamy, Yamuna; Ong, Kok Seng; Cornelis Metselaar, Hendrik Simon; Chee, Swee Yong; Lai, Koon Chun

    2017-07-28

    The composite PCM was prepared by blending polymethyl methacrylate (PMMA) and myristic acid (MA) in different weight percentages. The MA and PMMA were selected as PCM and supporting material, respectively. As liquid MA may leak out during the phase transition, this study proposes the use of two coatings, namely a polyacrylic coating and a conformal coating to overcome the leakage problem. Both coatings were studied in terms of the leakage test, chemical compatibility, thermal stability, morphology, and reliability. No leakage was found in the PCMs with coatings compared to those without under the same proportions of MA/PMMA, thus justifying the use of coatings in the present study. The chemically compatibility was confirmed by FTIR spectra: the functional groups of PCMs were in accordance with those of coatings. DSC showed that the coatings did not significantly change the melting and freezing temperatures, however, they improved the thermal stability of composite PCMs as seen in TGA analysis. Furthermore, the composite PCMs demonstrated good thermal reliability after 1000 times thermal cycling. The latent heat of melting reduced by only 0.16% and 1.02% for the PCMs coated with conformal coating and polyacrylic coating, respectively. Therefore, the proposed coatings can be considered in preparing fatty acid/PMMA blends attributed to the good stability, compatibility and leakage prevention.

  6. Crystal Structure and Conformational Change Mechanism of a Bacterial Nramp-Family Divalent Metal Transporter.

    Science.gov (United States)

    Bozzi, Aaron T; Bane, Lukas B; Weihofen, Wilhelm A; Singharoy, Abhishek; Guillen, Eduardo R; Ploegh, Hidde L; Schulten, Klaus; Gaudet, Rachelle

    2016-12-06

    The widely conserved natural resistance-associated macrophage protein (Nramp) family of divalent metal transporters enables manganese import in bacteria and dietary iron uptake in mammals. We determined the crystal structure of the Deinococcus radiodurans Nramp homolog (DraNramp) in an inward-facing apo state, including the complete transmembrane (TM) segment 1a (absent from a previous Nramp structure). Mapping our cysteine accessibility scanning results onto this structure, we identified the metal-permeation pathway in the alternate outward-open conformation. We investigated the functional impact of two natural anemia-causing glycine-to-arginine mutations that impaired transition metal transport in both human Nramp2 and DraNramp. The TM4 G153R mutation perturbs the closing of the outward metal-permeation pathway and alters the selectivity of the conserved metal-binding site. In contrast, the TM1a G45R mutation prevents conformational change by sterically blocking the essential movement of that helix, thus locking the transporter in an inward-facing state.

  7. Accelerated molecular dynamics and protein conformational change: a theoretical and practical guide using a membrane embedded model neurotransmitter transporter.

    Science.gov (United States)

    Gedeon, Patrick C; Thomas, James R; Madura, Jeffry D

    2015-01-01

    Molecular dynamics simulation provides a powerful and accurate method to model protein conformational change, yet timescale limitations often prevent direct assessment of the kinetic properties of interest. A large number of molecular dynamic steps are necessary for rare events to occur, which allow a system to overcome energy barriers and conformationally transition from one potential energy minimum to another. For many proteins, the energy landscape is further complicated by a multitude of potential energy wells, each separated by high free-energy barriers and each potentially representative of a functionally important protein conformation. To overcome these obstacles, accelerated molecular dynamics utilizes a robust bias potential function to simulate the transition between different potential energy minima. This straightforward approach more efficiently samples conformational space in comparison to classical molecular dynamics simulation, does not require advanced knowledge of the potential energy landscape and converges to the proper canonical distribution. Here, we review the theory behind accelerated molecular dynamics and discuss the approach in the context of modeling protein conformational change. As a practical example, we provide a detailed, step-by-step explanation of how to perform an accelerated molecular dynamics simulation using a model neurotransmitter transporter embedded in a lipid cell membrane. Changes in protein conformation of relevance to the substrate transport cycle are then examined using principle component analysis.

  8. Lidocaine action and conformational changes in cytoskeletal protein network in human red blood cells.

    Science.gov (United States)

    Nishiguchi, E; Hamada, N; Shindo, J

    1995-11-03

    The mechanism of action of lidocaine, which is commonly used clinically as a local anesthetic, was studied in human red blood cells. The influx of [14C]lidocaine through the cell membrane induced reversible transformation of human red blood cells from discocytes to stomatocytes. This change in shape depended on the lidocaine concentration and required both ATP and carbonic anhydrase. The lidocaine-induced shape change occurred as a result of spectrin aggregation, which altered the intracellular environment of the human red blood cells, mediated by carbonic anhydrase and activation of vacuolar type H(+)-ATPase (V-ATPase). Lidocaine controlled the influx of 22Na into the human red blood cells in a concentration-dependent manner. When incubated in media containing 6-chloro-9-[(4-diethylamino)-1-methyl-butyl]amino-2-methoxyacridine (mepacrine), an inhibitor of Na+ channels, human red blood cells changed shape from discocytes to stomatocytes and the intracellular pH decreased. This phenomenon was very similar to the shape change induced by lidocaine. These results suggest that the mode of action of lidocaine is related to a conformational change in the cytoskeletal protein network.

  9. Real-time visualization of conformational changes within single MloK1 cyclic nucleotide-modulated channels.

    Science.gov (United States)

    Rangl, Martina; Miyagi, Atsushi; Kowal, Julia; Stahlberg, Henning; Nimigean, Crina M; Scheuring, Simon

    2016-09-20

    Eukaryotic cyclic nucleotide-modulated (CNM) ion channels perform various physiological roles by opening in response to cyclic nucleotides binding to a specialized cyclic nucleotide-binding domain. Despite progress in structure-function analysis, the conformational rearrangements underlying the gating of these channels are still unknown. Here, we image ligand-induced conformational changes in single CNM channels from Mesorhizobium loti (MloK1) in real-time, using high-speed atomic force microscopy. In the presence of cAMP, most channels are in a stable conformation, but a few molecules dynamically switch back and forth (blink) between at least two conformations with different heights. Upon cAMP depletion, more channels start blinking, with blinking heights increasing over time, suggestive of slow, progressive loss of ligands from the tetramer. We propose that during gating, MloK1 transitions from a set of mobile conformations in the absence to a stable conformation in the presence of ligand and that these conformations are central for gating the pore.

  10. Conformational changes of active site of copper zinc superoxide dismutase can be detected sensitively by electron-transfer reaction

    Institute of Scientific and Technical Information of China (English)

    舒占永

    1996-01-01

    The electron-transfer (ET) reaction between Fe(CN)64- and copper zinc superoxide dismutase (CuZn-SOD) occurs at the active site of the enzyme. The ET parameters which are sensitive to the denaturation have been used to determine the conformational changes of the active site induced by guanidine hydrochloride and thermal denaturation. The decreases of ET rates for all the denatured enzyme samples reflect the collapse of the active cavity of enzyme in the unfolding processes. The interesting changes of ET amplitude for the enzyme denatured at different pH values suggest that electrostatic interaction plays an important role in the conformational changes of active site. From the results of the kinetic analyses, it is concluded that the conformational changes of the active site are parallel with the inactivation.

  11. Fluorescence study on ligand induced conformational changes of glutamine synthetase from Bacillus brevis Bb G1 under sporulating conditions

    Directory of Open Access Journals (Sweden)

    SUJA ABRAHAM

    2015-04-01

    Full Text Available Glutamine synthetase, an important enzyme of nitrogen metabolism, was purified under sporulating conditions (GSala. The effect of ligands on the tryptophan fluorescence of the purified enzyme GSala was investigated. With increasing concentrations of L-glutamine in GSala, a blue shift in emission maximum with an increase in fluorescence intensity and decrease in life times were observed compared to the emission maximum, fluorescence intensity and life times of GSala. With increasing concentrations of glycine in GSala, a shift in emission maximum, change in fluorescence intensity and change in lifetimes were observed compared to the emission maximum, fluorescence intensity and life times of GSala. These observations strongly support the possibility that GSala undergoes a conformational change on binding with ligands and each ligand produced different conformational changes in GSala. Also, different concentrations of each ligand produced different protein conformations in the enzyme GSala.

  12. Parallel dynamics and evolution: Protein conformational fluctuations and assembly reflect evolutionary changes in sequence and structure.

    Science.gov (United States)

    Marsh, Joseph A; Teichmann, Sarah A

    2014-02-01

    Protein structure is dynamic: the intrinsic flexibility of polypeptides facilitates a range of conformational fluctuations, and individual protein chains can assemble into complexes. Proteins are also dynamic in evolution: significant variations in secondary, tertiary and quaternary structure can be observed among divergent members of a protein family. Recent work has highlighted intriguing similarities between these structural and evolutionary dynamics occurring at various levels. Here we review evidence showing how evolutionary changes in protein sequence and structure are often closely related to local protein flexibility and disorder, large-scale motions and quaternary structure assembly. We suggest that these correspondences can be largely explained by neutral evolution, while deviations between structural and evolutionary dynamics can provide valuable functional insights. Finally, we address future prospects for the field and practical applications that arise from a deeper understanding of the intimate relationship between protein structure, dynamics, function and evolution.

  13. Protein adsorption on tailored substrates: long-range forces and conformational changes

    Energy Technology Data Exchange (ETDEWEB)

    Bellion, M; Santen, L [Department of Theoretical Physics, Saarland University, 66041 Saarbruecken (Germany); Mantz, H; Haehl, H; Quinn, A; Nagel, A; Gilow, C; Weitenberg, C; Schmitt, Y; Jacobs, K [Department of Experimental Physics, Saarland University, 66041 Saarbruecken (Germany)], E-mail: k.jacobs@physik.uni-saarland.de

    2008-10-08

    Adsorption of proteins onto solid surfaces is an everyday phenomenon that is not yet fully understood. To further the current understanding, we have performed in situ ellipsometry studies to reveal the adsorption kinetics of three different proteins, lysozyme, {alpha}-amylase and bovine serum albumin. As substrates we offer Si wafers with a controlled Si oxide layer thickness and a hydrophilic or hydrophobic surface functionalization, allowing the tailoring of the influence of short- and long-range interactions. Our studies show that not only the surface chemistry determines the properties of an adsorbed protein layer but also the van der Waals contributions of a composite substrate. We compare the experimental findings to results of a colloidal Monte Carlo approach that includes conformational changes of the adsorbed proteins induced by density fluctuations.

  14. Long-Lived Conformation Changes Induced by Electric Impulses in Biopolymers

    Science.gov (United States)

    Neumann, Eberhard; Katchalsky, Aharon

    1972-01-01

    Electric impulses are capable of inducing long-lived conformational changes in (metastable) biopolymers. Results of experiments with poly(A)·2 poly(U) and ribosomal RNA, which are known to develop metastabilities, are reported. A polarization mechanism is proposed to explain the structural transitions observed in the biopolymers exposed to the impulses. In accordance with this idea, the applied electric field (of about 20 kV/cm and decaying exponentially, with a decay time of about 10 μsec) induces large dipole moments by shifting the ionic atmosphere of multistranded polynucleotide helices. This shift, in turn, causes strand repulsion and partial unwinding. The fields used in our experiments are of the same order of magnitude as those in nerve impulses. The significance of the impulse experiments with regard to the question of biological memory recording is briefly discussed. PMID:4502948

  15. Protein adsorption on tailored substrates: long-range forces and conformational changes

    Science.gov (United States)

    Bellion, M.; Santen, L.; Mantz, H.; Hähl, H.; Quinn, A.; Nagel, A.; Gilow, C.; Weitenberg, C.; Schmitt, Y.; Jacobs, K.

    2008-10-01

    Adsorption of proteins onto solid surfaces is an everyday phenomenon that is not yet fully understood. To further the current understanding, we have performed in situ ellipsometry studies to reveal the adsorption kinetics of three different proteins, lysozyme, α-amylase and bovine serum albumin. As substrates we offer Si wafers with a controlled Si oxide layer thickness and a hydrophilic or hydrophobic surface functionalization, allowing the tailoring of the influence of short- and long-range interactions. Our studies show that not only the surface chemistry determines the properties of an adsorbed protein layer but also the van der Waals contributions of a composite substrate. We compare the experimental findings to results of a colloidal Monte Carlo approach that includes conformational changes of the adsorbed proteins induced by density fluctuations.

  16. DNA induces conformational changes in a recombinant human minichromosome maintenance complex.

    Science.gov (United States)

    Hesketh, Emma L; Parker-Manuel, Richard P; Chaban, Yuriy; Satti, Rabab; Coverley, Dawn; Orlova, Elena V; Chong, James P J

    2015-03-20

    ATP-dependent DNA unwinding activity has been demonstrated for recombinant archaeal homohexameric minichromosome maintenance (MCM) complexes and their yeast heterohexameric counterparts, but in higher eukaryotes such as Drosophila, MCM-associated DNA helicase activity has been observed only in the context of a co-purified Cdc45-MCM-GINS complex. Here, we describe the production of the recombinant human MCM (hMCM) complex in Escherichia coli. This protein displays ATP hydrolysis activity and is capable of unwinding duplex DNA. Using single-particle asymmetric EM reconstruction, we demonstrate that recombinant hMCM forms a hexamer that undergoes a conformational change when bound to DNA. Recombinant hMCM produced without post-translational modifications is functional in vitro and provides an important tool for biochemical reconstitution of the human replicative helicase.

  17. 牛半腱肌肉结缔组织胶原蛋白热力特性热诱导变化DSC分析%DSC Analysis of Heat-induced Changes in Thermal Characteristics of Connective Tissue Collagen from Beef Semitendinosus Muscle

    Institute of Scientific and Technical Information of China (English)

    常海军; 王强; 徐幸莲; 周光宏

    2011-01-01

    The main objective of this study was to investigate the heat-induced changes of thermal characteristics of connective tissue collagen from beef Semitendinosus muscle.Muscle samples were heated to an internal core temperature of 40,50,60,70,80 or 90 ℃ in a water bath and in a microwave oven respectively.The changes of filter residues content and thermal characteristics of connective tissue collagen were examined with internal core temperature.The results indicated that filter residues were increased during heating,and presented significant differences(P 0.05) at the internal core temperatures of 60,70 ℃ and 80 ℃ between water bath and microwave heated meat samples.Internal core temperatures ranging from 40 to 60 ℃ were critical heating temperatures which affect thermal shrinkage temperatures of connective tissue collagen for both water bath and micro-wave heated meat samples.The significant differences in thermal shrinkage temperatures between water bath and microwave heated beef muscle samples were attributed to the heat-induced changes in thermal characteristics of connective tissue collagen.%探讨牛半腱肌肉结缔组织胶原蛋白热力特性的热诱导变化。牛半腱肌肉分别采用水浴和微波加热到内部终点温度分别为40、50、60、70、80℃和90℃,研究结缔组织滤渣和热力特性在热处理过程中的变化。结果表明:结缔组织滤渣含量随着热处理温度的升高而增加,当加热温度分别为60、70℃和80℃时,结缔组织滤渣含量在两种热处理方式间存在显著差异(P〈0.05)。在两种热处理方式中,40℃至60℃的内部终点温度是影响结缔组织胶原蛋白热收缩温度的关键加热温度。热诱导的结缔组织胶原蛋白热力特性的变化是水浴和微波加热牛肉胶原蛋

  18. Conformational changes during pore formation by the perforin-related protein pleurotolysin.

    Directory of Open Access Journals (Sweden)

    Natalya Lukoyanova

    2015-02-01

    Full Text Available Membrane attack complex/perforin-like (MACPF proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM structure of the two-part, fungal toxin Pleurotolysin (Ply, together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB. These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ∼70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2. We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of

  19. Conformational changes during pore formation by the perforin-related protein pleurotolysin.

    Science.gov (United States)

    Lukoyanova, Natalya; Kondos, Stephanie C; Farabella, Irene; Law, Ruby H P; Reboul, Cyril F; Caradoc-Davies, Tom T; Spicer, Bradley A; Kleifeld, Oded; Traore, Daouda A K; Ekkel, Susan M; Voskoboinik, Ilia; Trapani, Joseph A; Hatfaludi, Tamas; Oliver, Katherine; Hotze, Eileen M; Tweten, Rodney K; Whisstock, James C; Topf, Maya; Saibil, Helen R; Dunstone, Michelle A

    2015-02-01

    Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ∼70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters

  20. Low affinity Ca2+-binding sites of calcineurin B mediate conformational changes in calcineurin A.

    Science.gov (United States)

    Yang, S A; Klee, C B

    2000-12-26

    Limited proteolysis of calcineurin in the presence of Ca(2+) suggested that its calmodulin-binding domain, readily degraded by proteases, was unfolded while calcineurin B was compactly folded [Hubbard, M. J., and Klee, C. B. (1989) Biochemistry 28, 1868-1874]. Moreover, in the crystal structure of calcineurin, with the four Ca(2+) sites of calcineurin B occupied, the calmodulin-binding domain is not visible in the electron density map [Kissinger, C. R., et al. (1995) Nature 378, 641-644]. Limited proteolysis of calcineurin in the presence of EGTA, shows that, when the low affinity sites of calcineurin B are not occupied, the calmodulin-binding domain is completely protected against proteolytic attack. Slow cleavages are, however, detected in the linker region between the calmodulin-binding and the autoinhibitory domains of calcineurin A. Upon prolonged exposure to the protease, selective cleavages in carboxyl-terminal end of the first helix and the central helix linker of calcineurin B and the calcineurin B-binding helix of calcineurin A are also detected. Thus, Ca(2+) binding to the low-affinity sites of calcineurin B affects the conformation of calcineurin B and induces a conformational change of the regulatory domain of calcineurin A, resulting in the exposure of the calmodulin-binding domain. This conformational change is needed for the partial activation of the enzyme in the absence of calmodulin and its full activation by calmodulin. A synthetic peptide corresponding to the calmodulin-binding domain is shown to interact with a peptide corresponding to the calcineurin B-binding domain, and this interaction is prevented by calcineurin B in the presence but not the absence of Ca(2+). These observations provide a mechanism to explain the dependence on Ca(2+) binding to calcineurin B for calmodulin activation and for the 10-20-fold increase in affinity of calcineurin for Ca(2+) upon removal of the regulatory domain by limited proteolysis [Stemmer, P. M., and Klee

  1. Mangiferin binding to serum albumin is non-saturable and induces conformational changes at high concentrations

    Energy Technology Data Exchange (ETDEWEB)

    Freitas, P.G.; Barbosa, A.F. [Biochemistry Laboratory, Institute of Exact Sciences, Federal University of Alfenas, Unifal-MG, R. Gabriel Monteiro da Silva, 700, 37130-000 Alfenas, MG (Brazil); Saraiva, L.A. [Phytochemistry and Medicinal Chemistry Laboratory, Institute of Exact Sciences, Unifal-MG (Brazil); Camps, I. [Physics Laboratory, Institute of Exact Sciences, Unifal-MG (Brazil); Silveira, N.J.F. da [Bioinformatics Laboratory, Institute of Exact Sciences, Unifal-MG (Brazil); Veloso, M.P. [Phytochemistry and Medicinal Chemistry Laboratory, Institute of Exact Sciences, Unifal-MG (Brazil); Santos, M.H., E-mail: poliany.santos@gmail.com [Phytochemistry and Medicinal Chemistry Laboratory, Institute of Exact Sciences, Unifal-MG (Brazil); Schneedorf, J.M., E-mail: zemasfs@gmail.com [Biochemistry Laboratory, Institute of Exact Sciences, Federal University of Alfenas, Unifal-MG, R. Gabriel Monteiro da Silva, 700, 37130-000 Alfenas, MG (Brazil)

    2012-11-15

    The binding interaction between mangiferin (MGF), which a natural xanthone isolated from mangoes, and bovine serum albumin (BSA) was studied with absorbance and fluorescence spectroscopy, cyclic voltammetry and molecular modeling. The data were analyzed to assess the binding mechanism, effect of pH and ionic strength, conformational changes in the protein and electrical charge transfer involved. The MGF-BSA complex exhibited positive cooperativity with a 1:1 stoichiometry (K{sub d}=0.38 mmol L{sup -1}) for the first binding site and a non-saturable binding at high ligand concentrations. Furthermore, the data also suggest an increase in drug bioavailability in the acidic region and relatively low ionic strength values, which are close to physiological levels. The data suggest a specific electrostatic interaction together with hydrophobic effects and H-bonding displayed in MGF binding to the BSA IIA subdomain. Synchronous fluorescence spectra indicate that there are conformational changes in the polypeptide backbone upon ligand binding. Cyclic voltammetry indicates that there is an irreversible charge transfer between MGF and BSA that is modulated by diffusion on the electrode surface, where two electrons are transferred. These results can help the knowledge of the pharmacokinetic activities of natural or chemical xanthone-based drugs. - Highlights: Black-Right-Pointing-Pointer The MGF-BSA complex exhibited positive cooperativity beyond 1:1 stoichiometry. Black-Right-Pointing-Pointer The interaction of MGF with BSA is non-saturable at higher ligand concentration. Black-Right-Pointing-Pointer The binding was accomplished by H-bonding, hydrophobic and electrostatic forces. Black-Right-Pointing-Pointer The apparent binding constant for MGF-BSA was 0.38 mmol L{sup -1}. Black-Right-Pointing-Pointer MGF binds electrostatically to BSA, different from a hydrophobic interaction to HSA.

  2. Postprandial apoE isoform and conformational changes associated with VLDL lipolysis products modulate monocyte inflammation.

    Directory of Open Access Journals (Sweden)

    Laura J den Hartigh

    Full Text Available Postprandial hyperlipemia, characterized by increased circulating very low-density lipoproteins (VLDL and circulating lipopolysaccharide (LPS, has been proposed as a mechanism of vascular injury. Our goal was to examine the interactions between postprandial lipoproteins, LPS, and apoE3 and apoE4 on monocyte activation.We showed that apoE3 complexed to phospholipid vesicles attenuates LPS-induced THP-1 monocyte cytokine expression, while apoE4 increases expression. ELISA revealed that apoE3 binds to LPS with higher affinity than apoE4. Electron paramagnetic resonance (EPR spectroscopy of site-directed spin labels placed on specific amino acids of apoE3 showed that LPS interferes with conformational changes normally associated with lipid binding. Specifically, compared to apoE4, apoE bearing the E3-like R112→Ser mutation displays increased self association when exposed to LPS, consistent with a stronger apoE3-LPS interaction. Additionally, lipolysis of fasting VLDL from normal human donors attenuated LPS-induced TNFα secretion from monocytes to a greater extent than postprandial VLDL, an effect partially reversed by blocking apoE. This effect was reproduced using fasting VLDL lipolysis products from e3/e3 donors, but not from e4/e4 subjects, suggesting that apoE3 on fasting VLDL prevents LPS-induced inflammation more readily than apoE4.Postprandial apoE isoform and conformational changes associated with VLDL dramatically modulate vascular inflammation.

  3. A picrotoxin-specific conformational change in the glycine receptor M2-M3 loop.

    Science.gov (United States)

    Hawthorne, Rebecca; Lynch, Joseph W

    2005-10-28

    The external loop linking the M2 and M3 transmembrane domains is crucial for coupling agonist binding to channel gating in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility scan previously showed that glycine activation increased the surface accessibility of 6 contiguous residues (Arg271-Lys276) toward the N-terminal end of the homomeric alpha1 GlyR M2-M3 loop. In the present study we used a similar approach to determine whether the allosteric antagonist, picrotoxin, could impose conformational changes to this domain that cannot be induced by varying agonist concentrations alone. Picrotoxin slowed the reaction rate of a sulfhydryl-containing compound (MTSET) with A272C, S273C, and L274C. Before interpreting this as a picrotoxin-specific conformational change, it was necessary to eliminate the possibility of steric competition between picrotoxin and MTSET. Accordingly, we showed that picrotoxin and the structurally unrelated blocker, bilobalide, were both trapped in the R271C GlyR in the closed state and that a point mutation to the pore-lining Thr6' residue abolished inhibition by both compounds. We also demonstrated that the picrotoxin dissociation rate was linearly related to the channel open probability. These observations constitute a strong case for picrotoxin binding in the pore. We thus conclude that the picrotoxin-specific effects on the M2-M3 loop are mediated allosterically. This suggests that the M2-M3 loop responds differently to the occupation of different binding sites.

  4. α-Helical to β-Helical Conformation Change in the C-Terminal of the Mammalian Prion Protein

    Science.gov (United States)

    Singh, Jesse; Whitford, Paul; Hayre, Natha; Cox, Daniel; Onuchic, José.

    2011-03-01

    We employ all-atom structure-based models with mixed basis contact maps to explore whether there are any significant geometric or energetic constraints limiting conjectured conformational transitions between the alpha-helical (α H) and the left handed beta helical (LHBH) conformations for the C-terminal (residues 166-226) of the mammalian prion protein. The LHBH structure has been proposed to describe infectious oligomers and one class of in vitro grown fibrils, as well as possibly self- templating the conversion of normal cellular prion protein to the infectious form. Our results confirm that the kinetics of the conformation change are not strongely limited by large scale geometry modification and there exists an overall preference for the LHBH conformation.

  5. Coarse-grained free energy functions for studying protein conformational changes: a double-well network model.

    Science.gov (United States)

    Chu, Jhih-Wei; Voth, Gregory A

    2007-12-01

    In this work, a double-well network model (DWNM) is presented for generating a coarse-grained free energy function that can be used to study the transition between reference conformational states of a protein molecule. Compared to earlier work that uses a single, multidimensional double-well potential to connect two conformational states, the DWNM uses a set of interconnected double-well potentials for this purpose. The DWNM free energy function has multiple intermediate states and saddle points, and is hence a "rough" free energy landscape. In this implementation of the DWNM, the free energy function is reduced to an elastic-network model representation near the two reference states. The effects of free energy function roughness on the reaction pathways of protein conformational change is demonstrated by applying the DWNM to the conformational changes of two protein systems: the coil-to-helix transition of the DB-loop in G-actin and the open-to-closed transition of adenylate kinase. In both systems, the rough free energy function of the DWNM leads to the identification of distinct minimum free energy paths connecting two conformational states. These results indicate that while the elastic-network model captures the low-frequency vibrational motions of a protein, the roughness in the free energy function introduced by the DWNM can be used to characterize the transition mechanism between protein conformations.

  6. Conformational changes in bovine lactoferrin induced by slow or fast temperature increases.

    Science.gov (United States)

    Schwarcz, Waleska D; Carnelocce, Lorena; Silva, Jerson L; Oliveira, Andréa C; Gonçalves, Rafael B

    2008-08-01

    Lactoferrin (LF) is an iron-binding protein present in several secreted substances, such as milk, and has broad antimicrobial and physiological properties. Because high temperatures may affect protein stability and its functional properties, we investigated the effect of heat on bovine LF structure and stability. The effects of temperatures used during the pasteurization process on LF and its relationship to protein functionality were studied. Conformational changes were monitored using spectroscopic techniques, such as circular dichroism (CD) and fluorescence spectroscopy. The CD data at 70 degrees C showed that LF's secondary structure is drastically and irreversibly affected when the temperature is gradually increased. The same effect is observed when the temperature is gradually raised from 25 degrees C to 105 degrees C and changes are monitored by tryptophan fluorescence emission. We also verified the effects of simulating the pasteurization process; LF remained well structured during the entire process and this result was not time-dependent. Owing to preservation of the secondary structure with changes in the tertiary structure, we thus believe that pasteurization might cause LF to change into an intermediate partially folded state. A better understanding of heat stability is important for the use of LF as a bioactive component in food.

  7. Differences in G-actin containing bound ATP or ADP: the Mg2+-induced conformational change requires ATP.

    Science.gov (United States)

    Frieden, C; Patane, K

    1985-07-16

    The role of adenosine 5'-triphosphate (ATP) in the Mg2+-induced conformational change of rabbit skeletal muscle G-actin has been investigated by comparing actin containing bound ADP with actin containing bound ATP. As previously described [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886], N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled G-actin containing ATP undergoes a time-dependent Mg2+-induced fluorescence change that reflects a conformational change in the actin. Addition of Mg2+ to labeled G-actin containing ADP gives no fluorescence change, suggesting that the conformational change does not occur. The fluorescence change can be restored on the addition of ATP. Examination of the time courses of these experiments suggests that ATP must replace ADP prior to the Mg2+-induced change. The Mg2+-induced polymerization of actin containing ADP is extraordinarily slow compared to that of actin containing ATP. The lack of the Mg2+-induced conformational change, which is an essential step in the Mg2+-induced polymerization, is probably the cause for the very slow polymerization of actin containing ADP. On the other hand, at 20 degrees C, at pH 8, and in 2 mM Mg2+, the elongation rate from the slow growing end of an actin filament, measured by using the protein brevin to block growth at the fast growing end, is only 4 times slower for actin containing ADP than for actin containing ATP.

  8. Cholesterol-dependent conformational changes of P-glycoprotein are detected by the 15D3 monoclonal antibody.

    Science.gov (United States)

    Gutay-Tóth, Zsuzsanna; Fenyvesi, Ferenc; Bársony, Orsolya; Szente, Lajos; Goda, Katalin; Szabó, Gábor; Bacsó, Zsolt

    2016-03-01

    The 15D3 mouse monoclonal antibody (mAb) binds an uncharacterized extracellular epitope of the ATP Binding Cassette (ABC) transporter human P-glycoprotein (Pgp). Depletion of cell plasma membrane cholesterol by using methyl-β-cyclodextrin or other chemically modified β-cyclodextrins decreased the Pgp binding affinity of 15D3 mAb. UIC2 mAb, which is known to distinguish two conformers of this ABC transporter, binds only a fraction of cell surface Pgps. UIC2 mAb non-reactive pools of Pgp can be identified with other extracellular mAbs such as 15D3. Cyclosporin A (CsA) can shift non-reactive Pgps into their UIC2-reactive conformation: a phenomenon called the "UIC2 shift". Competition studies proposed these two mAbs share overlapping epitopes and can reveal conformational changes of Pgp that correlate (r=0.97) with the cholesterol content of cells. An apparent increase in competition of these mAbs suggested a conformational change similar to those found in the presence of CsA. However, the reason turned out not to be the UIC2-shift because cholesterol removal from the plasma membrane (PM) reduced the amount of detectable Pgps by 15D3 mAb. This study showed that 15D3 mAb bound to a conformation sensitive epitope of Pgp that was responsive to PM cholesterol levels. These conformational changes were gradual and not as great as the changes observed between the two conformers recognized by the UIC2 mAb.

  9. Heat induced damage detection in composite materials by terahertz radiation

    Science.gov (United States)

    Radzieński, Maciej; Mieloszyk, Magdalena; Rahani, Ehsan Kabiri; Kundu, Tribikram; Ostachowicz, Wiesław

    2015-03-01

    In recent years electromagnetic Terahertz (THz) radiation or T-ray has been increasingly used for nondestructive evaluation of various materials such as polymer composites and porous foam tiles in which ultrasonic waves cannot penetrate but T-ray can. Most of these investigations have been limited to mechanical damage detection like inclusions, cracks, delaminations etc. So far only a few investigations have been reported on heat induced damage detection. Unlike mechanical damage the heat induced damage does not have a clear interface between the damaged part and the surrounding intact material from which electromagnetic waves can be reflected back. Difficulties associated with the heat induced damage detection in composite materials using T-ray are discussed in detail in this paper. T-ray measurements are compared for different levels of heat exposure of composite specimens.

  10. Single-Molecule Confocal FRET Microscopy to Dissect Conformational Changes in the Catalytic Cycle of DNA Topoisomerases.

    Science.gov (United States)

    Hartmann, S; Weidlich, D; Klostermeier, D

    2016-01-01

    Molecular machines undergo large-scale conformational changes during their catalytic cycles that are linked to their biological functions. DNA topoisomerases are molecular machines that interconvert different DNA topoisomers and resolve torsional stress that is introduced during cellular processes that involve local DNA unwinding. DNA gyrase catalyzes the introduction of negative supercoils into DNA in an ATP-dependent reaction. During its catalytic cycle, gyrase undergoes large-scale conformational changes that drive the supercoiling reaction. These conformational changes can be followed by single-molecule Förster resonance energy transfer (FRET). Here, we use DNA gyrase from Bacillus subtilis as an illustrative example to present strategies for the investigation of conformational dynamics of multisubunit complexes. We provide a brief introduction into single-molecule FRET and confocal microscopy, with a focus on practical considerations in sample preparation and data analysis. Different strategies in the preparation of donor-acceptor-labeled molecules suitable for single-molecule FRET experiments are outlined. The insight into the mechanism of DNA supercoiling by gyrase gained from single-molecule FRET experiment is summarized. The general strategies described here can also be applied to investigate conformational changes and their link to biological function of other multisubunit molecular machines. © 2016 Elsevier Inc. All rights reserved.

  11. Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site

    Directory of Open Access Journals (Sweden)

    Soares Alexei S

    2007-11-01

    Full Text Available Abstract Background Ricin is a potent toxin and known bioterrorism threat with no available antidote. The ricin A-chain (RTA acts enzymatically to cleave a specific adenine base from ribosomal RNA, thereby blocking translation. To understand better the relationship between ligand binding and RTA active site conformational change, we used a fragment-based approach to find a minimal set of bonding interactions able to induce rearrangements in critical side-chain positions. Results We found that the smallest ligand stabilizing an open conformer of the RTA active site pocket was an amide group, bound weakly by only a few hydrogen bonds to the protein. Complexes with small amide-containing molecules also revealed a switch in geometry from a parallel towards a splayed arrangement of an arginine-tryptophan cation-pi interaction that was associated with an increase and red-shift in tryptophan fluorescence upon ligand binding. Using the observed fluorescence signal, we determined the thermodynamic changes of adenine binding to the RTA active site, as well as the site-specific binding of urea. Urea binding had a favorable enthalpy change and unfavorable entropy change, with a ΔH of -13 ± 2 kJ/mol and a ΔS of -0.04 ± 0.01 kJ/(K*mol. The side-chain position of residue Tyr80 in a complex with adenine was found not to involve as large an overlap of rings with the purine as previously considered, suggesting a smaller role for aromatic stacking at the RTA active site. Conclusion We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the

  12. Molecular dynamics simulation study of conformational changes of transcription factor TFIIS during RNA polymerase II transcriptional arrest and reactivation.

    Directory of Open Access Journals (Sweden)

    Changsun Eun

    Full Text Available Transcription factor IIS (TFIIS is a protein known for catalyzing the cleavage reaction of the 3'-end of backtracked RNA transcript, allowing RNA polymerase II (Pol II to reactivate the transcription process from the arrested state. Recent structural studies have provided a molecular basis of protein-protein interaction between TFIIS and Pol II. However, the detailed dynamic conformational changes of TFIIS upon binding to Pol II and the related thermodynamic information are largely unknown. Here we use computational approaches to investigate the conformational space of TFIIS in the Pol II-bound and Pol II-free (unbound states. Our results reveal two distinct conformations of TFIIS: the closed and the open forms. The closed form is dominant in the Pol II-free (unbound state of TFIIS, whereas the open form is favorable in the Pol II-bound state. Furthermore, we discuss the free energy difference involved in the conformational changes between the two forms in the presence or absence of Pol II. Additionally, our analysis indicates that hydrophobic interactions and the protein-protein interactions between TFIIS and Pol II are crucial for inducing the conformational changes of TFIIS. Our results provide novel insights into the functional interplay between Pol II and TFIIS as well as mechanism of reactivation of Pol II transcription by TFIIS.

  13. Nucleic Acid-Dependent Conformational Changes in CRISPR-Cas9 Revealed by Site-Directed Spin Labeling.

    Science.gov (United States)

    Vazquez Reyes, Carolina; Tangprasertchai, Narin S; Yogesha, S D; Nguyen, Richard H; Zhang, Xiaojun; Rajan, Rakhi; Qin, Peter Z

    2017-06-01

    In a type II clustered regularly interspaced short palindromic repeats (CRISPR) system, RNAs that are encoded at the CRISPR locus complex with the CRISPR-associated (Cas) protein Cas9 to form an RNA-guided nuclease that cleaves double-stranded DNAs at specific sites. In recent years, the CRISPR-Cas9 system has been successfully adapted for genome engineering in a wide range of organisms. Studies have indicated that a series of conformational changes in Cas9, coordinated by the RNA and the target DNA, direct the protein into its active conformation, yet details on these conformational changes, as well as their roles in the mechanism of function of Cas9, remain to be elucidated. Here, nucleic acid-dependent conformational changes in Streptococcus pyogenes Cas9 (SpyCas9) were investigated using the method of site-directed spin labeling (SDSL). Single nitroxide spin labels were attached, one at a time, at one of the two native cysteine residues (Cys80 and Cys574) of SpyCas9, and the spin-labeled proteins were shown to maintain their function. X-band continuous-wave electron paramagnetic resonance spectra of the nitroxide attached at Cys80 revealed conformational changes of SpyCas9 that are consistent with a large-scale domain re-arrangement upon binding to its RNA partner. The results demonstrate the use of SDSL to monitor conformational changes in CRISPR-Cas9, which will provide key information for understanding the mechanism of CRISPR function.

  14. Pressure effects on the structure, kinetic, and thermodynamic properties of heat-induced aggregation of protein studied by FT-IR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Taniguchi, Y [Applied Chemistry Department, Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); Okuno, A [Research Department 3, Central Research, Bridgestone Co. Kodaira, Tokyo 187-8531 (Japan); Kato, M, E-mail: taniguti@sk.ritsumei.ac.j [Pharmaceutical Sciences Department, Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan)

    2010-03-01

    Pressure can retrain the heat-induced aggregation and dissociate the heat-induced aggregates. We observed the aggregation-preventing pressure effect and the aggregates-dissociating pressure effect to characterize the heat-induced aggregation of equine serum albumin (ESA) by FT-IR spectroscopy. The results suggest the {alpha}-helical structure collapses at the beginning of heat-induced aggregation through the swollen structure, and then the rearrangement of structure to the intermolecular {beta}-sheet takes place through partially unfolded structure. We determined the activation volume for the heat-induced aggregation ({Delta}V'' = +93 ml/mol) and the partial molar volume difference between native state and heat-induced aggregates ({Delta}V=+32 ml/mol). This positive partial molar volume difference suggests that the heat-induced aggregates have larger internal voids than the native structure. Moreover, the positive volume change implies that the formation of the intermolecular {beta}-sheet is unfavorable under high pressure.

  15. DBD dyes as fluorescence lifetime probes to study conformational changes in proteins.

    Science.gov (United States)

    Wawrzinek, Robert; Ziomkowska, Joanna; Heuveling, Johanna; Mertens, Monique; Herrmann, Andreas; Schneider, Erwin; Wessig, Pablo

    2013-12-16

    Previously, [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD)-based fluorophores used as highly sensitive fluorescence lifetime probes reporting on their microenvironmental polarity have been described. Now, a new generation of DBD dyes has been developed. Although they are still sensitive to polarity, in contrast to the former DBD dyes, they have extraordinary spectroscopic properties even in aqueous surroundings. They are characterized by long fluorescence lifetimes (10-20 ns), large Stokes shifts (≈100 nm), high photostabilities, and high quantum yields (>0.56). Here, the spectroscopic properties and synthesis of functionalized derivatives for labeling biological targets are described. Furthermore, thio-reactive maleimido derivatives of both DBD generations show strong intramolecular fluorescence quenching. This mechanism has been investigated and is found to undergo a photoelectron transfer (PET) process. After reaction with a thiol group, this fluorescence quenching is prevented, indicating successful bonding. Being sensitive to their environmental polarity, these compounds have been used as powerful fluorescence lifetime probes for the investigation of conformational changes in the maltose ATP-binding cassette transporter through fluorescence lifetime spectroscopy. The differing tendencies of the fluorescence lifetime change for both DBD dye generations promote their combination as a powerful toolkit for studying microenvironments in proteins.

  16. Ligand photo-isomerization triggers conformational changes in iGluR2 ligand binding domain.

    Directory of Open Access Journals (Sweden)

    Tino Wolter

    Full Text Available Neurological glutamate receptors bind a variety of artificial ligands, both agonistic and antagonistic, in addition to glutamate. Studying their small molecule binding properties increases our understanding of the central nervous system and a variety of associated pathologies. The large, oligomeric multidomain membrane protein contains a large and flexible ligand binding domains which undergoes large conformational changes upon binding different ligands. A recent application of glutamate receptors is their activation or inhibition via photo-switchable ligands, making them key systems in the emerging field of optochemical genetics. In this work, we present a theoretical study on the binding mode and complex stability of a novel photo-switchable ligand, ATA-3, which reversibly binds to glutamate receptors ligand binding domains (LBDs. We propose two possible binding modes for this ligand based on flexible ligand docking calculations and show one of them to be analogues to the binding mode of a similar ligand, 2-BnTetAMPA. In long MD simulations, it was observed that transitions between both binding poses involve breaking and reforming the T686-E402 protein hydrogen bond. Simulating the ligand photo-isomerization process shows that the two possible configurations of the ligand azo-group have markedly different complex stabilities and equilibrium binding modes. A strong but slow protein response is observed after ligand configuration changes. This provides a microscopic foundation for the observed difference in ligand activity upon light-switching.

  17. Temperature-Induced Protein Conformational Changes in Barley Root Plasma Membrane-Enriched Microsomes

    Science.gov (United States)

    Caldwell, Charles R.

    1987-01-01

    The membrane-bound proteins of barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes displayed fluorescence typical of protein-associated trytophan residues. The protein fluorescence intensity was sensitive to variations in sample temperature. The temperature-induced decline in protein fluorescence intensity was nonlinear with slope discontinuities at about 12 and 32°C. Detergents at levels above their critical micelle concentration enhanced protein fluorescence. Glutaraldehyde reduced protein fluorescence. Protein fluorescence polarization increased at temperatures above 30°C. Both the rate of tryptophan photoionization and the fluorescence intensity of the photoionization products suggested alterations in membrane protein conformation between 12 and 32°C. The quenching of the intrinsic protein fluorescence by acrylamide and potassium iodide indicated changes in accessibility of the extrinsic agents to the protein tryptophan residues beginning at about 14°C. The results indicate thermally induced changes in the dynamics of the membrane proteins over the temperature range of 12 to 32°C which could account for the complex temperature dependence of the barley root plasma membrane ATPase. PMID:16665545

  18. Changes of conformation and aggregation state induced by binding of lanthanide ions to insulin

    Institute of Scientific and Technical Information of China (English)

    程驿; 李荣昌; 王夔

    2002-01-01

    To clarify the mechanism of lanthanide ions (Ln3+) on the across-membrane transport of insulin and subsequent reducing blood glucose, the interactions of Ln3+with Zn-insulin and Zn-free insulin are investigated by spectroscopic methods. The results reveal that the binding of Ln3+ to insulin can induce its structure changes from secondary to quaternary structure, depending on the Ln3+ concentration. In the lower concentration, it triggers the conformational changes of insulin monomer in the binding region with insulin receptor (B(24-30)). It would affect insulin-insulin receptor interaction. Moreover, Ln3+ binding promotes the assembly of insulin monomer from dimer to polymer. The potency of Ln3+ in inducing insulin’s aggregation is stronger than that of Zn2+. Furthermore, the aggregation can be reversed partly by EDTA-treatment, indicating that it is not due to denaturation. Similar to Zn2+ effect, Ln3+ can stabilize insulin hexamer in a certain range of concentration, but is stronger than the former.

  19. Changes of conformation and aggregation state induced by binding of lanthanide ions to insulin

    Institute of Scientific and Technical Information of China (English)

    程驿; 李荣昌; 王夔

    2002-01-01

    To clarify the mechanism of lanthanide ions (Ln3+) on the across-membrane transport of insulin and subsequent reducing blood glucose, the interactions of Ln3+ with Zn-insulin and Zn-free insulin are investigated by spectroscopic methods. The results reveal that the binding of Ln3+ to insulin can induce its structure changes from secondary to quaternary structure, depending on the Ln3+ concentration. In the lower concentration, it triggers the conformational changes of insulin monomer in the binding region with insulin receptor (B(24-30)). It would affect insulin-insulin receptor interaction. Moreover, Ln3+ binding promotes the assembly of insulin monomer from dimer to polymer. The potency of Ln3+ in inducing insulin's aggregation is stronger than that of Zn2+. Furthermore, the aggregation can be reversed partly by EDTA-treatment, indicating that it is not due to denaturation. Similar to Zn2+ effect, Ln3+ can stabilize insulin hexamer in a certain range of concentration, but is stronger than the former.

  20. Magnitude of a conformational change in the glycine receptor beta1-beta2 loop is correlated with agonist efficacy

    DEFF Research Database (Denmark)

    Pless, Stephan Alexander; Lynch, Joseph W

    2009-01-01

    associated with the closed-flip transition in the alpha1-glycine receptor. We employed voltage-clamp fluorometry to compare ligand-binding domain conformational changes induced by the following agonists, listed from highest to lowest affinity and efficacy: glycine > beta-alanine > taurine. Voltage...

  1. Conformation of chromatin oligomers. A new argument for a change with the hexanucleosome.

    Science.gov (United States)

    Marion, C; Bezot, P; Hesse-Bezot, C; Roux, B; Bernengo, J C

    1981-11-01

    Quasielastic laser light scattering measurements have been made on chromatin oligomers to obtain information on the transition in their electrooptical properties, previously observed for the hexameric structures [Marion, C. and Roux, B. (1978) Nucleic Acids Res. 5, 4431-4449]. Translational diffusion coefficients were determined for mononucleosomes to octanucleosomes containing histone H1 over a range of ionic strength. At high ionic strength, oligomers show a linear dependence of the logarithm of diffusion coefficient upon the logarithm of number of nucleosomes. At low ionic strength a change occurs between hexamer and heptamer. Our results agree well with the recent sedimentation data of Osipova et al. [Eur. J. Biochem. (1980) 113, 183-188] and of Butler and Thomas [J. Mol. Biol. (1980) 140, 505-529] showing a change in stability with hexamer. Various models for the arrangements of nucleosomes in the superstructure of chromatin are discussed. All calculations clearly indicate a conformational change with the hexanucleosome and the results suggest that, at low ionic strength, the chromatin adopts a loosely helical structure of 28-nm diameter and 22-nm pitch. These results are also consistent with a discontinuity every sixth nucleosome, corresponding to a turn of the helix. This discontinuity may explain the recent electric dichroism data of Lee et al. [Biochemistry (1981) 20, 1438-1445]. The hexanucleosome structure which we have previously suggested, with the faces of nucleosomes arranged radially to the helical axis has been recently confirmed by Mc Ghee et al. [Cell (1980) 22, 87-96]. With an increase of ionic strength, the helix becomes more regular and compact with a slightly reduced outer diameter and a decreased pitch, the dimensions resembling those proposed for solenoid models.

  2. Large negative thermal expansion of a polymer driven by a submolecular conformational change.

    Science.gov (United States)

    Shen, Xingyuan; Viney, Christopher; Johnson, Erin R; Wang, Changchun; Lu, Jennifer Q

    2013-12-01

    Mechanoresponsive polymers hold great technological potential in drug delivery, 'smart' optical systems and microelectromechanical systems. However, hysteresis and fatigue (associated with large-scale polymer chain rearrangement) are often problematic. Here, we describe a polyarylamide film that contains s-dibenzocyclooctadiene (DBCOD), which can generate unconventional and completely reversible thermal contraction under low-energy stimulation. The films exhibit a giant negative thermal expansion coefficient of approximately -1,200 ppm K(-1) at ambient or near-ambient temperatures, much higher than any known negative-thermal-expansion materials under similar operating conditions. Mechanical characterization, calorimetry, spectroscopic analysis and density-functional theory calculations all point to the conformational change of the DBCOD moiety, from the thermodynamic global energy minimum (twist-boat) to a local minimum (chair), as the origin of this abnormal thermal shrinkage. This newly identified, low-energy-driven, thermally agile molecular subunit opens a new pathway to creating near-infrared-based macromolecular switches and motors, and for ambient thermal energy storage and conversion.

  3. RNA Ligase Structures Reveal the Basis for RNA Specificity and Conformational Changes that Drive Ligation Forward

    Energy Technology Data Exchange (ETDEWEB)

    Nandakumar,J.; Shuman, S.; Lima, C.

    2006-01-01

    T4 RNA ligase 2 (Rnl2) and kinetoplastid RNA editing ligases exemplify a family of RNA repair enzymes that seal 3'OH/5'PO{sub 4} nicks in duplex RNAs via ligase adenylylation (step 1), AMP transfer to the nick 5'PO{sub 4} (step 2), and attack by the nick 3'OH on the 5'-adenylylated strand to form a phosphodiester (step 3). Crystal structures are reported for Rnl2 at discrete steps along this pathway: the covalent Rnl2-AMP intermediate; Rnl2 bound to an adenylylated nicked duplex, captured immediately following step 2; and Rnl2 at an adenylylated nick in a state poised for step 3. These structures illuminate the stereochemistry of nucleotidyl transfer and reveal how remodeling of active-site contacts and conformational changes propel the ligation reaction forward. Mutational analysis and comparison of nick-bound structures of Rnl2 and human DNA ligase I highlight common and divergent themes of substrate recognition that can explain their specialization for RNA versus DNA repair.

  4. Changing the criterion for memory conformity in free recall and recognition.

    Science.gov (United States)

    Wright, Daniel B; Gabbert, Fiona; Memon, Amina; London, Kamala

    2008-02-01

    People's responses during memory studies are affected by what other people say. This memory conformity effect has been shown in both free recall and recognition. Here we examine whether accurate, inaccurate, and suggested answers are affected similarly when the response criterion is varied. In the first study, participants saw four pictures of detailed scenes and then discussed the content of these scenes with another participant who saw the same scenes, but with a couple of details changed. Participants were either told to recall everything they could and not to worry about making mistakes (lenient), or only to recall items if they were sure that they were accurate (strict). The strict instructions reduced the amount of inaccurate information reported that the other person suggested, but also reduced the number of accurate details recalled. In the second study, participants were shown a large set of faces and then their memory recognition was tested with a confederate on these and fillers. Here also, the criterion manipulation shifted both accurate and inaccurate responses, and those suggested by the confederate. The results are largely consistent with a shift in response criterion affecting accurate, inaccurate, and suggested information. In addition we varied the level of secrecy in the participants' responses. The effects of secrecy were complex and depended on the level of response criterion. Implications for interviewing eyewitnesses and line-ups are discussed.

  5. Crystal Structures of Human SIRT[subscript 3] Displaying Substrate-induced Conformational Changes

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Lei; Wei, Wentao; Jiang, Yaobin; Peng, Hao; Cai, Jianhua; Mao, Chen; Dai, Han; Choy, Wendy; Bemis, Jean E.; Jirousek, Michael R.; Milne, Jill C.; Westphal, Christoph H.; Perni, Robert B.; (Viva Biotech); (Medicilon); (GSK)

    2009-11-04

    SIRT3 is a major mitochondrial NAD{sup +}-dependent protein deacetylase playing important roles in regulating mitochondrial metabolism and energy production and has been linked to the beneficial effects of exercise and caloric restriction. SIRT3 is emerging as a potential therapeutic target to treat metabolic and neurological diseases. We report the first sets of crystal structures of human SIRT3, an apo-structure with no substrate, a structure with a peptide containing acetyl lysine of its natural substrate acetyl-CoA synthetase 2, a reaction intermediate structure trapped by a thioacetyl peptide, and a structure with the dethioacetylated peptide bound. These structures provide insights into the conformational changes induced by the two substrates required for the reaction, the acetylated substrate peptide and NAD+. In addition, the binding study by isothermal titration calorimetry suggests that the acetylated peptide is the first substrate to bind to SIRT3, before NAD{sup +}. These structures and biophysical studies provide key insight into the structural and functional relationship of the SIRT3 deacetylation activity.

  6. Changing speed-VMT distributions: the effects on emissions inventories and conformity.

    Science.gov (United States)

    Nanzetta, K; Niemeier, D; Utts, J M

    2000-03-01

    The emissions factor modeling component of the motor vehicle emissions inventory (MVEI) modeling suite is currently being revised by the California Air Resources Board (CARB). One of the proposed changes in modeling philosophy is a shift from using link-based travel activity data to trip-based travel data for preparing mobile emissions inventories. Also as part of the revisions, new speed correction factors (SCFs) will be developed by CARB for the revised model. The new SCFs will be derived from vehicle emissions on 15 new driving cycles, each constructed to represent a typical trip at a specific average speed. This paper discusses how the new SCFs will affect transportation conformity and emissions inventory development, and evaluates the differences in total emissions produced by trip-based and link-based distributions of speed and vehicle miles of travel (VMT). We simulated both link-based and trip-based speed-VMT distributions using travel data from the Sacramento and San Diego travel demand models. On the basis of the simulation results, there is reason to expect that mobile emissions inventories constructed using the proposed trip-based philosophy will differ markedly from those constructed in the current manner. Noting that results may vary by region, increases are expected in the CO and HC inventory levels, with concomitant decreases in the NOx mobile emissions inventories.

  7. Spectroscopic study of conformational changes in subdomain 1 of G-actin: influence of divalent cations.

    Science.gov (United States)

    Nyitrai, M; Hild, G; Belágyi, J; Somogyi, B

    1997-10-01

    Temperature dependence of the fluorescence intensity and anisotropy decay of N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to Cys374 of actin monomer was investigated to characterize conformational differences between Ca- and Mg-G-actin. The fluorescence lifetime is longer in Mg-G-actin than that in Ca-G-actin in the temperature range of 5-34 degrees C. The width of the lifetime distribution is smaller by 30% in Mg-saturated actin monomer at 5 degrees C, and the difference becomes negligible above 30 degrees C. The semiangle of the cone within which the fluorophore can rotate is larger in Ca-G-actin at all temperatures. Electron paramagnetic resonance measurements on maleimide spin-labeled (on Cys374) monomer actin gave evidence that exchange of Ca2+ for Mg2+ induced a rapid decrease in the mobility of the label immediately after the addition of Mg2+. These results suggest that the C-terminal region of the monomer becomes more rigid as a result of the replacement of Ca2+ by Mg2+. The change can be related to the difference between the polymerization abilities of the two forms of G-actin.

  8. Yeast mitochondrial RNAP conformational changes are regulated by interactions with the mitochondrial transcription factor.

    Science.gov (United States)

    Drakulic, Srdja; Wang, Liping; Cuéllar, Jorge; Guo, Qing; Velázquez, Gilberto; Martín-Benito, Jaime; Sousa, Rui; Valpuesta, José M

    2014-01-01

    Mitochondrial RNA polymerases (MtRNAPs) are members of the single-subunit RNAP family, the most well-characterized member being the RNAP from T7 bacteriophage. MtRNAPs are, however, functionally distinct in that they depend on one or more transcription factors to recognize and open the promoter and initiate transcription, while the phage RNAPs are capable of performing these tasks alone. Since the transcriptional mechanisms that are conserved in phage and mitochondrial RNAPs have been so effectively characterized in the phage enzymes, outstanding structure-mechanism questions concern those aspects that are distinct in the MtRNAPs, particularly the role of the mitochondrial transcription factor(s). To address these questions we have used both negative staining and cryo-EM to generate three-dimensional reconstructions of yeast MtRNAP initiation complexes with and without the mitochondrial transcription factor (MTF1), and of the elongation complex. Together with biochemical experiments, these data indicate that MTF1 uses multiple mechanisms to drive promoter opening, and that its interactions with the MtRNAP regulate the conformational changes undergone by the latter enzyme as it traverses the template strand.

  9. Conformational changes of the histidine ATP-binding cassette transporter studied by double electron-electron resonance spectroscopy.

    Science.gov (United States)

    Sippach, Michael; Weidlich, Daniela; Klose, Daniel; Abé, Christoph; Klare, Johann; Schneider, Erwin; Steinhoff, Heinz-Jürgen

    2014-07-01

    The conformational dynamics of the histidine ABC transporter HisQMP2 from Salmonella enterica serovar Typhimurium, reconstituted into liposomes, is studied by site-directed spin labeling and double electron-electron resonance spectroscopy in the absence of nucleotides, in the ATP-bound, and in the post-hydrolysis state. The results show that the inter-dimer distances as measured between the Q-loops of HisP2 in the intact transporter resemble those determined for the maltose transporter in all three states of the hydrolysis cycle. Only in the presence of liganded HisJ the closed conformation of the nucleotide binding sites is achieved revealing the transmembrane communication of the presence of substrate. Two conformational states can be distinguished for the periplasmic moiety of HisQMP2 as detected by differences in distributions of interspin distances between positions 86 and 96 or 104 and 197. The observed conformational changes are correlated to proposed open, semi-open and closed conformations of the nucleotide binding domains HisP2. Our results are in line with a rearrangement of transmembrane helices 4 and 4' of HisQM during the closed to the semi-open transition of HisP2 driven by the reorientation of the coupled helices 3a and 3b to occur upon hydrolysis. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Influence of pH on viscoelastic properties of heat-induced gels obtained with a β-Lactoglobulin fraction isolated from bovine milk whey hydrolysates.

    Science.gov (United States)

    Estévez, Natalia; Fuciños, Pablo; Bargiela, Verónica; Picó, Guillermo; Valetti, Nadia Woitovich; Tovar, Clara Asunción; Rúa, M Luisa

    2017-03-15

    A β-Lactoglobulin fraction (r-βLg) was isolated from whey hydrolysates produced with cardosins from Cynara cardunculus. The impact of the hydrolysis process on the r-βLg structure and the rheological properties of heat-induced gels obtained thereafter were studied at different pH values. Differences were observed between r-βLg and commercial β-Lg used as control. Higher values for the fluorescence emission intensity and red shifts of the emission wavelength of r-βLg suggested changes in its tertiary structure and more solvent-exposed tryptophan residues. Circular dichroism spectra also supported these evidences indicating that hydrolysis yielded an intermediate (non-native) β-Lg state. The thermal history of r-βLg through the new adopted conformation improved the microstructure of the gels at acidic pH. So, a new microstructure with better rheological characteristics (higher conformational flexibility and lower rigidity) and greater water holding ability was founded for r-βLg gel. These results were reflected in the microstructural analysis by scanning electron microscopy.

  11. Role of a GAG hinge in the nucleotide-induced conformational change governing nucleotide specificity by T7 DNA polymerase.

    Science.gov (United States)

    Jin, Zhinan; Johnson, Kenneth A

    2011-01-14

    A nucleotide-induced change in DNA polymerase structure governs the kinetics of polymerization by high fidelity DNA polymerases. Mutation of a GAG hinge (G542A/G544A) in T7 DNA polymerase resulted in a 1000-fold slower rate of conformational change, which then limited the rate of correct nucleotide incorporation. Rates of misincorporation were comparable to that seen for wild-type enzyme so that the net effect of the mutation was a large decrease in fidelity. We demonstrate that a presumably modest change from glycine to alanine 20 Å from the active site can severely restrict the flexibility of the enzyme structure needed to recognize and incorporate correct substrates with high specificity. These results emphasize the importance of the substrate-induced conformational change in governing nucleotide selectivity by accelerating the incorporation of correct base pairs but not mismatches.

  12. Microscopic insight into thermodynamics of conformational changes of SAP-SLAM complex in signal transduction cascade

    Science.gov (United States)

    Samanta, Sudipta; Mukherjee, Sanchita

    2017-04-01

    The signalling lymphocytic activation molecule (SLAM) family of receptors, expressed by an array of immune cells, associate with SLAM-associated protein (SAP)-related molecules, composed of single SH2 domain architecture. SAP activates Src-family kinase Fyn after SLAM ligation, resulting in a SLAM-SAP-Fyn complex, where, SAP binds the Fyn SH3 domain that does not involve canonical SH3 or SH2 interactions. This demands insight into this SAP mediated signalling cascade. Thermodynamics of the conformational changes are extracted from the histograms of dihedral angles obtained from the all-atom molecular dynamics simulations of this structurally well characterized SAP-SLAM complex. The results incorporate the binding induced thermodynamic changes of individual amino acid as well as the secondary structural elements of the protein and the solvent. Stabilization of the peptide partially comes through a strong hydrogen bonding network with the protein, while hydrophobic interactions also play a significant role where the peptide inserts itself into a hydrophobic cavity of the protein. SLAM binding widens SAP's second binding site for Fyn, which is the next step in the signal transduction cascade. The higher stabilization and less fluctuation of specific residues of SAP in the Fyn binding site, induced by SAP-SLAM complexation, emerge as the key structural elements to trigger the recognition of SAP by the SH3 domain of Fyn. The thermodynamic quantification of the protein due to complexation not only throws deeper understanding in the established mode of SAP-SLAM interaction but also assists in the recognition of the relevant residues of the protein responsible for alterations in its activity.

  13. Structural basis and kinetics of force-induced conformational changes of an αA domain-containing integrin.

    Directory of Open Access Journals (Sweden)

    Xue Xiang

    Full Text Available BACKGROUND: Integrin α(Lβ₂ (lymphocyte function-associated antigen, LFA-1 bears force upon binding to its ligand intercellular adhesion molecule 1 (ICAM-1 when a leukocyte adheres to vascular endothelium or an antigen presenting cell (APC during immune responses. The ligand binding propensity of LFA-1 is related to its conformations, which can be regulated by force. Three conformations of the LFA-1 αA domain, determined by the position of its α₇-helix, have been suggested to correspond to three different affinity states for ligand binding. METHODOLOGY/PRINCIPAL FINDINGS: The kinetics of the force-driven transitions between these conformations has not been defined and dynamically coupled to the force-dependent dissociation from ligand. Here we show, by steered molecular dynamics (SMD simulations, that the αA domain was successively transitioned through three distinct conformations upon pulling the C-terminus of its α₇-helix. Based on these sequential transitions, we have constructed a mathematical model to describe the coupling between the αA domain conformational changes of LFA-1 and its dissociation from ICAM-1 under force. Using this model to analyze the published data on the force-induced dissociation of single LFA-1/ICAM-1 bonds, we estimated the force-dependent kinetic rates of interstate transition from the short-lived to intermediate-lived and from intermediate-lived to long-lived states. Interestingly, force increased these transition rates; hence activation of LFA-1 was accelerated by pulling it via an engaged ICAM-1. CONCLUSIONS/SIGNIFICANCE: Our study defines the structural basis for mechanical regulation of the kinetics of LFA-1 αA domain conformational changes and relates these simulation results to experimental data of force-induced dissociation of single LFA-1/ICAM-1 bonds by a new mathematical model, thus provided detailed structural and kinetic characterizations for force-stabilization of LFA-1/ICAM-1 interaction.

  14. DSC Analysis of Heat-induced Changes of Thermal Characteristics for Perimy- sium and Endomysium Collagen from Beef semitendinosus Muscle%来自牛半腱肌肌束膜和肌内膜胶原蛋白热力学特性分析

    Institute of Scientific and Technical Information of China (English)

    常海军; 徐幸莲; 周光宏

    2012-01-01

    探讨牛半腱肌肉肌束膜和肌内膜胶原蛋白热力特性的热诱导变化。牛半腱肌肉分别采用水浴和微波加热到内部终点温度分别为20、40、50、60、70、80℃和90℃,用示差扫描量热法研究肌束膜和肌内膜胶原蛋白热力特性(起始、最高和最终热收缩温度)在热处理过程中的变化。结果表明:牛半腱肌肉肌束膜和肌内膜胶原蛋白的热收缩温度在两种热处理方式间都存在显著差异,在两种热处理方式中,内部终点温度60℃是影响肌束膜和肌内膜胶原蛋白热收缩温度的关键加热温度。热诱导的肌束膜和肌内膜胶原蛋白热力特性的变化是水浴和微波加热牛肉胶原蛋白热收缩温度存在差异的主要原因。%The main objective of this study was to investigate the heat-induced changes of thermal characteristics for perimysium and endomysium collagen from beef semitendinosus muscle. Muscle samples were heated to an internal core temperature of 20, 40, 50, 60, 70, 80 ℃ and 90 ℃ in water bath and in microwave oven respectively. The changes of thermal shrinkage temperatures (To: onset temperature; Tp: peak temperature; To: end temperature) of perimysium and endomysium collagen for beef semitendinosus muscle during heating were analyzed by Differential Scanning Calorimeter (DSC). The results indicated that the thermal shrinkage temperatures (To, Tp and To) of perimysium and endomysium collagen both showed significant differences at different internal core temperatures during water-bath and microwave heating. And an internal core temperature of 60 ℃ was the critical heating temperature which affects thermal shrinkage temperatures of perimysium and endomysium collagen for both waterbath and microwave heated meat. The significant differences in thermal shrinkage temperatures between water bath and microwave heated beef muscle samples were attributed to the heat-induced changes in thermal

  15. Probing Protein 3D Structures and Conformational Changes Using Electrochemistry-Assisted Isotope Labeling Cross-Linking Mass Spectrometry

    Science.gov (United States)

    Zheng, Qiuling; Zhang, Hao; Wu, Shiyong; Chen, Hao

    2016-05-01

    This study presents a new chemical cross-linking mass spectrometry (MS) method in combination with electrochemistry and isotope labeling strategy for probing both protein three-dimensional (3D) structures and conformational changes. For the former purpose, the target protein/protein complex is cross-linked with equal mole of premixed light and heavy isotope labeled cross-linkers carrying electrochemically reducible disulfide bonds (i.e., DSP-d0 and DSP-d8 in this study, DSP = dithiobis[succinimidyl propionate]), digested and then electrochemically reduced followed with online MS analysis. Cross-links can be quickly identified because of their reduced intensities upon electrolysis and the presence of doublet isotopic peak characteristics. In addition, electroreduction converts cross-links into linear peptides, facilitating MS/MS analysis to gain increased information about their sequences and modification sites. For the latter purpose of probing protein conformational changes, an altered procedure is adopted, in which the protein in two different conformations is cross-linked using DSP-d0 and DSP-d8 separately, and then the two protein samples are mixed in 1:1 molar ratio. The merged sample is subjected to digestion and electrochemical mass spectrometric analysis. In such a comparative cross-linking experiment, cross-links could still be rapidly recognized based on their responses to electrolysis. More importantly, the ion intensity ratios of light and heavy isotope labeled cross-links reveal the conformational changes of the protein, as exemplified by examining the effect of Ca2+ on calmodulin conformation alternation. This new cross-linking MS method is fast and would have high value in structural biology.

  16. {sup 13}C NMR detects conformational change in the 100-kD membrane transporter ClC-ec1

    Energy Technology Data Exchange (ETDEWEB)

    Abraham, Sherwin J.; Cheng, Ricky C.; Chew, Thomas A.; Khantwal, Chandra M. [Stanford University School of Medicine, Department of Molecular & Cellular Physiology (United States); Liu, Corey W. [Stanford University School of Medicine, Stanford Magnetic Resonance Laboratory (United States); Gong, Shimei; Nakamoto, Robert K. [University of Virginia, Department of Molecular Physiology and Biological Physics (United States); Maduke, Merritt, E-mail: maduke@stanford.edu [Stanford University School of Medicine, Department of Molecular & Cellular Physiology (United States)

    2015-04-15

    CLC transporters catalyze the exchange of Cl{sup −} for H{sup +} across cellular membranes. To do so, they must couple Cl{sup −} and H{sup +} binding and unbinding to protein conformational change. However, the sole conformational changes distinguished crystallographically are small movements of a glutamate side chain that locally gates the ion-transport pathways. Therefore, our understanding of whether and how global protein dynamics contribute to the exchange mechanism has been severely limited. To overcome the limitations of crystallography, we used solution-state {sup 13}C-methyl NMR with labels on methionine, lysine, and engineered cysteine residues to investigate substrate (H{sup +}) dependent conformational change outside the restraints of crystallization. We show that methyl labels in several regions report H{sup +}-dependent spectral changes. We identify one of these regions as Helix R, a helix that extends from the center of the protein, where it forms the part of the inner gate to the Cl{sup −}-permeation pathway, to the extracellular solution. The H{sup +}-dependent spectral change does not occur when a label is positioned just beyond Helix R, on the unstructured C-terminus of the protein. Together, the results suggest that H{sup +} binding is mechanistically coupled to closing of the intracellular access-pathway for Cl{sup −}.

  17. Conformational changes of ovine α-1-proteinase inhibitor: The influence of heparin binding

    Science.gov (United States)

    Gupta, Vivek Kumar; Gowda, Lalitha R.

    2008-11-01

    α-1-Proteinase inhibitor (α-1-PI), the archetypal serpin causes rapid, irreversible stoichiometric inhibition of redundant circulating serine proteases and is associated with emphysema, inflammatory response and maintenance of protease-inhibitor equilibrium in vascular and peri-vascular spaces. A homogenous preparation of heparin octasaccharide binds to ovine and human α-1-PI and enhances their protease inhibitory activity phenomenally. Size-exclusion chromatography and dynamic light scattering experiments reveal that ovine α-1-PI undergoes a decrease in the Stokes' radius upon heparin binding. A strong binding; characterizes this α-1-PI-heparin interaction as revealed by the binding constant ( Kα) 1.98 ± 0.2 × 10 -6 M and 2.1 ± 0.2 × 10 -6 M determined by fluorescence spectroscopy and equilibrium dialysis, respectively. The stoichiometry of heparin binding to ovine α-1-PI was 1.1 ± 0.2:1. The Stern-Volmer constants ( Ksv) for heparin activated ovine and human α-1-PI were found to be 5.13 × 10 -6 M and 5.67 × 10 -6 M, respectively, significantly higher than the native inhibitors. FTIR and CD spectroscopy project the systematic structural reorientations that α-1-PI undergoes upon heparin binding characterized by a decrease in α-helical content and a concomitant increase in β-turn and random coil elements. It is likely that these conformational changes result in the movement of the α-1-PI reactive site loop into an extended structure that is better poised to combat the cognate protease and accelerate the inhibition.

  18. The relationship between MRP1 activities and its NBD conformational changes

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    MIANS,a sulfhydryl-reactive fluorescence,was used to label the cysteines of MRP1(multidrug resistance protein),and the results indicated that an increase in fluorescence intensity and a large emission blue shift took place after two Cys residues of MRP1 reacted with MIANS,which demonstrated that labeled Cys residues in MRP1 reside in a relatively hydrophobic environment.The experimental results obtained from fluorescence resonance energy transfer further uncover that two Cys residues of MRP1 modified by MIANS located in the vicinity of its NBDs,of which one lies close to NBD1,and the other near NBD2.ATP,ADP and anticancer drugs can all reduce the rate of reaction of MRP1 with MIANS.The collisional quenchers,acrylamide,I-,and Cs+ were used to assess local environments of MIANS bound to MRP1 and the results showed that the region around the MIANS-labeled cysteine is positively charged.Both MIANS and NEM,which are sulfhydryl-reactive reagents,inhibited MRP1 ATPase activity,whereas anticancer drugs activated it.These results demonstrated that all nucleotides and drugs could induce changes in conformation of the NBDs in MRP1.Nucleotides can bind directly to NBDs,but drugs may react first with TMDs,which in turn alters the accessibility of the two Cys residues bound by MIANS and affects MRP1 ATPase activity,which is coupled with the transport of its substrates.Taken together,the above experimental results provide direct evidence for further study on the coupling of translocation of the transported species to hydrolysis of ATP in MRP1.

  19. Structural analysis of coxsackievirus A7 reveals conformational changes associated with uncoating.

    Science.gov (United States)

    Seitsonen, Jani J T; Shakeel, Shabih; Susi, Petri; Pandurangan, Arun P; Sinkovits, Robert S; Hyvönen, Heini; Laurinmäki, Pasi; Ylä-Pelto, Jani; Topf, Maya; Hyypiä, Timo; Butcher, Sarah J

    2012-07-01

    Coxsackievirus A7 (CAV7) is a rarely detected and poorly characterized serotype of the Enterovirus species Human enterovirus A (HEV-A) within the Picornaviridae family. The CAV7-USSR strain has caused polio-like epidemics and was originally thought to represent the fourth poliovirus type, but later evidence linked this strain to the CAV7-Parker prototype. Another isolate, CAV7-275/58, was also serologically similar to Parker but was noninfectious in a mouse model. Sequencing of the genomic region encoding the capsid proteins of the USSR and 275/58 strains and subsequent comparison with the corresponding amino acid sequences of the Parker strain revealed that the Parker and USSR strains are nearly identical, while the 275/58 strain is more distant. Using electron cryomicroscopy and three-dimensional image reconstruction, the structures of the CAV7-USSR virion and empty capsid were resolved to 8.2-Å and 6.1-Å resolutions, respectively. This is one of the first detailed structural analyses of the HEV-A species. Using homology modeling, reconstruction segmentation, and flexible fitting, we constructed a pseudoatomic T = 1 (pseudo T = 3) model incorporating the three major capsid proteins (VP1 to VP3), addressed the conformational changes of the capsid and its constituent viral proteins occurring during RNA release, and mapped the capsid proteins' variable regions to the structure. During uncoating, VP4 and RNA are released analogously to poliovirus 1, the interfaces of VP2 and VP3 are rearranged, and VP1 rotates. Variable regions in the capsid proteins were predicted to map mainly to the surface of VP1 and are thus likely to affect the tropism and pathogenicity of CAV7.

  20. Conformational Changes in the Orai1 C-Terminus Evoked by STIM1 Binding.

    Directory of Open Access Journals (Sweden)

    Leidamarie Tirado-Lee

    Full Text Available Store-operated CRAC channels regulate a wide range of cellular functions including gene expression, chemotaxis, and proliferation. CRAC channels consist of two components: the Orai proteins (Orai1-3, which form the ion-selective pore, and STIM proteins (STIM1-2, which form the endoplasmic reticulum (ER Ca2+ sensors. Activation of CRAC channels is initiated by the migration of STIM1 to the ER-plasma membrane (PM junctions, where it directly interacts with Orai1 to open the Ca2+-selective pores of the CRAC channels. The recent elucidation of the Drosophila Orai structure revealed a hexameric channel wherein the C-terminal helices of adjacent Orai subunits associate in an anti-parallel orientation. This association is maintained by hydrophobic interactions between the Drosophila equivalents of human Orai1 residues L273 and L276. Here, we used mutagenesis and chemical cross-linking to assess the nature and extent of conformational changes in the self-associated Orai1 C-termini during STIM1 binding. We find that linking the anti-parallel coiled-coils of the adjacent Orai1 C-termini through disulfide cross-links diminishes STIM1-Orai1 interaction, as assessed by FRET. Conversely, prior binding of STIM1 to the Orai1 C-terminus impairs cross-linking of the Orai1 C-termini. Mutational analysis indicated that a bend of the Orai1 helix located upstream of the self-associated coils (formed by the amino acid sequence SHK establishes an appropriate orientation of the Orai1 C-termini that is required for STIM1 binding. Together, our results support a model wherein the self-associated Orai1 C-termini rearrange modestly to accommodate STIM1 binding.

  1. Role of active site conformational changes in photocycle activation of the AppA BLUF photoreceptor.

    Science.gov (United States)

    Goyal, Puja; Hammes-Schiffer, Sharon

    2017-02-14

    Blue light using flavin adenine dinucleotide (BLUF) proteins are essential for the light regulation of a variety of physiologically important processes and serve as a prototype for photoinduced proton-coupled electron transfer (PCET). Free-energy simulations elucidate the active site conformations in the AppA (activation of photopigment and puc expression) BLUF domain before and following photoexcitation. The free-energy profile for interconversion between conformations with either Trp104 or Met106 closer to the flavin, denoted Trpin/Metout and Trpout/Metin, reveals that both conformations are sampled on the ground state, with the former thermodynamically favorable by ∼3 kcal/mol. These results are consistent with the experimental observation of both conformations. To analyze the proton relay from Tyr21 to the flavin via Gln63, the free-energy profiles for Gln63 rotation were calculated on the ground state, the locally excited state of the flavin, and the charge-transfer state associated with electron transfer from Tyr21 to the flavin. For the Trpin/Metout conformation, the hydrogen-bonding pattern conducive to the proton relay is not thermodynamically favorable on the ground state but becomes more favorable, corresponding to approximately half of the configurations sampled, on the locally excited state. The calculated energy gaps between the locally excited and charge-transfer states suggest that electron transfer from Tyr21 to the flavin is more facile for configurations conducive to proton transfer. When the active site conformation is not conducive to PCET from Tyr21, Trp104 can directly compete with Tyr21 for electron transfer to the flavin through a nonproductive pathway, impeding the signaling efficiency.

  2. NIR Laser Radiation Induced Conformational Changes and Tunneling Lifetimes of High-Energy Conformers of Amino Acids in Low-Temperature Matrices

    Science.gov (United States)

    Bazso, Gabor; Najbauer, Eszter E.; Magyarfalvi, Gabor; Tarczay, Gyorgy

    2013-06-01

    We review our recent results on combined matrix isolation FT-IR and NIR laser irradiation studies on glycine alanine, and cysteine. The OH and the NH stretching overtones of the low-energy conformers of these amino acids deposited in Ar, Kr, Xe, and N_{2} matrices were irradiated. At the expense of the irradiated conformer, other conformers were enriched and new, high-energy, formerly unobserved conformers were formed in the matrices. This enabled the separation and unambiguous assignment of the vibrational transitions of the different conformers. The main conversion paths and their efficiencies are described qualitatively showing that there are significant differences in different matrices. It was shown that the high-energy conformer decays in the matrix by H-atom tunneling. The lifetimes of the high-energy conformers in different matrices were measured. Based on our results we conclude that some theoretically predicted low-energy conformers of amino acids are likely even absent in low-energy matrices due to fast H-atom tunneling. G. Bazso, G. Magyarfalvi, G. Tarczay J. Mol. Struct. 1025 (Light-Induced Processes in Cryogenic Matrices Special Issue) 33-42 (2012). G. Bazso, G. Magyarfalvi, G. Tarczay J. Phys. Chem. A 116 (43) 10539-10547 (2012). G. Bazso, E. E. Najbauer, G. Magyarfalvi, G. Tarczay J. Phys. Chem. A in press, DOI: 10.1021/jp400196b. E. E. Najbauer, G. Bazso, G. Magyarfalvi, G. Tarczay in preparation.

  3. Conformational changes in proteins recovered from jumbo squid (Dosidicus gigas) muscle through pH shift washing treatments.

    Science.gov (United States)

    Cortés-Ruiz, Juan A; Pacheco-Aguilar, Ramón; Ramírez-Suárez, Juan C; Lugo-Sánchez, Maria E; García-Orozco, Karina D; Sotelo-Mundo, Rogerio R; Peña-Ramos, Aida

    2016-04-01

    Conformational and thermal-rheological properties of acidic (APC) and neutral (NPC) protein concentrates were evaluated and compared to those of squid (Dosidicus gigas) muscle proteins (SM). Surface hydrophobicity, sulfhydryl status, secondary structure profile, differential scanning calorimetry and oscillatory dynamic rheology were used to evaluate the effect of treatments on protein properties. Acidic condition during the washing process (APC) promoted structural and conformational changes in the protein present in the concentrate produced. These changes were enhanced during the heat setting of the corresponding sol. Results demonstrate that washing squid muscle under the proposed acidic conditions is a feasible technological alternative for squid-based surimi production improving its yield and gel-forming ability.

  4. Regulation of phenylalanine hydroxylase: conformational changes upon phenylalanine binding detected by hydrogen/deuterium exchange and mass spectrometry.

    Science.gov (United States)

    Li, Jun; Dangott, Lawrence J; Fitzpatrick, Paul F

    2010-04-20

    Phenylalanine acts as an allosteric activator of the tetrahydropterin-dependent enzyme phenylalanine hydroxylase. Hydrogen/deuterium exchange monitored by mass spectrometry has been used to gain insight into local conformational changes accompanying activation of rat phenylalanine hydroxylase by phenylalanine. Peptides in the regulatory and catalytic domains that lie in the interface between these two domains show large increases in the extent of deuterium incorporation from solvent in the presence of phenylalanine. In contrast, the effects of phenylalanine on the exchange kinetics of a mutant enzyme lacking the regulatory domain are limited to peptides surrounding the binding site for the amino acid substrate. These results support a model in which the N-terminus of the protein acts as an inhibitory peptide, with phenylalanine binding causing a conformational change in the regulatory domain that alters the interaction between the catalytic and regulatory domains.

  5. Effect of colloidal gold size on the conformational changes of adsorbed cytochrome c: probing by circular dichroism, UV-visible, and infrared spectroscopy.

    Science.gov (United States)

    Jiang, Xiue; Jiang, Junguang; Jin, Yongdong; Wang, Erkang; Dong, Shaojun

    2005-01-01

    The conformational changes of bovine heart cytochrome c (cyt c) induced by the adsorption on gold nanoparticles with different sizes have been investigated by electronic absorption, circular dichroism (CD), and Fourier transform infrared spectra. The combination of these techniques can give complementary information about adsorption-induced conformational changes. The results show that there are different conformational changes for cyt c adsorbed on gold nanoparticles with different sizes due to the different interaction forces between cyt c and gold nanoparticles. The colloidal gold concentration-dependent conformation distribution curves of cyt c obtained by analysis of CD spectra using the singular value decomposition least-squares method show that the coverage of cyt c on the gold nanoparticles surface also affects the conformational changes of the adsorbed cyt c.

  6. CARS and Raman spectroscopy of function-related conformational changes of chymotrypsin

    NARCIS (Netherlands)

    Brandt, N.N.; Chikishev, A.Yu.; Chikishev, A.Y.; Greve, Jan; Koroteev, N.I.; Otto, Cornelis; Sakodinskaya, I.K.; Sakodynskaya, I.K.

    2000-01-01

    We report on the comparative analysis of the conformation-sensitive bands of free enzyme (chymotrypsin), liganded enzyme (chymotrypsin anthranilate) and enzyme complex with 18-crown-6. The studies were carried out by Raman scattering spectroscopy and polarization-sensitive coherent anti-Stokes Raman

  7. From flexibility to function: Molecular dynamics simulations of conformational changes in chaperones and photoreceptors

    NARCIS (Netherlands)

    Singhal, K.

    2016-01-01

    Proteins are uniquely-shaped macromolecules that function as biological machines, and regulate a living cell’s behavior. Crucial to protein function is the folding of the polypeptide chain into a unique well-defined three-dimensional conformation. In complex cell environments, the spontaneous unassi

  8. Cation ion specifically induces a conformational change in trans-dehydroandrosterone - a solid-state NMR study.

    Science.gov (United States)

    Wang, Darong; Chen, Meiman; Chein, Rong-Jie; Ching, Wei-Min; Hung, Chen-Hsiung; Tzou, Der-Lii M

    2015-04-01

    In this work, we demonstrated that calcium (Ca(+2)) is able to induce a conformational change in trans-dehydroandrosterone (DHEA). To this respect, solid-state NMR spectroscopy was applied to a series of DHEA molecules that were incubated with Ca(+2) under different concentrations. The high-resolution (13)C NMR spectra of the DHEA/Ca(+2) mixtures exhibited two distinct sets of signals; one was attributed to DHEA in the free form, and the second set was due to the DHEA/Ca(+2) complex. Based on chemical shift isotropy and anisotropy analyses, we postulated that Ca(+2) might have associated with the oxygen attached to C17 via a lone-pair of electrons, which induced a conformational change in DHEA. Apart from Ca(+2), we also incubated DHEA with magnesium (Mg(+2)) to determine whether Mg(+2) was able to interact with DHEA in a similar manner to Ca(+2). We found that Mg(+2) was able to induce a conformational change in DHEA deviated from that of Ca(+2). These solid-state NMR observations indicate that DHEA is able to interact with cations, such as Mg(+2) and Ca(+2), with specificity.

  9. Detailing radio frequency heating induced by coronary stents: a 7.0 Tesla magnetic resonance study.

    Science.gov (United States)

    Santoro, Davide; Winter, Lukas; Müller, Alexander; Vogt, Julia; Renz, Wolfgang; Ozerdem, Celal; Grässl, Andreas; Tkachenko, Valeriy; Schulz-Menger, Jeanette; Niendorf, Thoralf

    2012-01-01

    The sensitivity gain of ultrahigh field Magnetic Resonance (UHF-MR) holds the promise to enhance spatial and temporal resolution. Such improvements could be beneficial for cardiovascular MR. However, intracoronary stents used for treatment of coronary artery disease are currently considered to be contra-indications for UHF-MR. The antenna effect induced by a stent together with RF wavelength shortening could increase local radiofrequency (RF) power deposition at 7.0 T and bears the potential to induce local heating, which might cause tissue damage. Realizing these constraints, this work examines RF heating effects of stents using electro-magnetic field (EMF) simulations and phantoms with properties that mimic myocardium. For this purpose, RF power deposition that exceeds the clinical limits was induced by a dedicated birdcage coil. Fiber optic probes and MR thermometry were applied for temperature monitoring using agarose phantoms containing copper tubes or coronary stents. The results demonstrate an agreement between RF heating induced temperature changes derived from EMF simulations versus MR thermometry. The birdcage coil tailored for RF heating was capable of irradiating power exceeding the specific-absorption rate (SAR) limits defined by the IEC guidelines by a factor of three. This setup afforded RF induced temperature changes up to +27 K in a reference phantom. The maximum extra temperature increase, induced by a copper tube or a coronary stent was less than 3 K. The coronary stents examined showed an RF heating behavior similar to a copper tube. Our results suggest that, if IEC guidelines for local/global SAR are followed, the extra RF heating induced in myocardial tissue by stents may not be significant versus the baseline heating induced by the energy deposited by a tailored cardiac transmit RF coil at 7.0 T, and may be smaller if not insignificant than the extra RF heating observed under the circumstances used in this study.

  10. A hydrogen peroxide-generating agent, 6-formylpterin, enhances heat-induced apoptosis.

    Science.gov (United States)

    Wada, S; Cui, Z-G; Kondo, T; Zhao, Q-L; Ogawa, R; Shoji, M; Arai, T; Makino, K; Furuta, I

    2005-05-01

    The enhancement of heat-induced apoptosis by 6-formylpterin, an intra-cellular generator of hydrogen peroxide (H2O2), was examined in human myelomonocytic lymphoma U937 cells. The cells were treated with either 6-formylpterin alone at a nontoxic concentration of 300 microM (37 degrees C), heat shock (44 degrees C per 20 min) alone or a combination of the two, then incubated at 37 degrees C for 6 h. Assessments of apoptosis, mitochondrial membrane potential and caspase-3 activation were performed by flow cytometry. Moreover, caspase-8 activation and changes in the intra-cellular Ca2+ concentration ([Ca2+]i) were examined. Bax, Bcl-2, Bcl-XL, Bid, cytochrome c and PKCd were detected by Western blotting. The induction of heat-induced apoptosis evaluated by morphological observation and DNA fragmentation were promoted by the addition of 6-formylpterin. Mitochondrial membrane potential was decreased and the activation of caspase-3 and -8 was enhanced in the cells treated with the combination. A decreased-expression of Bid was noted, although no significant changes in Bax, Bcl-2 and Bcl-XL expression were observed after the combined treatment. Furthermore, both the release of cytochrome c from mitochondria to cytosol and the translocation of PKCd from cytosol to mitochondria, which were induced by heat shock, were enhanced by the addition of 6-formylpterin. The number of cells with a higher [Ca2+]i was also increased by the addition of 6-formylpterin. These findings suggest that the increase in [Ca2+]i, the activation of the mitochondria-caspase dependent pathway and the translocation of PKCd to mitochondria play principal roles in the enhancement of heat-induced apoptosis by 6-FP.

  11. Detailing radio frequency heating induced by coronary stents: a 7.0 Tesla magnetic resonance study.

    Directory of Open Access Journals (Sweden)

    Davide Santoro

    Full Text Available The sensitivity gain of ultrahigh field Magnetic Resonance (UHF-MR holds the promise to enhance spatial and temporal resolution. Such improvements could be beneficial for cardiovascular MR. However, intracoronary stents used for treatment of coronary artery disease are currently considered to be contra-indications for UHF-MR. The antenna effect induced by a stent together with RF wavelength shortening could increase local radiofrequency (RF power deposition at 7.0 T and bears the potential to induce local heating, which might cause tissue damage. Realizing these constraints, this work examines RF heating effects of stents using electro-magnetic field (EMF simulations and phantoms with properties that mimic myocardium. For this purpose, RF power deposition that exceeds the clinical limits was induced by a dedicated birdcage coil. Fiber optic probes and MR thermometry were applied for temperature monitoring using agarose phantoms containing copper tubes or coronary stents. The results demonstrate an agreement between RF heating induced temperature changes derived from EMF simulations versus MR thermometry. The birdcage coil tailored for RF heating was capable of irradiating power exceeding the specific-absorption rate (SAR limits defined by the IEC guidelines by a factor of three. This setup afforded RF induced temperature changes up to +27 K in a reference phantom. The maximum extra temperature increase, induced by a copper tube or a coronary stent was less than 3 K. The coronary stents examined showed an RF heating behavior similar to a copper tube. Our results suggest that, if IEC guidelines for local/global SAR are followed, the extra RF heating induced in myocardial tissue by stents may not be significant versus the baseline heating induced by the energy deposited by a tailored cardiac transmit RF coil at 7.0 T, and may be smaller if not insignificant than the extra RF heating observed under the circumstances used in this study.

  12. Localized frustration and binding-induced conformational change in recognition of 5S RNA by TFIIIA zinc finger.

    Science.gov (United States)

    Tan, Cheng; Li, Wenfei; Wang, Wei

    2013-12-19

    Protein TFIIIA is composed of nine tandemly arranged Cys2His2 zinc fingers. It can bind either to the 5S RNA gene as a transcription factor or to the 5S RNA transcript as a chaperone. Although structural and biochemical data provided valuable information on the recognition between the TFIIIIA and the 5S DNA/RNA, the involved conformational motions and energetic factors contributing to the binding affinity and specificity remain unclear. In this work, we conducted MD simulations and MM/GBSA calculations to investigate the binding-induced conformational changes in the recognition of the 5S RNA by the central three zinc fingers of TFIIIA and the energetic factors that influence the binding affinity and specificity at an atomistic level. Our results revealed drastic interdomain conformational changes between these three zinc fingers, involving the exposure/burial of several crucial DNA/RNA binding residues, which can be related to the competition between DNA and RNA for the binding of TFIIIA. We also showed that the specific recognition between finger 4/finger 6 and the 5S RNA introduces frustrations to the nonspecific interactions between finger 5 and the 5S RNA, which may be important to achieve optimal binding affinity and specificity.

  13. Molecular simulations of conformation change and aggregation of HIV-1 Vpr13-33 on graphene oxide

    Science.gov (United States)

    Zeng, Songwei; Zhou, Guoquan; Guo, Jianzhong; Zhou, Feng; Chen, Junlang

    2016-04-01

    Recent experiments have reported that the fragment of viral protein R (Vpr), Vpr13-33, can assemble and change its conformation after adsorbed on graphene oxide (GO) and then reduce its cytotoxicity. This discovery is of great importance, since the mutation of Vpr13-33 can decrease the viral replication, viral load and delay the disease progression. However, the interactions between Vpr13-33 and GO at atomic level are still unclear. In this study, we performed molecular dynamics simulation to investigate the dynamic process of the adsorption of Vpr13-33 onto GO and the conformation change after aggregating on GO surface. We found that Vpr13-33 was adsorbed on GO surface very quickly and lost its secondary structure. The conformation of peptides-GO complex was highly stable because of π-π stacking and electrostatic interactions. When two peptides aggregated on GO, they did not dimerize, since the interactions between the two peptides were much weaker than those between each peptide and GO.

  14. Conformational change upon ligand binding and dynamics of the PDZ domain from leukemia-associated Rho guanine nucleotide exchange factor.

    Science.gov (United States)

    Liu, Jiangxin; Zhang, Jiahai; Yang, Yinshan; Huang, Hongda; Shen, Weiqun; Hu, Qi; Wang, Xingsheng; Wu, Jihui; Shi, Yunyu

    2008-06-01

    Leukemia-associated Rho guanine nucleotide exchange factor (LARG) is a RhoA-specific guanine nucleotide exchange factor (GEF) that can activate RhoA. The PDZ (PSD-95/Disc-large/ZO-1 homology) domain of LARG interacts with membrane receptors, which can relay extracellular signals to RhoA signal transduction pathways. Until now there is no structural and dynamic information about these interactions. Here we report the NMR structures of the LARG PDZ in the apo form and in complex with the plexin-B1 C-terminal octapeptide. Unobservable resonances of the residues in betaB/betaC and betaE/alphaB loops in apo state were observed in the complex state. A distinct region of the binding groove in the LARG PDZ was found to undergo conformational change compared with other PDZs. Analysis of the (15)N relaxation data using reduced spectral density mapping shows that the apo LARG PDZ (especially its ligand-binding groove) is flexible and exhibits internal motions on both picosecond to nanosecond and microsecond to millisecond timescales. Mutagenesis and thermodynamic studies indicate that the conformation of the betaB/betaC and betaE/alphaB loops affects the PDZ-peptide interaction. It is suggested that the conformational flexibility could facilitate the change of structures upon ligand binding.

  15. Non-invasive SFG spectroscopy: a tool to reveal the conformational change of grafted chains due to bacterial adhesion

    Science.gov (United States)

    Bulard, Emilie; Dubost, Henri; Fontaine-Aupart, Marie-Pierre; Zheng, Wanquan; Herry, Jean-Marie; Bellon-Fontaine, Marie-No"lle; Briandet, Romain; Bourguignon, Bernard

    2011-07-01

    In many fields such as biomedical or food industry, surface colonization by micro-organisms leads to biofilms formation that are tridimentional biostructures highly resistant to the action of antimicrobials, by mechanisms still unclear. In order to deepen our understanding of the initial interaction of bacteria cells with a solid surface, we analyze by in situ vibrational Sum Frequency Generation (SFG) spectroscopy the effect of the adhesion of hydrophilic Lactoccocus lactis bacteria and its hydrophobic mutants in distilled water on a self-assembled monolayer (SAM) of octadecanethiol (ODT) on a gold film. When a homogeneous bacterial monolayer is deposited on this ordered surface, SFG spectrum of the ODT SAM shows significant intensity changes from that in air or in water. Its modelling as a function of conformation allows to distinguish optical effects due to the water solution surrounding bacteria from conformational changes of the ODT SAM due to the presence of the bacteria cells. Futhermore, bacterial adhesion induces different measurable effects on the ODT SAM conformation, depending on the hydrophobic / hydrophilic character of the bacterial surface. Such a result deserves to be taken into account for the design of new materials with improved properties or to control biofilm formation.

  16. The reovirus sigma1 aspartic acid sandwich: a trimerization motif poised for conformational change.

    Science.gov (United States)

    Schelling, Pierre; Guglielmi, Kristen M; Kirchner, Eva; Paetzold, Bernhard; Dermody, Terence S; Stehle, Thilo

    2007-04-13

    Reovirus attachment protein sigma1 mediates engagement of receptors on the surface of target cells and undergoes dramatic conformational rearrangements during viral disassembly in the endocytic pathway. The sigma1 protein is a filamentous, trimeric molecule with a globular beta-barrel head domain. An unusual cluster of aspartic acid residues sandwiched between hydrophobic tyrosines is located at the sigma1 subunit interface. A 1.75-A structure of the sigma1 head domain now reveals two water molecules at the subunit interface that are held strictly in position and interact with neighboring residues. Structural and biochemical analyses of mutants affecting the aspartic acid sandwich indicate that these residues and the corresponding chelated water molecules act as a plug to block the free flow of solvent and stabilize the trimer. This arrangement of residues at the sigma1 head trimer interface illustrates a new protein design motif that may confer conformational mobility during cell entry.

  17. The Reovirus Sigmal Aspartic Acid Sandwich: A Trimerization Motif Poised for Conformational Change

    Energy Technology Data Exchange (ETDEWEB)

    Schelling,P.; Guglielml, K.; Kirchner, E.; Paetzold, b.; Dermody, T.; Stehle, T.

    2007-01-01

    Reovirus attachment protein {sigma}1 mediates engagement of receptors on the surface of target cells and undergoes dramatic conformational rearrangements during viral disassembly in the endocytic pathway. The {sigma}1 protein is a filamentous, trimeric molecule with a globular {beta}-barrel head domain. An unusual cluster of aspartic acid residues sandwiched between hydrophobic tyrosines is located at the {sigma}1 subunit interface. A 1.75 {angstrom} structure of the {sigma}1 head domain now reveals two water molecules at the subunit interface that are held strictly in position and interact with neighboring residues. Structural and biochemical analyses of mutants affecting the aspartic acid sandwich indicate that these residues and the corresponding chelated water molecules act as a plug to block the free flow of solvent and stabilize the trimer. This arrangement of residues at the {sigma}1 head trimer interface illustrates a new protein design motif that may confer conformational mobility during cell entry.

  18. Relationship between conformational changes in the dopamine transporter and cocaine-like subjective effects of uptake inhibitors

    DEFF Research Database (Denmark)

    Løland, Claus Juul; Desai, Rajeev I; Zou, Mu-Fa

    2007-01-01

    the subjective effects of different inhibitors with their molecular mode of interaction at the DAT. We determined how different inhibitors affected accessibility of the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl]-methanethiosulfonate to an inserted cysteine (I159C), which is accessible when...... at a mutant DAT (Y335A) characterized by a global change in the conformational equilibrium. We observed a close relationship between the decrease in potencies of inhibitors at this mutant and cocaine-like responding in rats trained to discriminate cocaine from saline injections. Our data suggest...... that chemically different DAT inhibitors stabilize distinct transporter conformations and that this in turn affects the cocaine-like subjective effects of these compounds in vivo....

  19. Conformational Change Observed in the Active Site of Class C β-Lactamase MOX-1 upon Binding to Aztreonam.

    Science.gov (United States)

    Oguri, Takuma; Ishii, Yoshikazu; Shimizu-Ibuka, Akiko

    2015-08-01

    We solved the crystal structure of the class C β-lactamase MOX-1 complexed with the inhibitor aztreonam at 1.9Å resolution. The main-chain oxygen of Ser315 interacts with the amide nitrogen of aztreonam. Surprisingly, compared to that in the structure of free MOX-1, this main-chain carboxyl changes its position significantly upon binding to aztreonam. This result indicates that the interaction between MOX-1 and β-lactams can be accompanied by conformational changes in the B3 β-strand main chain.

  20. Substrate and Inhibitor-Specific Conformational Changes in the Human Serotonin Transporter Revealed by Voltage-Clamp Fluorometry

    DEFF Research Database (Denmark)

    Söderhielm, Pella C; Andersen, Jacob; Munro, Lachlan

    2015-01-01

    of TM6, Ala419 in the interface between TM8 and extracellular loop (EL) 4, and Leu481 in EL5. The reporter positions were used for time-resolved measurement of conformational changes during 5-HT transport and binding of cocaine and the selective serotonin reuptake inhibitors fluoxetine and escitalopram....... At all reporter positions, fluorescence changes observed upon substrate application were distinctly different from those observed upon inhibitor application, with respect to relative amplitude or direction. Furthermore, escitalopram, fluoxetine, and cocaine induced a very similar pattern of fluorescent...

  1. Medicare determinations and income-related monthly adjustment amounts to Medicare Part B premiums; conforming changes to regulations. Final rule.

    Science.gov (United States)

    2014-03-01

    This final rule adopts, without change, the interim final rule with request for comments we published in the Federal Register on September 18, 2013. The interim final rule modified our rules regarding Medicare Part B income-related monthly adjustment amounts to conform to changes made to the Social Security Act (Act) and Internal Revenue Code by the Affordable Care Act. We also removed provisions that phased in income-related monthly adjustment amounts between 2007 and 2009 and updated a citation to reflect the transfer of authority for hearing appeals under title XVIII of the Act from the Social Security Administration to the Department of Health and Human Services.

  2. Regulation of phenylalanine hydroxylase: conformational changes upon phosphorylation detected by H/D exchange and mass spectrometry.

    Science.gov (United States)

    Li, Jun; Fitzpatrick, Paul F

    2013-07-15

    The enzyme phenylalanine hydroxylase catalyzes the hydroxylation of excess phenylalanine in the liver to tyrosine. The enzyme is regulated allosterically by phenylalanine and by phosphorylation of Ser16. Hydrogen/deuterium exchange monitored by mass spectrometry has been used to gain insight into any structural change upon phosphorylation. Peptides in both the catalytic and regulatory domains show increased deuterium incorporation into the phosphorylated protein. Deuterium is incorporated into fewer peptides than when the enzyme is activated by phenylalanine, and the incorporation is slower. This establishes that the conformational change upon phosphorylation of phenylalanine hydroxylase is different from and less extensive than that upon phenylalanine activation.

  3. Reordering hydrogen bonds using hamiltonian replica exchange enhances sampling of conformational changes in biomolecular systems.

    Science.gov (United States)

    Vreede, Jocelyne; Wolf, Maarten G; de Leeuw, Simon W; Bolhuis, Peter G

    2009-05-07

    Hydrogen bonds play an important role in stabilizing (meta-)stable states in protein folding. Hence, they can potentially be used as a way to bias these states in molecular simulation methods. Previously, Wolf et al. showed that applying repulsive and attractive hydrogen bond biasing potentials in an alternating way significantly accelerates the folding process (Wolf, M. G.; de Leeuw, S. W. Biophys. J. 2008, 94, 3742). As the biasing potentials are only active during a fixed time interval, this alternating scheme does not represent a thermodynamic equilibrium. In this work, we present a Hamiltonian replica exchange molecular dynamics (REMD) scheme that aims to shuffle and reorder hydrogen bonds in the protein backbone. We therefore apply adapted hydrogen bond potentials in a Hamiltonian REMD scheme, which we call hydrogen bond switching (HS). To compare the performance of the HS to a standard REMD method, we performed HS and temperature REMD simulations of a beta-heptapeptide in methanol. Both methods sample the conformational space to a similar extent. As the HS simulation required only five replicas, while the REMD simulation required 20 replicas, the HS method is significantly more efficient. We tested the HS method also on a larger system, 16-residue polyalanine in water. Both of the simulations starting from a completely unfolded and a folded conformation resulted in an ensemble with, apart from the starting structure, similar conformational minima. We can conclude that the HS method provides an efficient way to sample the conformational space of a protein, without requiring knowledge of the folded states beforehand. In addition, these simulations revealed that convergence was hampered by replicas having a preference for specific biasing potentials. As this sorting effect is inherent to any Hamiltonian REMD method, finding a solution will result in an additional increase in the efficiency of Hamiltonian REMD methods in general.

  4. Conformational Changes and Substrate Recognition in Pseudomonas aeruginosa d-Arginine Dehydrogenase

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Guoxing; Yuan, Hongling; Li, Congran; Lu, Chung-Dar; Gadda, Giovanni; Weber, Irene T. (GSU); (PUMC)

    2010-11-15

    DADH catalyzes the flavin-dependent oxidative deamination of D-amino acids to the corresponding {alpha}-keto acids and ammonia. Here we report the first X-ray crystal structures of DADH at 1.06 {angstrom} resolution and its complexes with iminoarginine (DADH{sub red}/iminoarginine) and iminohistidine (DADH{sub red}/iminohistidine) at 1.30 {angstrom} resolution. The DADH crystal structure comprises an unliganded conformation and a product-bound conformation, which is almost identical to the DADH{sub red}/iminoarginine crystal structure. The active site of DADH was partially occupied with iminoarginine product (30% occupancy) that interacts with Tyr53 in the minor conformation of a surface loop. This flexible loop forms an 'active site lid', similar to those seen in other enzymes, and may play an essential role in substrate recognition. The guanidinium side chain of iminoarginine forms a hydrogen bond interaction with the hydroxyl of Thr50 and an ionic interaction with Glu87. In the structure of DADH in complex with iminohistidine, two alternate conformations were observed for iminohistidine where the imidazole groups formed hydrogen bond interactions with the side chains of His48 and Thr50 and either Glu87 or Gln336. The different interactions and very distinct binding modes observed for iminoarginine and iminohistidine are consistent with the 1000-fold difference in k{sub cat}/K{sub m} values for D-arginine and D-histidine. Comparison of the kinetic data for the activity of DADH on different D-amino acids and the crystal structures in complex with iminoarginine and iminohistidine establishes that this enzyme is characterized by relatively broad substrate specificity, being able to oxidize positively charged and large hydrophobic D-amino acids bound within a flask-like cavity.

  5. Changes in conformation of myosin heads during the development of isometric contraction and rapid shortening in single frog muscle fibres.

    Science.gov (United States)

    Piazzesi, G; Reconditi, M; Dobbie, I; Linari, M; Boesecke, P; Diat, O; Irving, M; Lombardi, V

    1999-01-15

    1. Two-dimensional X-ray diffraction patterns were recorded at the European Synchrotron Radiation Facility from central segments of intact single muscle fibres of Rana temporaria with 5 ms time resolution during the development of isometric contraction. Shortening at ca 0.8 times the maximum velocity was also imposed at the isometric tetanus plateau. 2. The first myosin-based layer line (ML1) and the second myosin-based meridional reflection (M2), which are both strong in resting muscle, were completely abolished at the plateau of the isometric tetanus. The third myosin-based meridional reflection (M3), arising from the axial repeat of the myosin heads along the filaments, remained intense but its spacing changed from 14.34 to 14.56 nm. The intensity change of the M3 reflection, IM3, could be explained as the sum of two components, I14.34 and I14.56, arising from myosin head conformations characteristic of rest and isometric contraction, respectively. 3. The amplitudes (A) of the X-ray reflections, which are proportional to the fraction of myosin heads in each conformation, changed with half-times that were similar to that of isometric force development, which was 33.5 +/- 2. 0 ms (mean +/- s.d., 224 tetani from three fibres, 4 C), measured from the end of the latent period. We conclude that the myosin head conformation changes synchronously with force development, at least within the 5 ms time resolution of these measurements. 4. The changes in the X-ray reflections during rapid shortening have two temporal components. The rapid decrease in intensity of the 14.56 nm reflection at the start of shortening is likely to be due to tilting of myosin heads attached to actin. The slower changes in the other reflections were consistent with a return to the resting conformation of the myosin heads that was about 60 % complete after shortening of 70 nm per half-sarcomere.

  6. Exploring transition pathway and free-energy profile of large-scale protein conformational change by combining normal mode analysis and umbrella sampling molecular dynamics.

    Science.gov (United States)

    Wang, Jinan; Shao, Qiang; Xu, Zhijian; Liu, Yingtao; Yang, Zhuo; Cossins, Benjamin P; Jiang, Hualiang; Chen, Kaixian; Shi, Jiye; Zhu, Weiliang

    2014-01-09

    Large-scale conformational changes of proteins are usually associated with the binding of ligands. Because the conformational changes are often related to the biological functions of proteins, understanding the molecular mechanisms of these motions and the effects of ligand binding becomes very necessary. In the present study, we use the combination of normal-mode analysis and umbrella sampling molecular dynamics simulation to delineate the atomically detailed conformational transition pathways and the associated free-energy landscapes for three well-known protein systems, viz., adenylate kinase (AdK), calmodulin (CaM), and p38α kinase in the absence and presence of respective ligands. For each protein under study, the transient conformations along the conformational transition pathway and thermodynamic observables are in agreement with experimentally and computationally determined ones. The calculated free-energy profiles reveal that AdK and CaM are intrinsically flexible in structures without obvious energy barrier, and their ligand binding shifts the equilibrium from the ligand-free to ligand-bound conformation (population shift mechanism). In contrast, the ligand binding to p38α leads to a large change in free-energy barrier (ΔΔG ≈ 7 kcal/mol), promoting the transition from DFG-in to DFG-out conformation (induced fit mechanism). Moreover, the effect of the protonation of D168 on the conformational change of p38α is also studied, which reduces the free-energy difference between the two functional states of p38α and thus further facilitates the conformational interconversion. Therefore, the present study suggests that the detailed mechanism of ligand binding and the associated conformational transition is not uniform for all kinds of proteins but correlated to their respective biological functions.

  7. A conformational change within the WAVE2 complex regulates its degradation following cellular activation

    Science.gov (United States)

    Joseph, Noah; Biber, Guy; Fried, Sophia; Reicher, Barak; Levy, Omer; Sabag, Batel; Noy, Elad; Barda-Saad, Mira

    2017-01-01

    WASp family Verprolin-homologous protein-2 (WAVE2), a member of the Wiskott-Aldrich syndrome protein (WASp) family of actin nucleation promoting factors, is a central regulator of actin cytoskeleton polymerization and dynamics. Multiple signaling pathways operate via WAVE2 to promote the actin-nucleating activity of the actin-related protein 2/3 (Arp2/3) complex. WAVE2 exists as a part of a pentameric protein complex known as the WAVE regulatory complex (WRC), which is unstable in the absence of its individual proteins. While the involvement of WAVE2 in actin polymerization has been well documented, its negative regulation mechanism is poorly characterized to date. Here, we demonstrate that WAVE2 undergoes ubiquitylation in a T-cell activation dependent manner, followed by proteasomal degradation. The WAVE2 ubiquitylation site was mapped to lysine 45, located at the N-terminus where WAVE2 binds to the WRC. Using Förster resonance energy transfer (FRET), we reveal that the autoinhibitory conformation of the WRC maintains the stability of WAVE2 in resting cells; the release of autoinhibition following T-cell activation facilitates the exposure of WAVE2 to ubiquitylation, leading to its degradation. The dynamic conformational structures of WAVE2 during cellular activation dictate its degradation. PMID:28332566

  8. Ligand Binding and Conformational Changes in the Purine-Binding Riboswitch Aptamer Domains

    Science.gov (United States)

    Noeske, Jonas; Buck, Janina; Wöhnert, Jens; Schwalbe, Harald

    Riboswitches are highly structured mRNA elements that regulate gene expression upon specific binding of small metabolite molecules. The purine-binding riboswitches bind different purine ligands by forming both canonical Watson—Crick and non-canonical intermolecular base pairs, involving a variety of hydrogen bonds between the riboswitch aptamer domain and the purine ligand. Here, we summarize work on the ligand binding modes of both purine-binding aptamer domains, their con-formational characteristics in the free and ligand-bound forms, and their ligand-induced folding. The adenine- and guanine-binding riboswitch aptamer domains display different conformations in their free forms, despite nearly identical nucleotide loop sequences that form a loop—loop interaction in the ligand-bound forms. Interestingly, the stability of helix II is crucial for the formation of the loop—loop interaction in the free form. A more stable helix II in the guanine riboswitch leads to a preformed loop—loop interaction in its free form. In contrast, a less stable helix II in the adenine riboswitch results in a lack of this loop—loop interaction in the absence of ligand and divalent cations.

  9. The mechanism of heat-induced damage of endothelial cells and its effect on vital organs

    Directory of Open Access Journals (Sweden)

    Lei SU

    2017-06-01

    Full Text Available As an important organ of the human body, vascular endothelial cells (VECs play a vital role in heat stress-induced tissue damage. Its integrity not only serves as a barrier for maintaining vascular permeability but also has major impact on cellular structure and function during acute phase response to heat stress. In heat stroke, a series of acute and complicated pathophysiological changes, including microcirculation change, damage VECs and thereby induce or aggravate multiple organ dysfunction syndrome (MODS. Meanwhile, studies have shown that, during heat stroke, VECs are the major responding cells and one of the most common cells that experience morphological and functional changes. Therefore, VECs damage might be an important mechanism involved in heat stroke. This article reviews the mechanism of heat-induced damage of VECs and its effect on vital organs. DOI: 10.11855/j.issn.0577-7402.2017.04.01

  10. Structural studies of Saccharomyces cerevesiae mitochondrial NADP-dependent isocitrate dehydrogenase in different enzymatic states reveal substantial conformational changes during the catalytic reaction.

    Science.gov (United States)

    Peng, Yingjie; Zhong, Chen; Huang, Wei; Ding, Jianping

    2008-09-01

    Isocitrate dehydrogenases (IDHs) catalyze oxidative decarboxylation of isocitrate (ICT) into alpha-ketoglutarate (AKG). We report here the crystal structures of Saccharomyces cerevesiae mitochondrial NADP-IDH Idp1p in binary complexes with coenzyme NADP, or substrate ICT, or product AKG, and in a quaternary complex with NADPH, AKG, and Ca(2+), which represent different enzymatic states during the catalytic reaction. Analyses of these structures identify key residues involved in the binding of these ligands. Comparisons among these structures and with the previously reported structures of other NADP-IDHs reveal that eukaryotic NADP-IDHs undergo substantial conformational changes during the catalytic reaction. Binding or release of the ligands can cause significant conformational changes of the structural elements composing the active site, leading to rotation of the large domain relative to the small and clasp domains along two hinge regions (residues 118-124 and residues 284-287) while maintaining the integrity of its secondary structural elements, and thus, formation of at least three distinct overall conformations. Specifically, the enzyme adopts an open conformation when bound to NADP, a quasi-closed conformation when bound to ICT or AKG, and a fully closed conformation when bound to NADP, ICT, and Ca(2+) in the pseudo-Michaelis complex or with NADPH, AKG, and Ca(2+) in the product state. The conformational changes of eukaryotic NADP-IDHs are quite different from those of Escherichia coli NADP-IDH, for which significant conformational changes are observed only between two forms of the apo enzyme, suggesting that the catalytic mechanism of eukaryotic NADP-IDHs is more complex than that of EcIDH, and involves more fine-tuned conformational changes.

  11. Structure and dynamics of water in crowded environments slows down peptide conformational changes

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Cheng; Prada-Gracia, Diego; Rao, Francesco, E-mail: francesco.rao@frias.uni-freiburg.de [Freiburg Institute for Advanced Studies, School of Soft Matter Research, Albertstrasse 19, 79104 Freiburg im Breisgau (Germany)

    2014-07-28

    The concentration of macromolecules inside the cell is high with respect to conventional in vitro experiments or simulations. In an effort to characterize the effects of crowding on the thermodynamics and kinetics of disordered peptides, molecular dynamics simulations were run at different concentrations by varying the number of identical weakly interacting peptides inside the simulation box. We found that the presence of crowding does not influence very much the overall thermodynamics. On the other hand, peptide conformational dynamics was found to be strongly affected, resulting in a dramatic slowing down at larger concentrations. The observation of long lived water bridges between peptides at higher concentrations points to a nontrivial role of the solvent in the altered peptide kinetics. Our results reinforce the idea for an active role of water in molecular crowding, an effect that is expected to be relevant for problems influenced by large solvent exposure areas like in intrinsically disordered proteins.

  12. Changes in the conformational state of hemoglobin in hemodialysed patients with chronic renal failure.

    Science.gov (United States)

    Pieniazek, Anna; Gwozdzinski, Krzysztof

    2015-01-01

    The aim of this study was to evaluate the properties of internal components of erythrocytes in chronic renal failure (CRF) patients undergoing hemodialysis (HD) in comparison to control subjects. For investigation of conformational state of hemoglobin and nonheme proteins (NHP) the maleimide spin label (MSL) in electron paramagnetic resonance (EPR) was applied. The studies were performed using MSL in whole cells and hemolysate as well as proteins separated by ion exchange chromatography and checked by electrophoresis. Additionally the level of -SH groups in hemolysate and isolated internal proteins of CRF erythrocytes was determined using 4,4'-dithiodipyridine. All measurements were performed before and after hemodialysis. Oxidative stress accompanying CRF/hemodialysed patients caused a significant decrease in the mobility of internal components inside erythrocytes indicated by MSL (P hemoglobins and internal nonheme proteins in erythrocytes of CRF patients.

  13. Conformation change of opiorphin derivates. A theoretical study of the radical initiated epimerization of opiorphin

    Science.gov (United States)

    Szórád, János J.; Faragó, Eszter P.; Rágyanszki, Anita; Cimino, Franco A.; Fiser, Béla; Owen, Michael C.; Jójárt, Balázs; Morgado, Claudio A.; Szőri, Milán; Jensen, Svend J. Knak; Csizmadia, Imre G.; Viskolcz, Béla

    2015-04-01

    The global minimum (GM) structure of opiorphin (QRFSR) and its seven derivatives (qRFSR, QrFSR, QRfSR, QRFsR, QRFSr, QRYSR, QRySR) were explored using molecular mechanics method. Based on STRIDE analysis, most of the conformers possessed no secondary structures. Among global minima, only QrFSR epimer contained a β-turn formed between glutamine (Q1) and serine (S4) residues. The most common hydrogen bonds were formed between the 2nd arginine (R2) and glutamine (Q1) which also appeared in some GMs. According to DFT calculations (ωB97X-D/cc-pVTZ//B3LYP/6-31G(d)), the overall epimerization of the GM structures is a downhill process in the presence of an rad OH/H2O2 redox system.

  14. Heat-inducible RNAi for gene functional analysis in plants.

    Science.gov (United States)

    Masclaux, Frédéric; Galaud, Jean-Philippe

    2011-01-01

    Controlling gene expression during plant development is an efficient method to explore gene function and RNA interference (RNAi) is now considered as a powerful technology for gene functional analysis. However, constitutive gene silencing cannot be used with genes involved in fundamental processes such as embryo viability or plant growth and alternative silencing strategies avoiding these limitations should be preferred. Tissue-specific and inducible promoters, able to control gene expression at spatial and/or temporal level, can be used to circumvent viability problems. In this chapter, after a rapid overview of the inducible promoters currently used for transgenic approaches in plants, we describe a method we have developed to study gene function by heat-inducible RNAi. This system is easy to use and complementary to those based on chemical gene inducer treatments and might be useful for both research and biotechnological applications.

  15. Conformational Changes in the GM-CSF Receptor Suggest a Molecular Mechanism for Affinity Conversion and Receptor Signaling.

    Science.gov (United States)

    Broughton, Sophie E; Hercus, Timothy R; Nero, Tracy L; Dottore, Mara; McClure, Barbara J; Dhagat, Urmi; Taing, Houng; Gorman, Michael A; King-Scott, Jack; Lopez, Angel F; Parker, Michael W

    2016-08-02

    The GM-CSF, IL-3, and IL-5 receptors constitute the βc family, playing important roles in inflammation, autoimmunity, and cancer. Typical of heterodimeric type I cytokine receptors, signaling requires recruitment of the shared subunit to the initial cytokine:α subunit binary complex through an affinity conversion mechanism. This critical process is poorly understood due to the paucity of crystal structures of both binary and ternary receptor complexes for the same cytokine. We have now solved the structure of the binary GM-CSF:GMRα complex at 2.8-Å resolution and compared it with the structure of the ternary complex, revealing distinct conformational changes. Guided by these differences we performed mutational and functional studies that, importantly, show GMRα interactions playing a major role in receptor signaling while βc interactions control high-affinity binding. These results support the notion that conformational changes underlie the mechanism of GM-CSF receptor activation and also suggest how related type I cytokine receptors signal.

  16. A Quantitative bgl Operon Model for E. coli Requires BglF Conformational Change for Sugar Transport

    Science.gov (United States)

    Chopra, Paras; Bender, Andreas

    The bgl operon is responsible for the metabolism of β-glucoside sugars such as salicin or arbutin in E. coli. Its regulatory system involves both positive and negative feedback mechanisms and it can be assumed to be more complex than that of the more closely studied lac and trp operons. We have developed a quantitative model for the regulation of the bgl operon which is subject to in silico experiments investigating its behavior under different hypothetical conditions. Upon administration of 5mM salicin as an inducer our model shows 80-fold induction, which compares well with the 60-fold induction measured experimentally. Under practical conditions 5-10mM inducer are employed, which is in line with the minimum inducer concentration of 1mM required by our model. The necessity of BglF conformational change for sugar transport has been hypothesized previously, and in line with those hypotheses our model shows only minor induction if conformational change is not allowed. Overall, this first quantitative model for the bgl operon gives reasonable predictions that are close to experimental results (where measured). It will be further refined as values of the parameters are determined experimentally. The model was developed in Systems Biology Markup Language (SBML) and it is available from the authors and from the Biomodels repository [www.ebi.ac.uk/biomodels].

  17. Conformational change in the C form of palmitic acid investigated by Raman spectroscopy and X-ray diffraction

    Science.gov (United States)

    de Sousa, F. F.; Nogueira, C. E. S.; Freire, P. T. C.; Moreira, S. G. C.; Teixeira, A. M. R.; de Menezes, A. S.; Mendes Filho, J.; Saraiva, G. D.

    2016-05-01

    Fatty acids are substances found in most living beings in nature. Here we report the effect of the low temperature in the vibrational and structural properties of the C form of palmitic acid, a fatty acid with 16 carbon atoms. The Raman spectra were obtained in the temperature interval from 300 to 18 K in the spectral range between 30 and 3100 cm- 1. The assignment of the duly observed bands was done based on the density functional theory. On cooling, the main changes observed in the lattice mode region of the Raman spectra were interpreted as a conformational modification undergone by the palmitic acid molecules in the unit cell. The X-ray diffraction measurements were obtained from 290 to 80 K showing a slight modification in the lattice parameters at about 210 K. Differential scanning calorimetry (DSC) measurements were recorded between 150 and 300 K and no enthalpic anomaly in the DSC thermogram was observed. These techniques provided strong evidence of the conformational change in the molecules of palmitic acid at low temperatures.

  18. Substrate-Linked Conformational Change in the Periplasmic Component of a Cu(I)/Ag(I) Efflux System

    Energy Technology Data Exchange (ETDEWEB)

    Bagai, I.; Liu, W.; Rensing, C.; Blackburn, N.J.; McEvoy, M.M.

    2009-06-02

    Gram-negative bacteria utilize dual membrane resistance nodulation division-type efflux systems to export a variety of substrates. These systems contain an essential periplasmic component that is important for assembly of the protein complex. We show here that the periplasmic protein CusB from the Cus copper/silver efflux system has a critical role in Cu(I) and Ag(I) binding. Isothermal titration calorimetry experiments demonstrate that one Ag(I) ion is bound per CusB molecule with high affinity. X-ray absorption spectroscopy data indicate that the metal environment is an all-sulfur 3-coordinate environment. Candidates for the metal-coordinating residues were identified from sequence analysis, which showed four conserved methionine residues. Mutations of three of these methionine residues to isoleucine resulted in significant effects on CusB metal binding in vitro. Cells containing these CusB variants also show a decrease in their ability to grow on copper-containing plates, indicating an important functional role for metal binding by CusB. Gel filtration chromatography demonstrates that upon binding metal, CusB undergoes a conformational change to a more compact structure. Based on these structural and functional effects of metal binding, we propose that the periplasmic component of resistance nodulation division-type efflux systems plays an active role in export through substrate-linked conformational changes.

  19. Conformational changes of the H+-ATPase from Escherichia coli upon nucleotide binding detected by single molecule fluorescence.

    Science.gov (United States)

    Börsch, M; Turina, P; Eggeling, C; Fries, J R; Seidel, C A; Labahn, A; Gräber, P

    1998-10-23

    Using a confocal fluorescence microscope with an avalanche photodiode as detector, we studied the fluorescence of the tetramethylrhodamine labeled F1 part of the H+-ATPase from Escherichia coli, EF1, carrying the gammaT106-C mutation [Aggeler, J.A. and Capaldi, R.A. (1992) J. Biol. Chem. 267, 21355-21359] in aqueous solution upon excitation with a mode-locked argon ion laser at 528 nm. The diffusion of the labeled EF1 through the confocal volume gives rise to photon bursts, which were analyzed with fluorescence correlation spectroscopy, resulting in a diffusion coefficient of 3.3 x 10(-7) cm2 s(-1). In the presence of nucleotides the diffusion coefficient increases by about 15%. This effect indicates a change of the shape and/or the volume of the enzyme upon binding of nucleotides, i.e. fluorescence correlation spectroscopy with single EF1 molecules allows the detection of conformational changes.

  20. Interaction of fisetin with human serum albumin by fluorescence, circular dichroism spectroscopy and DFT calculations: binding parameters and conformational changes

    Energy Technology Data Exchange (ETDEWEB)

    Matei, Iulia; Ionescu, Sorana [Department of Physical Chemistry, Faculty of Chemistry, University of Bucharest, Bd. Regina Elisabeta 4-12, 030018 Bucharest (Romania); Hillebrand, Mihaela, E-mail: mihh@gw-chimie.math.unibuc.ro [Department of Physical Chemistry, Faculty of Chemistry, University of Bucharest, Bd. Regina Elisabeta 4-12, 030018 Bucharest (Romania)

    2011-08-15

    The interaction between fisetin, an antioxidant and neuroprotective flavonoid, and human serum albumin (HSA) is investigated by means of fluorescence (steady-state, synchronous, time-resolved) and circular dichroism (CD) spectroscopy. The formation of a 1:1 complex with a constant of about 10{sup 5} M{sup -1} was evidenced. Foerster's resonance energy transfer and competitive binding with site markers warfarin and ibuprofen were considered and discussed. Changes in the CD band of HSA indicate a decrease in the {alpha}-helix content upon binding. An induced CD signal for bound fisetin was observed and rationalized in terms of density functional theory calculations. - Highlights: > Fisetin-BSA system was studied by fluorescence spectroscopy. > Binding parameters, association constant and number of sites were estimated. > Binding site of fisetin was identified by competitive experiments. > Conformational changes in HSA and fisetin were evidenced by circular dichroism. > TDDFT calculated CD spectra supported the experimental data.

  1. Luminescence detection of cysteine based on Ag⁺-mediated conformational change of terbium ion-promoted G-quadruplex.

    Science.gov (United States)

    Tan, Hongliang; Tang, Gonge; Ma, Chanjiao; Li, Qian

    2016-02-18

    In this work, we developed a simple and sensitive method for the detection of cysteine (Cys) by employing terbium ion (Tb(3+))-promoted G-qudraplex (G4/Tb) as a luminescent probe, which is based on Ag(+)-mediated conformational change of G4/Tb. Due to Ag(+) is able to compete with Tb(3+) to bind guanine at G4, the presence of Ag(+) can lead to the formation of G4/Tb-Ag(+) complex and disrupt the structure of G4/Tb. Meanwhile, the binding of Ag(+) with G4/Tb will also cause the alteration of the excited state of G4 and more efficient energy transfer from G4 to Tb(3+), enhancing the luminescence of G4/Tb. However, upon the addition of Cys, Ag(+) will be released from G4/Tb-Ag(+) complex because of the high affinity of Cys to Ag(+). This results in the re-formation of the conformation of G4/Tb and the decrease of the luminescence of G4/Tb. So, Ag(+)-enhanced luminescence of G4/Tb is associated with its conformational transformation. As a luminescent probe for Cys, G4/Tb not only shows excellent selectivity and sensitivity with a detection limit of 20 nM, but also possesses the features of simple preparation, easy reproducibility, and eliminating the interferences from background fluorescence. We envision that the presented strategy might provide new insight into the biosensing applications of lanthanide complex. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Luminescence detection of cysteine based on Ag{sup +}-mediated conformational change of terbium ion-promoted G-quadruplex

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Hongliang, E-mail: hltan@jxnu.edu.cn; Tang, Gonge; Ma, Chanjiao; Li, Qian

    2016-02-18

    In this work, we developed a simple and sensitive method for the detection of cysteine (Cys) by employing terbium ion (Tb{sup 3+})-promoted G-qudraplex (G4/Tb) as a luminescent probe, which is based on Ag{sup +}-mediated conformational change of G4/Tb. Due to Ag{sup +} is able to compete with Tb{sup 3+} to bind guanine at G4, the presence of Ag{sup +} can lead to the formation of G4/Tb–Ag{sup +} complex and disrupt the structure of G4/Tb. Meanwhile, the binding of Ag{sup +} with G4/Tb will also cause the alteration of the excited state of G4 and more efficient energy transfer from G4 to Tb{sup 3+}, enhancing the luminescence of G4/Tb. However, upon the addition of Cys, Ag{sup +} will be released from G4/Tb–Ag{sup +} complex because of the high affinity of Cys to Ag{sup +}. This results in the re-formation of the conformation of G4/Tb and the decrease of the luminescence of G4/Tb. So, Ag{sup +}-enhanced luminescence of G4/Tb is associated with its conformational transformation. As a luminescent probe for Cys, G4/Tb not only shows excellent selectivity and sensitivity with a detection limit of 20 nM, but also possesses the features of simple preparation, easy reproducibility, and eliminating the interferences from background fluorescence. We envision that the presented strategy might provide new insight into the biosensing applications of lanthanide complex. - Highlights: • A G4/Tb-based luminescent probe for Cys detection was developed for the first time. • The binding and remove of Ag{sup +} mediate the luminescence of G4/Tb. • The luminescent ​sensor showed excellent selectivity and high sensitivity to Cys.

  3. Phosphorylation by cAMP-dependent protein kinase causes a conformational change in the R domain of the cystic fibrosis transmembrane conductance regulator.

    Science.gov (United States)

    Dulhanty, A M; Riordan, J R

    1994-04-05

    Individuals with cystic fibrosis have a defect in the CFTR protein, a chloride channel regulated by cAMP-dependent protein kinase (PKA). The majority of the phosphorylation sites of PKA are located in the R domain of CFTR. It has been postulated that this domain may act as a gate for the chloride channel. Of the many possible mechanisms whereby the R domain could gate the channel, including interdomain interactions, charge distribution, or conformational change, we investigated the possibility that phosphorylation leads to conformational changes in the R domain. To test this hypothesis, a protocol for purification of human R domain peptide synthesized in a bacterial expression system was developed. Purified R domain was phosphorylated by PKA, and CD spectra were obtained. As a result of phosphorylation by PKA, a significant spectral change, indicative of a reduction in the alpha-helical content, was found. CD spectra of the R domain of a shark homologue of CFTR indicated similar changes in conformation as a result of phosphorylation by PKA. In contrast, phosphorylation of the human R domain by PKC, which has only a small influence on CFTR channel activity, failed to elicit CD spectral changes, indicating no conformational change comparable to those induced by PKA phosphorylation. These observations provide the first structural characterization of the R domain and suggest that the gating of the CFTR chloride channel by PKA may involve a conformational change in the R domain.

  4. Conformational changes in stratum corneum lipids by effect of bicellar systems.

    Science.gov (United States)

    Rodríguez, Gelen; Barbosa-Barros, Lucyanna; Rubio, Laia; Cócera, Mercedes; Díez, Avencia; Estelrich, Joan; Pons, Ramon; Caelles, Jaume; De la Maza, Alfonso; López, Olga

    2009-09-15

    Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was applied to study the effects of the bicelles formed by dimyristoyl-glycero-phosphocholine (DMPC) and dihexanoyl-glycero-phosphocholine (DHPC) in porcine stratum corneum (SC) in vitro. A comparison of skin samples treated and untreated with bicelles at different temperatures was carried out. The analysis of variations after treatment in the position of the symmetric CH2 stretching, CH2 scissoring, and CH2 rocking vibrations reported important information about the effect of bicelles on the skin. Bicellar systems caused a phase transition from the gel or solid state to the liquid crystalline state in the lipid conformation of SC, reflecting the major order-disorder transition from hexagonally packed to disordered chains. Grazing incidence small and wide X-ray scattering (GISAXS and GIWAXS) techniques confirmed this effect of bicelles on the SC. These results are probably related to with the permeabilizing effect previously described for the DMPC/DHPC bicelles.

  5. Structural evidence for conformational changes of Delta class glutathione transferases after ligand binding.

    Science.gov (United States)

    Wongsantichon, Jantana; Robinson, Robert C; Ketterman, Albert J

    2012-05-01

    We report four new crystal structures for Delta class glutathione transferases from insects. We compare these new structures as well as several previously reported structures to determine that structural transitions can be observed with ligand binding. These transitions occurred in the regions around the active site entrance, including alpha helix 2, C-terminus of alpha helix 4 including the loop to helix 5 and the C-terminus of helix 8. These structural movements have been reported or postulated to occur for several other glutathione transferase classes; however, this is the first report showing structural evidence of all these movements occurring, in this case in Delta class glutathione transferases. These fluctuations also can be observed occurring within a single structure as there is ligand bound in only one subunit and each subunit is undergoing different conformational transitions. The structural comparisons show reorganizations occur both pre- and post-GSH ligand binding communicated through the subunit interface of the quaternary assembly. Movements of these positions would allow 'breathing' of the active site for substrate entrance, topological rearrangement for varying substrate specificity and final product release.

  6. Computational Recipe for Efficient Description of Large-Scale Conformational Changes in Biomolecular Systems.

    Science.gov (United States)

    Moradi, Mahmoud; Tajkhorshid, Emad

    2014-07-01

    Characterizing large-scale structural transitions in biomolecular systems poses major technical challenges to both experimental and computational approaches. On the computational side, efficient sampling of the configuration space along the transition pathway remains the most daunting challenge. Recognizing this issue, we introduce a knowledge-based computational approach toward describing large-scale conformational transitions using (i) nonequilibrium, driven simulations combined with work measurements and (ii) free energy calculations using empirically optimized biasing protocols. The first part is based on designing mechanistically relevant, system-specific reaction coordinates whose usefulness and applicability in inducing the transition of interest are examined using knowledge-based, qualitative assessments along with nonequilirbrium work measurements which provide an empirical framework for optimizing the biasing protocol. The second part employs the optimized biasing protocol resulting from the first part to initiate free energy calculations and characterize the transition quantitatively. Using a biasing protocol fine-tuned to a particular transition not only improves the accuracy of the resulting free energies but also speeds up the convergence. The efficiency of the sampling will be assessed by employing dimensionality reduction techniques to help detect possible flaws and provide potential improvements in the design of the biasing protocol. Structural transition of a membrane transporter will be used as an example to illustrate the workings of the proposed approach.

  7. Structure of a lipid A phosphoethanolamine transferase suggests how conformational changes govern substrate binding.

    Science.gov (United States)

    Anandan, Anandhi; Evans, Genevieve L; Condic-Jurkic, Karmen; O'Mara, Megan L; John, Constance M; Phillips, Nancy J; Jarvis, Gary A; Wills, Siobhan S; Stubbs, Keith A; Moraes, Isabel; Kahler, Charlene M; Vrielink, Alice

    2017-02-28

    Multidrug-resistant (MDR) gram-negative bacteria have increased the prevalence of fatal sepsis in modern times. Colistin is a cationic antimicrobial peptide (CAMP) antibiotic that permeabilizes the bacterial outer membrane (OM) and has been used to treat these infections. The OM outer leaflet is comprised of endotoxin containing lipid A, which can be modified to increase resistance to CAMPs and prevent clearance by the innate immune response. One type of lipid A modification involves the addition of phosphoethanolamine to the 1 and 4' headgroup positions by phosphoethanolamine transferases. Previous structural work on a truncated form of this enzyme suggested that the full-length protein was required for correct lipid substrate binding and catalysis. We now report the crystal structure of a full-length lipid A phosphoethanolamine transferase from Neisseria meningitidis, determined to 2.75-Å resolution. The structure reveals a previously uncharacterized helical membrane domain and a periplasmic facing soluble domain. The domains are linked by a helix that runs along the membrane surface interacting with the phospholipid head groups. Two helices located in a periplasmic loop between two transmembrane helices contain conserved charged residues and are implicated in substrate binding. Intrinsic fluorescence, limited proteolysis, and molecular dynamics studies suggest the protein may sample different conformational states to enable the binding of two very different- sized lipid substrates. These results provide insights into the mechanism of endotoxin modification and will aid a structure-guided rational drug design approach to treating multidrug-resistant bacterial infections.

  8. Changes in the Conformational State of Hemoglobin in Hemodialysed Patients with Chronic Renal Failure

    Directory of Open Access Journals (Sweden)

    Anna Pieniazek

    2015-01-01

    Full Text Available The aim of this study was to evaluate the properties of internal components of erythrocytes in chronic renal failure (CRF patients undergoing hemodialysis (HD in comparison to control subjects. For investigation of conformational state of hemoglobin and nonheme proteins (NHP the maleimide spin label (MSL in electron paramagnetic resonance (EPR was applied. The studies were performed using MSL in whole cells and hemolysate as well as proteins separated by ion exchange chromatography and checked by electrophoresis. Additionally the level of –SH groups in hemolysate and isolated internal proteins of CRF erythrocytes was determined using 4,4′-dithiodipyridine. All measurements were performed before and after hemodialysis. Oxidative stress accompanying CRF/hemodialysed patients caused a significant decrease in the mobility of internal components inside erythrocytes indicated by MSL (P < 0.02. The significant decrease in mobility of spin labeled HbA1c and HbA both before and after HD (P < 0.0002 as well as in nonheme proteins before hemodialysis (P < 0.05 versus control was indicated. Decrease in mobility of internal components of erythrocytes was accompanied by loss of thiols before and after hemodialysis versus control in NHP (P < 0.05, HbA1c (P < 0.0002, and HbA (P < 0.0005. These findings showed oxidative influence of hemodialysis on hemoglobins and internal nonheme proteins in erythrocytes of CRF patients.

  9. Detection of conformational changes in immunoglobulin G using isothermal titration calorimetry with low-molecular-weight probes.

    Science.gov (United States)

    Rispens, Theo; Lakemond, Catriona M M; Derksen, Ninotska I L; Aalberse, Rob C

    2008-09-15

    Proteins for therapeutic use may contain small amounts of partially misfolded monomeric precursors to postproduction aggregation. To detect these misfolded proteins in the presence of an excess of properly folded protein, fluorescent probes such as 8-anilino-1-naphthalene sulfonate (ANS) are commonly used. We investigated the possibility of using isothermal titration calorimetry (ITC) to improve the detection of this type of conformational change using hydrophobic probes. As a case study, conformational changes in human polyclonal immunoglobulin G (IgG) were monitored by measuring the enthalpies of binding of ANS using ITC. Results were compared with those using fluorescence spectroscopy. IgG heated at 63 degrees C was used as a model system for "damaged" IgG. Heat-treated IgG can be detected already at levels below 5% with both ITC and fluorescence. However, ITC allows a much wider molar probe-to-protein ratio to be sampled. In particular, using reverse titration experiments (allowing high probe-to-protein ratios not available to fluorescence spectroscopy), an increase in the number of binding sites with a K(d)>10 mM was observed for heat-treated IgG, reflecting subtle changes in structure. Both ITC and fluorescence spectroscopy showed low background signals for native IgG. The nature of the background signals was not clear from the fluorescence measurements. However, further analysis of the ITC background signals shows that a fraction (8%) binds ANS with a dissociation constant of approximately 0.2 mM. Measurements were also carried out at pH 4.5. Precipitation of IgG was induced by ANS at concentrations above 0.5 mM, interfering with the ITC measurements. Instead, with the nonfluorescent probes 4-amino-1-naphthalene sulfonate and 1-naphthalene sulfonate, no precipitation is observed. These probes yield differences in the enthalpies of binding to heated and nonheated IgG similar to ANS. The data illustrate that ITC with low-molecular-weight probes is a versatile

  10. Reversible conformational change in herpes simplex virus glycoprotein B with fusion-from-without activity is triggered by mildly acidic pH

    Directory of Open Access Journals (Sweden)

    Nicola Anthony V

    2010-12-01

    Full Text Available Abstract Background The pre-fusion form of the herpes simplex virus (HSV fusion protein gB undergoes pH-triggered conformational change in vitro and during viral entry (Dollery et al., J. Virol. 84:3759-3766, 2010. The antigenic structure of gB from the fusion-from-without (FFWO strain of HSV-1, ANG path, resembles wild type gB that has undergone pH-triggered changes. Together, changes in the antigenic and oligomeric conformation of gB correlate with fusion activity. We tested whether the pre-fusion form of FFWO gB undergoes altered conformational change in response to low pH. Results A pH of 5.5 - 6.0 altered the conformation of Domains I and V of FFWO gB, which together comprise the functional region containing the hydrophobic fusion loops. The ANG path gB oligomer was altered at a similar pH. All changes were reversible. In wild type HSV lacking the UL45 protein, which has been implicated in gB-mediated fusion, gB still underwent pH-triggered changes. ANG path entry was inactivated by pretreatment of virions with low pH. Conclusion The pre-fusion conformation of gB with enhanced fusion activity undergoes alteration in antigenic structure and oligomeric conformation in response to acidic pH. We propose that endosomal pH triggers conformational change in mutant gB with FFWO activity in a manner similar to wild type. Differences apart from this trigger may account for the increased fusion activity of FFWO gB.

  11. Conformal Coating of a Phase Change Material on Ordered Plasmonic Nanorod Arrays for Broadband All-Optical Switching.

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Peijun; Weimer, Matthew S.; Emery, Jonathan D.; Diroll, Benjamin T.; Chen, Xinqi; Hock, Adam S.; Chang, Robert P. H.; Martinson, Alex B. F.; Schaller, Richard D.

    2017-01-01

    Actively tunable optical transmission through artificial metamaterials holds great promise for next-generation nanophotonic devices and metasurfaces. Plasmonic nanostructures and phase change materials have been extensively studied to this end due to their respective strong interactions with light and tunable dielectric constants under external stimuli. Seamlessly integrating plasmonic components with phase change materials, as demonstrated in the present work, can facilitate phase change by plasmonically enabled light confinement and meanwhile make use of the high sensitivity of plasmon resonances to the variation of dielectric constant associated with the phase change. The hybrid platform here is composed of plasmonic indium tin-oxide nanorod arrays (ITO-NRAs) conformally coated with an ultrathin layer of a prototypical phase change material, vanadium dioxide (VO2), which enables all-optical modulation of the infrared as well as the visible spectral ranges. The interplay between the intrinsic plasmonic nonlinearity of ITO-NRAs and the phase transition induced permittivity change of VO2 gives rise to spectral and temporal responses that cannot be achieved with individual material components alone.

  12. Conformal Nets II: Conformal Blocks

    Science.gov (United States)

    Bartels, Arthur; Douglas, Christopher L.; Henriques, André

    2017-03-01

    Conformal nets provide a mathematical formalism for conformal field theory. Associated to a conformal net with finite index, we give a construction of the `bundle of conformal blocks', a representation of the mapping class groupoid of closed topological surfaces into the category of finite-dimensional projective Hilbert spaces. We also construct infinite-dimensional spaces of conformal blocks for topological surfaces with smooth boundary. We prove that the conformal blocks satisfy a factorization formula for gluing surfaces along circles, and an analogous formula for gluing surfaces along intervals. We use this interval factorization property to give a new proof of the modularity of the category of representations of a conformal net.

  13. Conformal Nets II: Conformal Blocks

    Science.gov (United States)

    Bartels, Arthur; Douglas, Christopher L.; Henriques, André

    2017-08-01

    Conformal nets provide a mathematical formalism for conformal field theory. Associated to a conformal net with finite index, we give a construction of the `bundle of conformal blocks', a representation of the mapping class groupoid of closed topological surfaces into the category of finite-dimensional projective Hilbert spaces. We also construct infinite-dimensional spaces of conformal blocks for topological surfaces with smooth boundary. We prove that the conformal blocks satisfy a factorization formula for gluing surfaces along circles, and an analogous formula for gluing surfaces along intervals. We use this interval factorization property to give a new proof of the modularity of the category of representations of a conformal net.

  14. Conformation Changes N-terminal Involvement and cGMP Signal Relay in the Phosphodiesterase-5 GAF Domain

    Energy Technology Data Exchange (ETDEWEB)

    H Wang; H Robinson; H Ke

    2011-12-31

    The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, which may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98-147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes.

  15. Cyanotryptophans as Novel Fluorescent Probes for Studying Protein Conformational Changes and DNA-Protein Interaction.

    Science.gov (United States)

    Talukder, Poulami; Chen, Shengxi; Roy, Basab; Yakovchuk, Petro; Spiering, Michelle M; Alam, Mohammad P; Madathil, Manikandadas M; Bhattacharya, Chandrabali; Benkovic, Stephen J; Hecht, Sidney M

    2015-12-29

    Described herein are the syntheses and photophysical characterization of three novel cyanotryptophans, and their efficient incorporation into proteins as fluorescent probes. Photophysical characteristics indicated that each was significantly brighter and red-shifted in fluorescence emission relative to tryptophan. Each analogue was used to activate a suppressor tRNA transcript and was incorporated with good efficiency into two different positions (Trp22 and Trp74) of Escherichia coli dihydrofolate reductase (ecDHFR). The Trp analogues could be monitored selectively in the presence of multiple native Trp residues in DHFR. 6-CNTrp (A) formed an efficient Förster resonance energy transfer (FRET) pair with l-(7-hydroxycoumarin-4-yl)ethylglycine (HCO, D) at position 17. Further, 6-CNTrp (A) was incorporated into two DNA binding proteins, including the Klenow fragment of DNA polymerase I and an RNA recognition motif (RRM2) of heterogeneous nuclear ribonucleoprotein L-like (hnRNP LL). Using these proteins, we demonstrated the use of FRET involving A as a fluorescence donor and benzo[g]quinazoline-2,4-(1H,3H)-dione 2'-deoxyriboside (Tf) or 4-aminobenzo[g]quinazoline-2-one 2'-deoxyriboside (Cf) as fluorescent acceptors to study the binding interaction of the Klenow fragment with duplex DNA oligomers (labeled with Tf), or the domain-specific association between hnRNP LL and the BCL2 i-motif DNA (labeled with Cf). Thus, the non-natural amino acid could be used as a FRET partner for studying protein-nucleic acid interactions. Together, these findings demonstrate the potential utility of 6-CNTrp (A) as a fluorescence donor for the study of protein conformational events.

  16. Joule heating induced transient temperature field and its effects on electroosmosis in a microcapillary packed with microspheres.

    Science.gov (United States)

    Kang, Y; Yang, C; Huang, X

    2005-08-02

    The Joule heating induced transient temperature field and its effect on the electroosmotic flow in a capillary packed with microspheres is analyzed numerically using the control-volume-based finite difference method. The model incorporates the coupled momentum equation for the electroosmotic velocity, the energy equations for the Joule heating induced temperature distributions in both the packed column and the capillary wall, and the mass and electric current continuity equations. The temperature-dependent physical properties of the electrolyte solution are taken into consideration. The characteristics of the Joule heating induced transient development of temperature and electroosmotic flow fields are studied. Specifically, the simulation shows that the presence of Joule heating causes a noticeable axial temperature gradient in the thermal entrance region and elevates a significant temperature increment inside the microcapillary. The temperature changes in turn greatly affect the electroosmotic velocity by means of the temperature-dependent fluid viscosity, dielectric constant, and local electric field strength. Furthermore, the model predicts an induced pressure gradient to counterbalance the axial variation of the electroosmotic velocity so as to maintain the fluid mass continuity. In addition, under specific conditions, the present model is validated by comparing with the existing analytical model and experimental data from the literature.

  17. Heat- and pH-induced BSA conformational changes, hydrogel formation and application as 3D cell scaffold.

    Science.gov (United States)

    Navarra, Giovanna; Peres, Chiara; Contardi, Marco; Picone, Pasquale; San Biagio, Pier Luigi; Di Carlo, Marta; Giacomazza, Daniela; Militello, Valeria

    2016-09-15

    Aggregation and gelation of globular proteins can be an advantage to generate new forms of nanoscale biomaterials based on the fibrillar architecture. Here, we report results obtained by exploiting the proteins' natural tendency to self-organize in 3D network, for the production of new material based on BSA for medical application. In particular, at five different pH values the conformational and structural changes of the BSA during all the steps of the thermal aggregation and gelation have been analyzed by FTIR spectroscopy. The macroscopic mechanical properties of these hydrogels have been obtained by rheological measurements. The microscopic structure of the gels have been studied by AFM and SEM images to have a picture of their different spatial arrangement. Finally, the use of the BSA hydrogels as scaffold has been tested in two different cell cultures.

  18. Light-induced conformational changes of the chromophore and the protein in phytochromes: bacterial phytochromes as model systems.

    Science.gov (United States)

    Scheerer, Patrick; Michael, Norbert; Park, Jung Hee; Nagano, Soshichiro; Choe, Hui-Woog; Inomata, Katsuhiko; Borucki, Berthold; Krauss, Norbert; Lamparter, Tilman

    2010-04-26

    Recombinant phytochromes Agp1 and Agp2 from Agrobacterium tumefaciens are used as model phytochromes for biochemical and biophysical studies. In biliverdin binding phytochromes the site for covalent attachment of the chromophore lies in the N-terminal region of the protein, different from plant phytochromes. The issue which stereochemistry the chromophore adopts in the so-called Pr and Pfr forms is addressed by using a series of locked chromophores which form spectrally characteristic adducts with Agp1 and Agp2. Studies on light-induced conformational changes of Agp1 give an insight into how the intrinsic histidine kinase is modulated by light. Comparison of the crystal structure of an Agp1 fragment with other phytochrome crystal structures supports the idea that a light induced rearrangement of subunits within the homodimer modulates the activity of the kinase.

  19. pH-dependent conformational changes in the HCV NS3 protein modulate its ATPase and helicase activities.

    Science.gov (United States)

    Ventura, Gustavo Tavares; Costa, Emmerson Corrêa Brasil da; Capaccia, Anne Miranda; Mohana-Borges, Ronaldo

    2014-01-01

    The hepatitis C virus (HCV) infects 170 to 200 million people worldwide and is, therefore, a major health problem. The lack of efficient treatments that specifically target the viral proteins or RNA and its high chronicity rate make hepatitis C the cause of many deaths and hepatic transplants annually. The NS3 protein is considered an important target for the development of anti-HCV drugs because it is composed of two domains (a serine protease in the N-terminal portion and an RNA helicase/NTPase in the C-terminal portion), which are essential for viral replication and proliferation. We expressed and purified both the NS3 helicase domain (NS3hel) and the full-length NS3 protein (NS3FL) and characterized pH-dependent structural changes associated with the increase in their ATPase and helicase activities at acidic pH. Using intrinsic fluorescence experiments, we have observed that NS3hel was less stable at pH 6.4 than at pH 7.2. Moreover, binding curves using an extrinsic fluorescent probe (bis-ANS) and ATPase assays performed under different pH conditions demonstrated that the hydrophobic clefts of NS3 are significantly more exposed to the aqueous medium at acidic pH. Using fluorescence spectroscopy and anisotropy assays, we have also observed more protein interaction with DNA upon pH acidification, which suggests that the hydrophobic clefts exposure on NS3 might be related to a loss of stability that could lead it to adopt a more open conformation. This conformational change at acidic pH would stimulate both its ATPase and helicase activities, as well as its ability to bind DNA. Taken together, our results indicate that the NS3 protein adopts a more open conformation due to acidification from pH 7.2 to 6.4, resulting in a more active form at a pH that is found near Golgi-derived membranes. This increased activity could better allow NS3 to carry out its functions during HCV replication.

  20. pH-dependent conformational changes in the HCV NS3 protein modulate its ATPase and helicase activities.

    Directory of Open Access Journals (Sweden)

    Gustavo Tavares Ventura

    Full Text Available The hepatitis C virus (HCV infects 170 to 200 million people worldwide and is, therefore, a major health problem. The lack of efficient treatments that specifically target the viral proteins or RNA and its high chronicity rate make hepatitis C the cause of many deaths and hepatic transplants annually. The NS3 protein is considered an important target for the development of anti-HCV drugs because it is composed of two domains (a serine protease in the N-terminal portion and an RNA helicase/NTPase in the C-terminal portion, which are essential for viral replication and proliferation. We expressed and purified both the NS3 helicase domain (NS3hel and the full-length NS3 protein (NS3FL and characterized pH-dependent structural changes associated with the increase in their ATPase and helicase activities at acidic pH. Using intrinsic fluorescence experiments, we have observed that NS3hel was less stable at pH 6.4 than at pH 7.2. Moreover, binding curves using an extrinsic fluorescent probe (bis-ANS and ATPase assays performed under different pH conditions demonstrated that the hydrophobic clefts of NS3 are significantly more exposed to the aqueous medium at acidic pH. Using fluorescence spectroscopy and anisotropy assays, we have also observed more protein interaction with DNA upon pH acidification, which suggests that the hydrophobic clefts exposure on NS3 might be related to a loss of stability that could lead it to adopt a more open conformation. This conformational change at acidic pH would stimulate both its ATPase and helicase activities, as well as its ability to bind DNA. Taken together, our results indicate that the NS3 protein adopts a more open conformation due to acidification from pH 7.2 to 6.4, resulting in a more active form at a pH that is found near Golgi-derived membranes. This increased activity could better allow NS3 to carry out its functions during HCV replication.

  1. The crystal structure of the D-alanine-D-alanine ligase from Acinetobacter baumannii suggests a flexible conformational change in the central domain before nucleotide binding.

    Science.gov (United States)

    Huynh, Kim-Hung; Hong, Myoung-ki; Lee, Clarice; Tran, Huyen-Thi; Lee, Sang Hee; Ahn, Yeh-Jin; Cha, Sun-Shin; Kang, Lin-Woo

    2015-11-01

    Acinetobacter baumannii, which is emerging as a multidrug-resistant nosocomial pathogen, causes a number of diseases, including pneumonia, bacteremia, meningitis, and skin infections. With ATP hydrolysis, the D-alanine-D-alanine ligase (DDL) catalyzes the synthesis of D-alanyl-D-alanine, which is an essential component of bacterial peptidoglycan. In this study, we determined the crystal structure of DDL from A. baumannii (AbDDL) at a resolution of 2.2 Å. The asymmetric unit contained six protomers of AbDDL. Five protomers had a closed conformation in the central domain, while one protomer had an open conformation in the central domain. The central domain with an open conformation did not interact with crystallographic symmetry-related protomers and the conformational change of the central domain was not due to crystal packing. The central domain of AbDDL can have an ensemble of the open and closed conformations before the binding of substrate ATP. The conformational change of the central domain is important for the catalytic activity and the detail information will be useful for the development of inhibitors against AbDDL and putative antibacterial agents against A. baumannii. The AbDDL structure was compared with that of other DDLs that were in complex with potent inhibitors and the catalytic activity of AbDDL was confirmed using enzyme kinetics assays.

  2. Conformal house

    DEFF Research Database (Denmark)

    Ryttov, Thomas Aaby; Sannino, Francesco

    2010-01-01

    fixed point. As a consistency check we recover the previously investigated bounds of the conformal windows when restricting to a single matter representation. The earlier conformal windows can be imagined to be part now of the new conformal house. We predict the nonperturbative anomalous dimensions...... at the infrared fixed points. We further investigate the effects of adding mass terms to the condensates on the conformal house chiral dynamics and construct the simplest instanton induced effective Lagrangian terms...

  3. Structures of Prostacyclin Synthase and Its Complexes with Substrate Analog and Inhibitor Reveal a Ligand-specific Heme Conformation Change*s

    Science.gov (United States)

    Li, Yi-Ching; Chiang, Chia-Wang; Yeh, Hui-Chun; Hsu, Pei-Yung; Whitby, Frank G.; Wang, Lee-Ho; Chan, Nei-Li

    2008-01-01

    Prostacyclin synthase (PGIS) is a cytochrome P450 (P450) enzyme that catalyzes production of prostacyclin from prostaglandin H2. PGIS is unusual in that it catalyzes an isomerization rather than a monooxygenation, which is typical of P450 enzymes. To understand the structural basis for prostacyclin biosynthesis in greater detail, we have determined the crystal structures of ligand-free, inhibitor (minoxidil)-bound and substrate analog U51605-bound PGIS. These structures demonstrate a stereo-specific substrate binding and suggest features of the enzyme that facilitate isomerization. Unlike most microsomal P450s, where large substrate-induced conformational changes take place at the distal side of the heme, conformational changes in PGIS are observed at the proximal side and in the heme itself. The conserved and extensive heme propionate-protein interactions seen in all other P450s, which are largely absent in the ligand-free PGIS, are recovered upon U51605 binding accompanied by water exclusion from the active site. In contrast, when minoxidil binds, the propionate-protein interactions are not recovered and water molecules are largely retained. These findings suggest that PGIS represents a divergent evolution of the P450 family, in which a heme barrier has evolved to ensure strict binding specificity for prostaglandin H2, leading to a radical-mediated isomerization with high product fidelity. The U51605-bound structure also provides a view of the substrate entrance and product exit channels. PMID:18032380

  4. Conformational changes in reconstituted skeletal muscle thin filaments observed by fluorescence spectroscopy

    OpenAIRE

    MIKI, Masao

    2007-01-01

    The cyclic interaction of myosin and actin coupled ATP hydrolysis generates the mechanical force of muscle contraction. During this process, the system passes through several steps. One of these is thought to be identical to the stable rigor complex formed by myosin and actin in the absence of ATP. This cyclic interaction is regulated by changes in tropomyosin (Tm) and troponin (Tn) located on the actin filament in response alterations in intracellular Ca2+ concentration (Ebashi et al., 1969)...

  5. Targeted molecular dynamics reveals overall common conformational changes upon hybrid domain swing-out in beta3 integrins.

    Science.gov (United States)

    Provasi, Davide; Murcia, Marta; Coller, Barry S; Filizola, Marta

    2009-11-01

    The beta3 integrin family members alphaIIbeta3 and alphaVbeta3 signal bidirectionally through long-range allosteric changes, including a transition from a bent unliganded-closed low-affinity state to an extended liganded-open high-affinity state. To obtain an atomic-level description of this transition in an explicit solvent, we carried out targeted molecular dynamics simulations of the headpieces of alphaIIbeta3 and alphaVbeta3 integrins. Although minor differences were observed between these receptors, our results suggest a common transition pathway in which the hybrid domain swing-out is accompanied by conformational changes within the beta3 betaA (I-like) domain that propagate through the alpha7 helix C-terminus, and are followed by the alpha7 helix downward motion and the opening of the beta6-alpha7 loop. Breaking of contact interactions between the beta6-alpha7 loop and the alpha1 helix N-terminus results in helix straightening, internal rearrangements of the specificity determining loop (SDL), movement of the beta1-alpha1 loop toward the metal ion dependent adhesion site (MIDAS), and final changes at the interfaces between the beta3 betaA (I-like) domain and either the hybrid or the alpha beta-propeller domains. Taken together, our results suggest novel testable hypotheses of intradomain and interdomain interactions responsible for beta3 integrin activation.

  6. Structural analysis of prolyl oligopeptidases using molecular docking and dynamics: insights into conformational changes and ligand binding.

    Directory of Open Access Journals (Sweden)

    Swati Kaushik

    Full Text Available Prolyl oligopeptidase (POP is considered as an important pharmaceutical target for the treatment of numerous diseases. Despite enormous studies on various aspects of POPs structure and function still some of the questions are intriguing like conformational dynamics of the protein and interplay between ligand entry/egress. Here, we have used molecular modeling and docking based approaches to unravel questions like differences in ligand binding affinities in three POP species (porcine, human and A. thaliana. Despite high sequence and structural similarity, they possess different affinities for the ligands. Interestingly, human POP was found to be more specific, selective and incapable of binding to a few planar ligands which showed extrapolation of porcine POP in human context is more complicated. Possible routes for substrate entry and product egress were also investigated by detailed analyses of molecular dynamics (MD simulations for the three proteins. Trajectory analysis of bound and unbound forms of three species showed differences in conformational dynamics, especially variations in β-propeller pore size, which was found to be hidden by five lysine residues present on blades one and seven. During simulation, β-propeller pore size was increased by ∼2 Å in porcine ligand-bound form which might act as a passage for smaller product movement as free energy barrier was reduced, while there were no significant changes in human and A. thaliana POPs. We also suggest that these differences in pore size could lead to fundamental differences in mode of product egress among three species. This analysis also showed some functionally important residues which can be used further for in vitro mutagenesis and inhibitor design. This study can help us in better understanding of the etiology of POPs in several neurodegenerative diseases.

  7. Calcium-dependent conformational change and thermal stability of the isolated PsbO protein detected by FTIR spectroscopy.

    Science.gov (United States)

    Heredia, P; De Las Rivas, J

    2003-10-14

    The structure and function of the photosystem II PsbO extrinsic protein is under intense research, being an essential part of the biomolecular engine that carries out water oxidation and oxygen production. This paper presents a structural analysis of the isolated PsbO protein by FTIR spectroscopy, reporting detailed secondary structure quantification and changes in the secondary structure content of the protein attributed to the effect of calcium (Ca(2+)). Measurements in H(2)O and D(2)O have allowed us to see the effect of calcium on the conformation of the protein. The results indicate that (i) the protein presents a major content of beta-structure (i.e., beta-sheet, beta-strands, beta-turns) as detected by the infrared bands at 1624-1625, 1678-1679, 1688-1689 cm(-1), which account for about 38% in water and 33% in heavy water, in the presence of calcium; and (ii) the amount of this beta-structure fraction increases 7-10% in the absence of calcium, with a concomitant decrease in loops and nonordered structure. The thermal denaturation profile of the protein in the presence of calcium showed low stability with T(m) approximately 56 degrees C. This profile also shows a second phase of denaturation above 60 degrees C and the appearance of aggregation signals above 70 degrees C. Our observations indicate that calcium is able to modify the conformation of the protein at least in solution and confirm that PsbO is mainly a beta-protein where beta-sheet is the major ordered secondary structure element of the protein core.

  8. PNIPAM grafted chains at the silicon/water interface: temperature-dependent conformational changes and protein adsorption

    Science.gov (United States)

    Yim, Hyun; Kent, Michael; Huber, Dale; Shin, Kwanwoo; Satija, Sushil; Majewski, Jaroslaw; Smith, Greg

    2002-03-01

    Poly(N-isopropyl acrylamide) (PNIPAM) is perhaps the most well known member of the class of responsive polymers. It has a lower critical solution temperature (LCST) at about 31 oC. This very sharp transition ( 5 oC) is attributed to the disruption of the hydrogen bonding of water molecules around the amide group of the side chain. In this work we investigate the conformation of grafted PNIPAM chains at the silicon/water interface using neutron reflection (NR). Grafted PNIPAM layers were prepared both by reacting COOH-terminated PNIPAM to OH-terminated self-assembled monolayers on silicon ("grafting to" method) and by polymerizing NIPAM monomers from silicon ("grafting from" method). Detailed concentration profiles of PNIPAM layers in D2O and d-acetone are determined by NR. A range of PNIPAM molecular weights from 33K to 220K have been examined. Surprisingly, whereas the samples all have a cloud point at about 31 oC in aqeuous solution, we find little change in the conformation of the grafted PNIPAM chains with temperature. A bilayer profile is required to fit the data for all PNIPAM samples in D2O, but a smoothly decaying one layer profile for the PNIPAM samples in d-acetone. The PNIPAM chains are more collapsed in d-acetone than in D2O. The adsorption of myoglobin to the grafted PNIPAM layers as a function of temperature will be also discussed. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy under contract CE-AC04-94AL85000.

  9. Coordination, microprotonation equilibria and conformational changes of myo-inositol hexakisphosphate with pertinence to its biological function.

    Science.gov (United States)

    Veiga, Nicolás; Torres, Julia; Macho, Israel; Gómez, Kerman; González, Gabriel; Kremer, Carlos

    2014-11-21

    Within all the eukaryotic cells there is an important group of biomolecules that has been potentially related to signalling functions: the myo-inositol phosphates (InsPs). In nature, the most abundant member of this family is the so called InsP6 (phytate, L(12-)), for which our group has strived in the past to elucidate its intricate chemical behaviour. In this work we expand on our earlier findings, shedding light on the inframolecular details of its protonation and complexation processes. We evaluate systematically the chemical performance of InsP6 in the presence and absence of alkali and alkaline earth metal ions, through (31)P NMR measurements, in a non-interacting medium and over a wide pH range. The analysis of the titration curves by means of a model based on the cluster expansion method allows us to describe in detail the distribution of the different protonated microspecies of the ligand. With the aid of molecular modelling tools, we assess the energetic and geometrical characteristics of the protonation sequence and the conformational transition suffered by InsP6 as the pH changes. By completely characterizing the protonation pattern, conformation and geometry of the metal complexes, we unveil the chemical and structural basis behind the influence that the physiologically relevant cations, Na(+), K(+), Mg(2+) and Ca(2+) have over the phytate chemical reactivity. This information is essential in the process of gaining reliable structural knowledge about the most important InsP6 species in the in vitro and in vivo experiments, and how these features modulate their probable biological functions.

  10. Evidence for distinct sodium-, dopamine-, and cocaine-dependent conformational changes in transmembrane segments 7 and 8 of the dopamine transporter

    DEFF Research Database (Denmark)

    Norregaard, Lene; Loland, Claus Juul; Gether, Ulrik

    2003-01-01

    . Inhibitors such as cocaine did not alter the effect of MTSET in M371C. The protection of M371C inactivation by dopamine required Na+. Because dopamine binding is believed to be Na+-independent, this suggests that dopamine induces a transport-associated conformational change that decreases the reactivity of M......371C with MTSET. In contrast to M371C, cocaine decreased the reaction rate of A399C with MTSET, whereas dopamine had no effect. The protection by cocaine can either reflect that Ala-399 lines the cocaine binding crevice or that cocaine induces a conformational change that decreases the reactivity of A...

  11. Redox-dependent conformational changes in eukaryotic cytochromes revealed by paramagnetic NMR spectroscopy.

    Science.gov (United States)

    Volkov, Alexander N; Vanwetswinkel, Sophie; Van de Water, Karen; van Nuland, Nico A J

    2012-03-01

    Cytochrome c (Cc) is a soluble electron carrier protein, transferring reducing equivalents between Cc reductase and Cc oxidase in eukaryotes. In this work, we assessed the structural differences between reduced and oxidized Cc in solution by paramagnetic NMR spectroscopy. First, we have obtained nearly-complete backbone NMR resonance assignments for iso-1-yeast Cc and horse Cc in both oxidation states. These were further used to derive pseudocontact shifts (PCSs) arising from the paramagnetic haem group. Then, an extensive dataset comprising over 450 measured PCSs and high-resolution X-ray and solution NMR structures of both proteins were used to define the anisotropic magnetic susceptibility tensor, Δχ. For most nuclei, the PCSs back-calculated from the Δχ tensor are in excellent agreement with the experimental PCS values. However, several contiguous stretches-clustered around G41, N52, and A81-exhibit large deviations both in yeast and horse Cc. This behaviour is indicative of redox-dependent structural changes, the extent of which is likely conserved in the protein family. We propose that the observed discrepancies arise from the changes in protein dynamics and discuss possible functional implications.

  12. Nanoparticle charge-transfer interactions induce surface dependent conformational changes in apolipoprotein biocorona

    CERN Document Server

    Raghavendra, Achyut J; Brown, Jared M; Podilaa, Ramakrishna

    2016-01-01

    Upon introduction into a biological system, engineered nanomaterials (ENMs) rapidly associate with a variety of biomolecules such as proteins and lipids to form a biocorona. The presence of biocorona influences nano-bio interactions considerably, and could ultimately result in altered biological responses. Apolipoprotein A-I (ApoA-I), the major constituent of high-density lipoprotein (HDL), is one of the most prevalent proteins found in ENM-biocorona irrespective of ENM nature, size, and shape. Given the importance of ApoA-I in HDL and cholesterol transport, it is necessary to understand the mechanisms of ApoA-I adsorption and the associated structural changes for assessing consequences of ENM exposure. Here, we used a comprehensive array of microscopic and spectroscopic tools to elucidate the interactions between ApoA-I and 100 nm Ag nanoparticles (AgNPs) with four different surface functional groups. We found that the protein adsorption and secondary structural changes are highly dependent on the surface fu...

  13. Colloidal Stability & Conformational Changes in β-Lactoglobulin: Unfolding to Self-Assembly

    Directory of Open Access Journals (Sweden)

    Steven Blake

    2015-08-01

    Full Text Available A detailed understanding of the mechanism of unfolding, aggregation, and associated rheological changes is developed in this study for β-Lactoglobulin at different pH values through concomitant measurements utilizing dynamic light scattering (DLS, optical microrheology, Raman spectroscopy, and differential scanning calorimetry (DSC. The diffusion interaction parameter kD emerges as an accurate predictor of colloidal stability for this protein consistent with observed aggregation trends and rheology. Drastic aggregation and gelation were observed at pH 5.5. Under this condition, the protein’s secondary and tertiary structures changed simultaneously. At higher pH (7.0 and 8.5, oligomerizaton with no gel formation occurred. For these solutions, tertiary structure and secondary structure transitions were sequential. The low frequency Raman data, which is a good indicator of hydrogen bonding and structuring in water, has been shown to exhibit a strong correlation with the rheological evolution with temperature. This study has, for the first time, demonstrated that this low frequency Raman data, in conjunction with the DSC endotherm, can be been utilized to deconvolve protein unfolding and aggregation/gelation. These findings can have important implications for the development of protein-based biotherapeutics, where the formulation viscosity, aggregation, and stability strongly affects efficacy or in foods where protein structuring is critical for functional and sensory performance.

  14. A thin and conformal metasurface for illusion acoustics of rapidly changing profiles

    Science.gov (United States)

    Dubois, Marc; Shi, Chengzhi; Wang, Yuan; Zhang, Xiang

    2017-04-01

    Recently developed metasurfaces have been used for surface engineering applications. However, the ability to cloak or mimic reflective surfaces with a large in-plane phase gradient remains unexplored. One major challenge is that even with a small incidence angle, the strong acoustic impedance variation induced by the random height profile creates additional scattering and increases the complexity of the analysis and design of the metasurface. Here, we introduce an acoustic metasurface with 1/12 wavelength thickness to realize an acoustic carpet cloak for a randomly rapid-change surface and a virtual acoustic diffuser from a flat surface using a set of Helmholtz resonators. The limitation of the metasurface for large phase gradient application is explored based on a nonlocal model that considers the contributions from neighboring surface profiles. This study extends the integration of smart acoustic surface and may find applications of surface engineering such as in architectural acoustics.

  15. Radiation induced chromatin conformation changes analysed by fluorescent localization microscopy, statistical physics, and graph theory.

    Science.gov (United States)

    Zhang, Yang; Máté, Gabriell; Müller, Patrick; Hillebrandt, Sabina; Krufczik, Matthias; Bach, Margund; Kaufmann, Rainer; Hausmann, Michael; Heermann, Dieter W

    2015-01-01

    It has been well established that the architecture of chromatin in cell nuclei is not random but functionally correlated. Chromatin damage caused by ionizing radiation raises complex repair machineries. This is accompanied by local chromatin rearrangements and structural changes which may for instance improve the accessibility of damaged sites for repair protein complexes. Using stably transfected HeLa cells expressing either green fluorescent protein (GFP) labelled histone H2B or yellow fluorescent protein (YFP) labelled histone H2A, we investigated the positioning of individual histone proteins in cell nuclei by means of high resolution localization microscopy (Spectral Position Determination Microscopy = SPDM). The cells were exposed to ionizing radiation of different doses and aliquots were fixed after different repair times for SPDM imaging. In addition to the repair dependent histone protein pattern, the positioning of antibodies specific for heterochromatin and euchromatin was separately recorded by SPDM. The present paper aims to provide a quantitative description of structural changes of chromatin after irradiation and during repair. It introduces a novel approach to analyse SPDM images by means of statistical physics and graph theory. The method is based on the calculation of the radial distribution functions as well as edge length distributions for graphs defined by a triangulation of the marker positions. The obtained results show that through the cell nucleus the different chromatin re-arrangements as detected by the fluorescent nucleosomal pattern average themselves. In contrast heterochromatic regions alone indicate a relaxation after radiation exposure and re-condensation during repair whereas euchromatin seemed to be unaffected or behave contrarily. SPDM in combination with the analysis techniques applied allows the systematic elucidation of chromatin re-arrangements after irradiation and during repair, if selected sub-regions of nuclei are

  16. Allosteric communication in myosin V: from small conformational changes to large directed movements.

    Directory of Open Access Journals (Sweden)

    M Cecchini

    Full Text Available The rigor to post-rigor transition in myosin, a consequence of ATP binding, plays an essential role in the Lymn-Taylor functional cycle because it results in the dissociation of the actomyosin complex after the powerstroke. On the basis of the X-ray structures of myosin V, we have developed a new normal mode superposition model for the transition path between the two states. Rigid-body motions of the various subdomains and specific residues at the subdomain interfaces are key elements in the transition. The allosteric communication between the nucleotide binding site and the U50/L50 cleft is shown to result from local changes due to ATP binding, which induce large amplitude motions that are encoded in the structure of the protein. The triggering event is the change in the interaction of switch I and the P-loop, which is stabilized by ATP binding. The motion of switch I, which is a relatively rigid element of the U50 subdomain, leads directly to a partial opening of the U50/L50 cleft; the latter is expected to weaken the binding of myosin to actin. The calculated transition path demonstrates the nature of the subdomain coupling and offers an explanation for the mutual exclusion of ATP and actin binding. The mechanism of the uncoupling of the converter from the motor head, an essential part of the transition, is elucidated. The origin of the partial untwisting of the central beta-sheet in the rigor to post-rigor transition is described.

  17. Quantifying Allosteric Communication via Both Concerted Structural Changes and Conformational Disorder with CARDS.

    Science.gov (United States)

    Singh, Sukrit; Bowman, Gregory R

    2017-04-11

    Allosteric (i.e., long-range) communication within proteins is crucial for many biological processes, such as the activation of signaling cascades in response to specific stimuli. However, the physical basis for this communication remains unclear. Existing computational methods for identifying allostery focus on the role of concerted structural changes, but recent experimental work demonstrates that disorder is also an important factor. Here, we introduce the Correlation of All Rotameric and Dynamical States (CARDS) framework for quantifying correlations between both the structure and disorder of different regions of a protein. To quantify disorder, we draw inspiration from methods for quantifying "dynamic heterogeneity" from chemical physics to classify segments of a dihedral's time evolution as being in either ordered or disordered regimes. To demonstrate the utility of this approach, we apply CARDS to the Catabolite Activator Protein (CAP), a transcriptional activator that is regulated by Cyclic Adenosine MonoPhosphate (cAMP) binding. We find that CARDS captures allosteric communication between the two cAMP-Binding Domains (CBDs). Importantly, CARDS reveals that this coupling is dominated by disorder-mediated correlations, consistent with NMR experiments that establish allosteric coupling between the CBDs occurs without a concerted structural change. CARDS also recapitulates an enhanced role for disorder in the communication between the DNA-Binding Domains (DBDs) and CBDs in the S62F variant of CAP. Finally, we demonstrate that using CARDS to find communication hotspots identifies regions of CAP that are in allosteric communication without foreknowledge of their identities. Therefore, we expect CARDS to be of great utility for both understanding and predicting allostery.

  18. Combined probes of X-ray scattering and optical spectroscopy reveal how global conformational change is temporally and spatially linked to local structural perturbation in photoactive yellow protein.

    Science.gov (United States)

    Kim, Tae Wu; Yang, Cheolhee; Kim, Youngmin; Kim, Jong Goo; Kim, Jeongho; Jung, Yang Ouk; Jun, Sunhong; Lee, Sang Jin; Park, Sungjun; Kosheleva, Irina; Henning, Robert; van Thor, Jasper J; Ihee, Hyotcherl

    2016-04-07

    Real-time probing of structural transitions of a photoactive protein is challenging owing to the lack of a universal time-resolved technique that can probe the changes in both global conformation and light-absorbing chromophores of the protein. In this work, we combine time-resolved X-ray solution scattering (TRXSS) and transient absorption (TA) spectroscopy to investigate how the global conformational changes involved in the photoinduced signal transduction of photoactive yellow protein (PYP) is temporally and spatially related to the local structural change around the light-absorbing chromophore. In particular, we examine the role of internal proton transfer in developing a signaling state of PYP by employing its E46Q mutant (E46Q-PYP), where the internal proton transfer is inhibited by the replacement of a proton donor. The comparison of TRXSS and TA spectroscopy data directly reveals that the global conformational change of the protein, which is probed by TRXSS, is temporally delayed by tens of microseconds from the local structural change of the chromophore, which is probed by TA spectroscopy. The molecular shape of the signaling state reconstructed from the TRXSS curves directly visualizes the three-dimensional conformations of protein intermediates and reveals that the smaller structural change in E46Q-PYP than in wild-type PYP suggested by previous studies is manifested in terms of much smaller protrusion, confirming that the signaling state of E46Q-PYP is only partially developed compared with that of wild-type PYP. This finding provides direct evidence of how the environmental change in the vicinity of the chromophore alters the conformational change of the entire protein matrix.

  19. Protein-peptide molecular docking with large-scale conformational changes: the p53-MDM2 interaction

    Science.gov (United States)

    Ciemny, Maciej Pawel; Debinski, Aleksander; Paczkowska, Marta; Kolinski, Andrzej; Kurcinski, Mateusz; Kmiecik, Sebastian

    2016-12-01

    Protein-peptide interactions are often associated with large-scale conformational changes that are difficult to study either by classical molecular modeling or by experiment. Recently, we have developed the CABS-dock method for flexible protein-peptide docking that enables large-scale rearrangements of the protein chain. In this study, we use CABS-dock to investigate the binding of the p53-MDM2 complex, an element of the cell cycle regulation system crucial for anti-cancer drug design. Experimental data suggest that p53-MDM2 binding is affected by significant rearrangements of a lid region - the N-terminal highly flexible MDM2 fragment; however, the details are not clear. The large size of the highly flexible MDM2 fragments makes p53-MDM2 intractable for exhaustive binding dynamics studies using atomistic models. We performed extensive dynamics simulations using the CABS-dock method, including large-scale structural rearrangements of MDM2 flexible regions. Without a priori knowledge of the p53 peptide structure or its binding site, we obtained near-native models of the p53-MDM2 complex. The simulation results match well the experimental data and provide new insights into the possible role of the lid fragment in p53 binding. The presented case study demonstrates that CABS-dock methodology opens up new opportunities for protein-peptide docking with large-scale changes of the protein receptor structure.

  20. Temperature-Induced Protein Conformational Changes in Barley Root Plasma Membrane-Enriched Microsomes: II. Intrinsic Protein Fluorescence.

    Science.gov (United States)

    Caldwell, C R

    1987-07-01

    The membrane-bound proteins of barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes displayed fluorescence typical of protein-associated trytophan residues. The protein fluorescence intensity was sensitive to variations in sample temperature. The temperature-induced decline in protein fluorescence intensity was nonlinear with slope discontinuities at about 12 and 32 degrees C. Detergents at levels above their critical micelle concentration enhanced protein fluorescence. Glutaraldehyde reduced protein fluorescence. Protein fluorescence polarization increased at temperatures above 30 degrees C. Both the rate of tryptophan photoionization and the fluorescence intensity of the photoionization products suggested alterations in membrane protein conformation between 12 and 32 degrees C. The quenching of the intrinsic protein fluorescence by acrylamide and potassium iodide indicated changes in accessibility of the extrinsic agents to the protein tryptophan residues beginning at about 14 degrees C. The results indicate thermally induced changes in the dynamics of the membrane proteins over the temperature range of 12 to 32 degrees C which could account for the complex temperature dependence of the barley root plasma membrane ATPase.

  1. Redox- and pH-linked conformational changes in triheme cytochrome PpcA from Geobacter sulfurreducens.

    Science.gov (United States)

    Morgado, Leonor; Bruix, Marta; Pokkuluri, P Raj; Salgueiro, Carlos A; Turner, David L

    2017-01-15

    The periplasmic triheme cytochrome PpcA from Geobacter sulfurreducens is highly abundant; it is the likely reservoir of electrons to the outer surface to assist the reduction of extracellular terminal acceptors; these include insoluble metal oxides in natural habitats and electrode surfaces from which electricity can be harvested. A detailed thermodynamic characterization of PpcA showed that it has an important redox-Bohr effect that might implicate the protein in e(-)/H(+) coupling mechanisms to sustain cellular growth. This functional mechanism requires control of both the redox state and the protonation state. In the present study, isotope-labeled PpcA was produced and the three-dimensional structure of PpcA in the oxidized form was determined by NMR. This is the first solution structure of a G. sulfurreducens cytochrome in the oxidized state. The comparison of oxidized and reduced structures revealed that the heme I axial ligand geometry changed and there were other significant changes in the segments near heme I. The pH-linked conformational rearrangements observed in the vicinity of the redox-Bohr center, both in the oxidized and reduced structures, constitute the structural basis for the differences observed in the pKa values of the redox-Bohr center, providing insights into the e(-)/H(+) coupling molecular mechanisms driven by PpcA in G. sulfurreducens. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  2. DNA and IκBα both induce long range conformational changes in NFκB.

    Science.gov (United States)

    Ramsey, Kristen M; Dembinski, Holly E; Chen, Wei; Ricci, Clarisse G; Komives, Elizabeth A

    2017-02-26

    We recently discovered that IκBα enhances the rate of release of NFκB from DNA target sites in a process we have termed molecular stripping. Coarse-grained molecular dynamics simulations of the stripping pathway revealed two mechanisms for the enhanced release rate; the negatively charged PEST region of IκBα electrostatically repels the DNA, and binding of IκBα appears to twist the NFκB heterodimer so that DNA can no longer bind. Here we report amide hydrogen/deuterium exchange data that reveals long-range allosteric changes in the NFκB (RelA-p50) heterodimer induced by DNA or IκBα binding. The data suggest that the two immunoglobulin-like subdomains of each Rel-homology region, which are connected by a flexible linker in the heterodimer, communicate in such a way that when DNA binds to the N-terminal DNA binding domains, the nuclear localization signal becomes more highly exchanging. Conversely, when IκBα binds to the dimerization domains, amide exchange throughout the DNA binding domains is decreased as if the entire domain is becoming globally stabilized. The results help understand how the subtle mechanism of molecular stripping actually occurs.

  3. Structures of minimal catalytic fragments of topoisomerase V reveals conformational changes relevant for DNA binding.

    Science.gov (United States)

    Rajan, Rakhi; Taneja, Bhupesh; Mondragón, Alfonso

    2010-07-14

    Topoisomerase V is an archaeal type I topoisomerase that is unique among topoisomerases due to presence of both topoisomerase and DNA repair activities in the same protein. It is organized as an N-terminal topoisomerase domain followed by 24 tandem helix-hairpin-helix (HhH) motifs. Structural studies have shown that the active site is buried by the (HhH) motifs. Here we show that the N-terminal domain can relax DNA in the absence of any HhH motifs and that the HhH motifs are required for stable protein-DNA complex formation. Crystal structures of various topoisomerase V fragments show changes in the relative orientation of the domains mediated by a long bent linker helix, and these movements are essential for the DNA to enter the active site. Phosphate ions bound to the protein near the active site helped model DNA in the topoisomerase domain and show how topoisomerase V may interact with DNA.

  4. Multiple spectroscopic studies of the structural conformational changes of human serum albumin—Essential oil based nanoemulsions conjugates

    Energy Technology Data Exchange (ETDEWEB)

    Sekar, Gajalakshmi; Sugumar, Saranya; Mukherjee, Amitava; Chandrasekaran, Natarajan, E-mail: nchandra40@hotmail.com

    2015-05-15

    Nanoemulsions have numerous biomedical applications. For the first time, we have investigated the effects of orange and eucalyptus essential oil based nanoemulsions towards the structural aspect of human serum albumin (HSA). Quenching effect of nanoemulsion against the intrinsic fluorescence potential of tryptophan and tyrosine residues were evidenced from the fluorescence spectroscopic analysis. Static quenching mechanism was found to lead the binding of HSA–nanoemulsion systems. Synchronous and three dimensional spectroscopic studies have revealed the possible changes to the aromatic environment of HSA by the nanoemulsion. UV–Visible spectroscopic studies have confirmed the existence of the ground state complex formation between HSA and the surface of nanoemulsions by exhibiting the hyper-chromic effect in a concentration dependant manner. FTIR spectroscopy revealed the slight alteration in the Amide I, II and III bands of HSA after interaction. FT-Raman spectroscopy showed the decrease in the Raman intensity of the aromatic amino acid residues and shift in the amide bands of HSA upon binding with the nanoemulsion. Dichoric band obtained from the far UV-CD spectra at 208 and 222 nm of HSA showed the corresponding decrease in the alpha-helical contents upon interaction with nanoemulsions. Near UV-CD spectra also showed the prominent changes in the aromatic positions of the amino acid residues of HSA on binding with nanoemulsions. The above study has extrapolated the side effect analysis of the nanoemulsions in pharmaceutical applications in vitro in reference to their interaction with serum proteins. - Highlights: • Orange and eucalyptus oil based nanoemulsions were formulated and characterized. • UV–Visible spectroscopy confirmed the ground state complex formation. • Fluorescence spectroscopy confirmed the molecular conformational changes. • FTIR spectroscopy deep-rooted the alteration in the amide bands of HSA. • FT-Raman spectroscopy established

  5. TonB induces conformational changes in surface-exposed loops of FhuA, outer membrane receptor of Escherichia coli.

    Science.gov (United States)

    James, Karron J; Hancock, Mark A; Moreau, Violaine; Molina, Franck; Coulton, James W

    2008-10-01

    FhuA, outer membrane receptor of Escherichia coli, transports hydroxamate-type siderophores into the periplasm. Cytoplasmic membrane-anchored TonB transduces energy to FhuA to facilitate siderophore transport. Because the N-terminal cork domain of FhuA occludes the C-terminal beta-barrel lumen, conformational changes must occur to enable siderophore passage. To localize conformational changes at an early stage of the siderophore transport cycle, four anti-FhuA monoclonal antibodies (mAbs) were purified to homogeneity, and the epitopes that they recognize were determined by phage display. We mapped continuous and discontinuous epitopes to outer surface-exposed loops 3, 4, and 5 and to beta-barrel strand 14. To probe for conformational changes of FhuA, surface plasmon resonance measured mAb binding to FhuA in its apo- and siderophore-bound states. Changes in binding kinetics were observed for mAbs whose epitopes were mapped to outer surface-exposed loops. Further, we measured mAb binding in the absence and presence of TonB. After forming immobilized FhuA-TonB complexes, changes in kinetics of mAb binding to FhuA were even more pronounced compared with kinetics of binding in the absence of TonB. Measurement of extrinsic fluorescence of the dye MDCC conjugated to residue 336 in outer surface-exposed loop 4 revealed 33% fluorescence quenching upon ferricrocin binding and up to 56% quenching upon TonB binding. Binding of mAbs to apo- and ferricrocin-bound FhuA complemented by fluorescence spectroscopy studies showed that their cognate epitopes on loops 3, 4, and 5 undergo conformational changes upon siderophore binding. Further, our data demonstrate that TonB binding promotes conformational changes in outer surface-exposed loops of FhuA.

  6. Transportation Conformity

    Science.gov (United States)

    This section provides information on: current laws, regulations and guidance, policy and technical guidance, project-level conformity, general information, contacts and training, adequacy review of SIP submissions

  7. Probing structural differences between PrPC and PrPSc by surface nitration and acetylation: evidence of conformational change in the C-terminus

    Science.gov (United States)

    We used two chemical modifiers, tetranitromethane (TNM) and acetic anhydride, which specifically target accessible tyrosine and lysine residues, respectively, to modify Syrian hamster recombinant PrP(90-231) (rPrP) and PrP27-30, aiming at finding locations of conformational change. Modified proteins...

  8. Correlation of Conformational Changes and Protein Degradation with Loss of Lysozyme Activity Due to Chlorine Dioxide Treatment.

    Science.gov (United States)

    Ooi, Beng Guat; Branning, Sharon Alyssa

    2016-12-13

    Chlorine dioxide (ClO2) is a potent oxidizing agent used for the treatment of drinking water and decontamination of facilities and equipment. The purpose of this research is to elucidate the manner in which ClO2 destroys proteins by studying the effects of ClO2 on lysozyme. The degree of enzyme activity lost can be correlated to the treatment time and levels of the ClO2 used. Lysozyme activity was drastically reduced to 45.3% of original enzyme activity when exposed to 4.3 mM ClO2 in the sample after 3 h. Almost all activities were lost in 3 h after exposure to higher ClO2 concentrations of up to 16.8 and 21.9 mM. Changes in protein conformation and amount as a result of ClO2 treatment were determined using the Raman spectroscopy and gel electrophoresis. Raman shifts and the alteration of spectral features observed in the ClO2-treated lysozyme samples are associated with loss of the α-helix secondary structure, tertiary structure, and disulfide bond. Progressive degradation of the denatured lysozyme by increasing levels of chlorine dioxide was also observed in gel electrophoresis. Hence, ClO2 can effectively cause protein denaturation and degradation resulting in loss of enzyme activity.

  9. Building-block architecture of botulinum toxin complex: Conformational changes provide insights into the hemagglutination ability of the complex

    Directory of Open Access Journals (Sweden)

    Tomonori Suzuki

    2017-03-01

    Full Text Available Clostridium botulinum produces the botulinum neurotoxin (BoNT. Previously, we provided evidence for the “building-block” model of botulinum toxin complex (TC. In this model, a single BoNT is associated with a single nontoxic nonhemagglutinin (NTNHA, yielding M-TC; three HA-70 molecules are attached and form M-TC/HA-70, and one to three “arms” of the HA-33/HA-17 trimer (two HA-33 and one HA-17 further bind to M-TC/HA-70 via HA-17 and HA-70 binding, yielding one-, two-, and three-arm L-TC. Of all TCs, only the three-arm L-TC caused hemagglutination. In this study, we determined the solution structures for the botulinum TCs using small-angle X-ray scattering (SAXS. The mature three-arm L-TC exhibited the shape of a “bird spreading its wings”, in contrast to the model having three “arms”, as revealed by transmission electron microscopy. SAXS images indicated that one of the three arms of the HA-33/HA-17 trimer bound to both HA-70 and BoNT. Taken together, these findings regarding the conformational changes in the building-block architecture of TC may explain why only three-arm L-TC exhibited hemagglutination.

  10. A Bio-computing Analysis of the Resting-to-pulsed Conformational Changes in Cytochrome c Oxidase

    Directory of Open Access Journals (Sweden)

    T Alleyne

    2015-09-01

    Full Text Available Cytochrome c oxidase (Cox accepts electrons from its substrate, cytochrome c and passes these to oxygen, which is reduced to water. Kinetic studies show that an active form of the enzyme (pulsed and a slower form (resting exists. More efficient internal electron transfer and the switching of the enzyme’s oxygen/ligand binding site between opened and closed positions are said to account for the different rates of reduction. We employed bio-computing to analyse the structure of the oxygen/ligand binding site of bovine Cox under different redox states; a comparison with Thermus thermophilus Cox was also conducted. The study detected that the ligand binding site of Cox is exposed to the contents of the intermembrane space, and that the side chain of haem a3, located at the enzyme’s oxygen/ligand binding site, approached Pro-69 and Ile-34 in faraway subunit-II. However, no open-to-closed gating structures were detected at the ligand binding site. We concluded that the resting-to-pulse transition in Cox does not involve opening-up of the ligand binding site. We propose that the rates of ligand/oxygen/cyanide binding are partly controlled by “queuing” near the binding site and that the binding of oxygen to haem a3-CuB triggers the resting-to-pulsed transition via long-range conformational changes.

  11. Methylglyoxal mediated conformational changes in histone H2A-generation of carboxyethylated advanced glycation end products.

    Science.gov (United States)

    Mir, Abdul Rouf; uddin, Moin; Alam, Khursheed; Ali, Asif

    2014-08-01

    Methylglyoxal, an oxo-aldehyde has been implicated as a potential precursor in non enzymatic glycation reactions. Its role in the modification of extra cellular proteins has been extensively reported, but little is known about its modification of nuclear proteins, like histones. Here, we report the methylglyoxal induced modification of histone H2A which forms an essential part of intact core nucleosome. In this study commercially available histone H2A was subjected to in vitro non-enzymatic glycation by methylglyoxal. The structural alterations in the histone were characterised by various biophysical and biochemical techniques. The modified histone showed hyperchromicity at 276nm, loss in intrinsic tyrosine fluorescence intensity at 305nm along with a red shift, cross linking and dimer formation in SDS PAGE, induction of α-helix in CD spectroscopy, reduced hydrophobicity in ANS binding studies, accumulation of AGE products, increased carbonyl content, and appearance of a novel peak showing carboxyethylation complemented by a shift in amide I and amide II bands in ATR-FTIR spectroscopy. The modified histone exhibited increased melting temperatures (Tm) and enhanced heat capacities (Cp) in differential scanning calorimetric analysis. The results suggest that methylglyoxal significantly altered the structure of the nuclear histone H2A by non enzymatic glycation reaction. The conformational changes in histone H2A may influence the chromatin integrity which may have implications in various pathological conditions.

  12. Effect of Drying Methods on Protein and DNA Conformation Changes in Lactobacillus rhamnosus GG Cells by Fourier Transform Infrared Spectroscopy.

    Science.gov (United States)

    Hlaing, Mya M; Wood, Bayden R; McNaughton, Don; Ying, DanYang; Dumsday, Geoff; Augustin, Mary Ann

    2017-03-01

    Microencapsulation protects cells against environmental stress encountered during the production of probiotics, which are used as live microbial food ingredients. Freeze-drying and spray-drying are used in the preparation of powdered microencapsulated probiotics. This study examines the ability of Fourier transform infrared (FTIR) spectroscopy to detect differences in cells exposed to freeze-drying and spray-drying of encapsulated Lactobacillus rhamnosus GG cells. The FTIR analysis clearly demonstrated there were more significant molecular changes in lipid, fatty acid content, protein, and DNA conformation of nonencapsulated compared to encapsulated bacterial cells. The technique was also able to differentiate between spray-dried and freeze-dried cells. The results also revealed the extent of protection from a protein-carbohydrate-based encapsulant matrix on the cells depending on the type drying process. The extent of this protection to the dehydration stress was shown to be less in spray-dried cells than in freeze-dried cells. This suggests that FTIR could be used as a rapid, noninvasive, and real-time measurement technique to detect detrimental drying effects on cells.

  13. Allosteric regulation by cooperative conformational changes of actin filaments drives mutually exclusive binding with cofilin and myosin.

    Science.gov (United States)

    Ngo, Kien Xuan; Umeki, Nobuhisa; Kijima, Saku T; Kodera, Noriyuki; Ueno, Hiroaki; Furutani-Umezu, Nozomi; Nakajima, Jun; Noguchi, Taro Q P; Nagasaki, Akira; Tokuraku, Kiyotaka; Uyeda, Taro Q P

    2016-10-20

    Heavy meromyosin (HMM) of myosin II and cofilin each binds to actin filaments cooperatively and forms clusters along the filaments, but it is unknown whether the two cooperative bindings are correlated and what physiological roles they have. Fluorescence microscopy demonstrated that HMM-GFP and cofilin-mCherry each bound cooperatively to different parts of actin filaments when they were added simultaneously in 0.2 μM ATP, indicating that the two cooperative bindings are mutually exclusive. In 0.1 mM ATP, the motor domain of myosin (S1) strongly inhibited the formation of cofilin clusters along actin filaments. Under this condition, most actin protomers were unoccupied by S1 at any given moment, suggesting that transiently bound S1 alters the structure of actin filaments cooperatively and/or persistently to inhibit cofilin binding. Consistently, cosedimentation experiments using copolymers of actin and actin-S1 fusion protein demonstrated that the fusion protein affects the neighboring actin protomers, reducing their affinity for cofilin. In reciprocal experiments, cofilin-actin fusion protein reduced the affinity of neighboring actin protomers for S1. Thus, allosteric regulation by cooperative conformational changes of actin filaments contributes to mutually exclusive cooperative binding of myosin II and cofilin to actin filaments, and presumably to the differential localization of both proteins in cells.

  14. Connecting global change science with communities: About the conformation of a social network for early warnings in Colombia

    Science.gov (United States)

    Arias, P. A.; Vidal, L. M.; Serna, A. M.; Vieira, C.; Machado, J.; Cadavid, C. A.

    2014-12-01

    The risk associated with natural and social phenomena has notably increased in modern societies. On the other hand, socio-natural hazards have increased and diversified, in association with economic development. During 2010 and 2011, Colombia faced one of the most severe wet seasons in decades. One of the most significant impacts of this flood emergency was the demonstration of poor preparedness of communities, local authorities, and regional and national authorities to confront situations of large coverage. The emergencies occurred during 2010 and 2011, induced in association with a strong La Niña event, immediately demanded environmental and risk management authorities to provide communities with basic tools to understand the dynamics associated with excesses of rainfall and mitigate the possible impacts in their populations. For this reason, the Regional Autonomous Corporation of Central Antioquia, Colombia (CORANTIOQUIA) funded a project aimed to the design and conformation of a social network for early warnings of events associated to floods, torrential floods, and mass movements in 80 municipalities of the department of Antioquia, Colombia. For the execution of this project, the Corporation invited the Faculty of Engineering of the University of Antioquia. This talk aims to socialize this inititative that looked for integrating scientific and technical knowledge with popular knowledge in order to provide Latin American communities with tools to mitigate the possible impacts of global change.

  15. Conformational change of glutathione-S-transferase by its co-expression with prion domain of yeast Ure2p

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The Ure2 protein from Saccharomyces cerevisisae has a changeable structure similar to that ofrnammalian prion protein. Its N-terminal is the prion domain (PrD) consisting of 65 amino acids which plays a critical role in yeast prion development. In this study, PrD gene was recombinated with glutathione-S-transferase(GST) gene, and a soluble GST-PrD(sGST-PrD) fusion protein was expressed in E. coli. sGST-PrD could spontaneously polymerize into amyloid fibrils in vitro, displaying typical β-sheet-type structure; it had increased resistance to proteinase K and exhibited amvloid-like optical properties. Moreover, the aggregated GST-PrD(aGST-PrD) could induce sGST-PrD to aggregate into fibrils. These results indicate that PrD could change the conformation of GST moiety in a recombinant protein with PrD to form a prion-like chimeric protein, which proves that PrD has the ability to mediate a prion-like conversion of other proteins fused with it.

  16. Sensitivity of photoelectron diffraction to conformational changes of adsorbed molecules: Tetra-tert-butyl-azobenzene/Au(111

    Directory of Open Access Journals (Sweden)

    A. Schuler

    2017-01-01

    Full Text Available Electron diffraction is a standard tool to investigate the atomic structure of surfaces, interfaces, and adsorbate systems. In particular, photoelectron diffraction is a promising candidate for real-time studies of structural dynamics combining the ultimate time resolution of optical pulses and the high scattering cross-sections for electrons. In view of future time-resolved experiments from molecular layers, we studied the sensitivity of photoelectron diffraction to conformational changes of only a small fraction of molecules in a monolayer adsorbed on a metallic substrate. 3,3′,5,5′-tetra-tert-butyl-azobenzene served as test case. This molecule can be switched between two isomers, trans and cis, by absorption of ultraviolet light. X-ray photoelectron diffraction patterns were recorded from tetra-tert-butyl-azobenzene/Au(111 in thermal equilibrium at room temperature and compared to patterns taken in the photostationary state obtained by exposing the surface to radiation from a high-intensity helium discharge lamp. Difference patterns were simulated by means of multiple-scattering calculations, which allowed us to determine the fraction of molecules that underwent isomerization.

  17. Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.

    Science.gov (United States)

    Nakamichi, Yusuke; Oiki, Sayoko; Mikami, Bunzo; Murata, Kousaku; Hashimoto, Wataru

    2016-08-01

    Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.

  18. At odds with the group: changes in lateralization and escape performance reveal conformity and conflict in fish schools.

    Science.gov (United States)

    Chivers, Douglas P; McCormick, Mark I; Allan, Bridie J M; Mitchell, Matthew D; Gonçalves, Emanuel J; Bryshun, Reid; Ferrari, Maud C O

    2016-10-26

    Many vertebrates are known to show behavioural lateralization, whereby they differentially use one side of their body or either of their bilateral organs or limbs. Behavioural lateralization often manifests in a turning bias in fishes, with some individuals showing a left bias and others a right bias. Such biases could be the source of considerable conflict in fish schools given that there may be considerable social pressure to conform to the group to maintain effective group evasion. Here, we show that predation pressure is a major determinant of the degree of lateralization, both in a relative and absolute sense, in yellow-and-blueback fusiliers (Caesio teres), a schooling fish common on coral reefs. Wild-caught fish showed a bias for right turning. When predation pressure was experimentally elevated or relaxed, the strength of lateralization changed. Higher predation pressure resulted in an increase in the strength of lateralization. Individuals that exhibited the same turning bias as the majority of individuals in their group had improved escape performance compared with individuals that were at odds with the group. Moreover, individuals that were right-biased had improved escape performance, compared with left-biased ones. Plasticity in lateralization might be an important evolutionary consequence of the way gregarious species respond to predators owing to the probable costs associated with this behaviour.

  19. Mechanism of Substrate Recognition And PLP-Induced Conformational Changes in II-Diaminopimelate Aminotransferase From Arabidopsis Thaliana

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, N.; Clay, M.D.; Belkum, M.J.van; Cherney, M.M.; Vederas, J.C.; James, M.N.G.

    2009-05-26

    . Lastly, an apo-AtDAP-AT structure missing PLP revealed details of conformational changes induced by PLP binding and substrate entry into the active site.

  20. A molecular dynamics study of the BACE1 conformational change from Apo to closed form induced by hydroxyethylamine derived compounds.

    Science.gov (United States)

    Gueto-Tettay, Carlos; Zuchniarz, Joshua; Fortich-Seca, Yeyson; Gueto-Tettay, Luis Roberto; Drosos-Ramirez, Juan Carlos

    2016-11-01

    BACE1 is an aspartyl protease which is a therapeutic target for Alzheimer's disease (AD) because of its participation in the rate-limiting step in the production of Aβ-peptide, the accumulation of which produces senile plaques and, in turn, the neurodegenerative effects associated with AD. The active site of this protease is composed in part by two aspartic residues (Asp93 and Asp289). Additionally, the catalytic site has been found to be covered by an antiparallel hairpin loop called the flap. The dynamics of this flap are fundamental to the catalytic function of the enzyme. When BACE1 is inactive (Apo), the flap adopts an open conformation, allowing a substrate or inhibitor to access the active site. Subsequent interaction with the ligand induces flap closure and the stabilization of the macromolecular complex. Further, the protonation state of the aspartic dyad is affected by the chemical nature of the species entering the active site, so that appropriate selection of protonation states for the ligand and the catalytic residues will permit the elucidation of the inhibitory pathway for BACE1. In the present study, comparative analysis of different combinations of protonation states for the BACE1-hydroxyethylamine (HEA) system is reported. HEAs are potent inhibitors of BACE1 with favorable pharmacological and kinetic properties, as well as oral bioavailability. The results of Molecular Dynamics (MD) simulations and population density calculations using 8 different parameters demonstrate that the LnAsp289 configuration (HEA with a neutral amine and the Asp289 residue protonated) is the only one which permits the expected conformational change in BACE1, from apo to closed form, after flap closure. Additionally, differences in their capacities to establish and maintain interactions with residues such as Asp93, Gly95, Thr133, Asp289, Gly291, and Asn294 during this step allow differentiation among the inhibitory activities of the HEAs. The results and methodology here

  1. Design of a nanocarrier with regulated drug release ability utilizing a reversible conformational transition of a peptide, responsive to slight changes in pH.

    Science.gov (United States)

    Murai, Kazuki; Higuchi, Masahiro; Kinoshita, Takatoshi; Nagata, Kenji; Kato, Katsuya

    2013-07-21

    We investigated the drug releasing behavior of a novel nanocarrier system, utilizing a peptide to act as a nanogate to the mesopore, on a mesoporous silica nanoparticle. The surface peptide on mesoporous silica displayed pH-dependant mesopore cap-uncap switching behavior, enabled by the reversible β-sheet-to-random coil conformational transition resulting from slight pH changes between 8.0 and 6.0. The peptide adopted a β-sheet structure under weakly basic conditions (pH 8.0) and a random coil conformation under weakly acidic conditions (pH 6.0). We demonstrated the pH-dependant regulation of the material's drug release property by the reversible conformational transition of the surface peptide. Under basic pH conditions, the drug release from the nanocarrier was significantly inhibited. However, under acidic pH conditions, the drug in the mesopore was gradually released.

  2. Conformational changes at the highly reactive cystein and lysine regions of skeletal muscle myosin induced by formation of transition state analogues.

    Science.gov (United States)

    Maruta, S; Homma, K; Ohki, T

    1998-09-01

    Myosin forms stable ternary complexes with Mg2+-ADP and phosphate analogues of aluminum fluoride (AlF4-), beryllium fluoride (BeFn), and scandium fluoride (ScFn). These complexes are distinct from each other and may mimic different transient states in the ATPase cycle [Maruta et al. (1993) J. Biol. Chem. 268, 7093-7100]. Regions of skeletal muscle myosin containing the highly reactive residues Cys 707 (SH1), Cys 697 (SH2), and lysine 83 (RLR) dramatically alter their local conformation when myosin hydrolyzes ATP, and these changes may reflect formation of a series of transient intermediates during ATP hydrolysis. We used the fluorescent probes 4-fluoro-7-sulfamoylbezofurazan, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid, and trinitrobenzene-sulfonate, which bind to SH1, SH2, and RLR, respectively, to examine differences in local conformations within myosin.ADP.phosphate analogue (BeFn, Vi, AlF4-, and ScFn) complexes. It was observed that the ternary complexes had SH1 conformations similar to those seen on S-1 in the presence of ATP. In contrast, local conformations in the SH2 and RLR regions of S-1.ADP.BeFn were different from those in corresponding regions of S-1.ADP.AlF4- or ScFn. These results suggest that SH1 and SH2 move distinctly during ATP hydrolysis and that the local conformations of the SH2 and RLR regions more sensitively reflect different transient states.

  3. Workers’ Conformism

    Directory of Open Access Journals (Sweden)

    Nikolay Ivantchev

    2013-10-01

    Full Text Available Conformism was studied among 46 workers with different kinds of occupations by means of two modified scales measuring conformity by Santor, Messervey, and Kusumakar (2000 – scale for perceived peer pressure and scale for conformism in antisocial situations. The hypothesis of the study that workers’ conformism is expressed in a medium degree was confirmed partly. More than a half of the workers conform in a medium degree for taking risk, and for the use of alcohol and drugs, and for sexual relationships. More than a half of the respondents conform in a small degree for anti-social activities (like a theft. The workers were more inclined to conform for risk taking (10.9%, then – for the use of alcohol, drugs and for sexual relationships (8.7%, and in the lowest degree – for anti-social activities (6.5%. The workers who were inclined for the use of alcohol and drugs tended also to conform for anti-social activities.

  4. Conformational changes and slow dynamics through microsecond polarized atomistic molecular simulation of an integral Kv1.2 ion channel.

    Directory of Open Access Journals (Sweden)

    Pär Bjelkmar

    2009-02-01

    Full Text Available Structure and dynamics of voltage-gated ion channels, in particular the motion of the S4 helix, is a highly interesting and hotly debated topic in current membrane protein research. It has critical implications for insertion and stabilization of membrane proteins as well as for finding how transitions occur in membrane proteins-not to mention numerous applications in drug design. Here, we present a full 1 micros atomic-detail molecular dynamics simulation of an integral Kv1.2 ion channel, comprising 120,000 atoms. By applying 0.052 V/nm of hyperpolarization, we observe structural rearrangements, including up to 120 degrees rotation of the S4 segment, changes in hydrogen-bonding patterns, but only low amounts of translation. A smaller rotation ( approximately 35 degrees of the extracellular end of all S4 segments is present also in a reference 0.5 micros simulation without applied field, which indicates that the crystal structure might be slightly different from the natural state of the voltage sensor. The conformation change upon hyperpolarization is closely coupled to an increase in 3(10 helix contents in S4, starting from the intracellular side. This could support a model for transition from the crystal structure where the hyperpolarization destabilizes S4-lipid hydrogen bonds, which leads to the helix rotating to keep the arginine side chains away from the hydrophobic phase, and the driving force for final relaxation by downward translation is partly entropic, which would explain the slow process. The coordinates of the transmembrane part of the simulated channel actually stay closer to the recently determined higher-resolution Kv1.2 chimera channel than the starting structure for the entire second half of the simulation (0.5-1 micros. Together with lipids binding in matching positions and significant thinning of the membrane also observed in experiments, this provides additional support for the predictive power of microsecond-scale membrane

  5. Binding of lipoic acid induces conformational change and appearance of a new binding site in methylglyoxal modified serum albumin.

    Science.gov (United States)

    Suji, George; Khedkar, Santosh A; Singh, Sreelekha K; Kishore, Nand; Coutinho, Evans C; Bhor, Vikrant M; Sivakami, S

    2008-06-01

    The binding of lipoic acid (LA), to methylglyoxal (MG) modified BSA was studied using isothermal titration calorimetry in combination with enzyme kinetics and molecular modelling. The binding of LA to BSA was sequential with two sites, one with higher binding constant and another comparatively lower. In contrast the modified protein showed three sequential binding sites with a reduction in affinity at the high affinity binding site by a factor of 10. CD results show appreciable changes in conformation of the modified protein as a result of binding to LA. The inhibition of esterase like activity of BSA by LA revealed that it binds to site II in domain III of BSA. The pH dependence of esterase activity of native BSA indicated a catalytic group with a pK(a) = 7.9 +/- 0.1, assigned to Tyr411 with the conjugate base stabilised by interaction with Arg410. Upon modification by MG, this pK(a) increased to 8.13. A complex obtained by docking of LA to BSA and BSA in which Arg410 is modified to hydroimidazolone showed that the long hydrocarbon chain of lipoic acid sits in a cavity different from the one observed for unmodified BSA. The molecular electrostatic potential showed that the modification of Arg410 reduced the positive electrostatic potential around the protein-binding site. Thus it can be concluded that the modification of BSA by MG resulted in altered ligand binding characteristics due to changes in the internal geometry and electrostatic potential at the binding site.

  6. Extensive structural change of the envelope protein of dengue virus induced by a tuned ionic strength: conformational and energetic analyses

    Science.gov (United States)

    Degrève, Léo; Fuzo, Carlos A.; Caliri, Antonio

    2012-12-01

    The Dengue has become a global public health threat, with over 100 million infections annually; to date there is no specific vaccine or any antiviral drug. The structures of the envelope (E) proteins of the four known serotype of the dengue virus (DENV) are already known, but there are insufficient molecular details of their structural behavior in solution in the distinct environmental conditions in which the DENVs are submitted, from the digestive tract of the mosquito up to its replication inside the host cell. Such detailed knowledge becomes important because of the multifunctional character of the E protein: it mediates the early events in cell entry, via receptor endocytosis and, as a class II protein, participates determinately in the process of membrane fusion. The proposed infection mechanism asserts that once in the endosome, at low pH, the E homodimers dissociate and insert into the endosomal lipid membrane, after an extensive conformational change, mainly on the relative arrangement of its three domains. In this work we employ all-atom explicit solvent Molecular Dynamics simulations to specify the thermodynamic conditions in that the E proteins are induced to experience extensive structural changes, such as during the process of reducing pH. We study the structural behavior of the E protein monomer at acid pH solution of distinct ionic strength. Extensive simulations are carried out with all the histidine residues in its full protonated form at four distinct ionic strengths. The results are analyzed in detail from structural and energetic perspectives, and the virtual protein movements are described by means of the principal component analyses. As the main result, we found that at acid pH and physiological ionic strength, the E protein suffers a major structural change; for lower or higher ionic strengths, the crystal structure is essentially maintained along of all extensive simulations. On the other hand, at basic pH, when all histidine residues are in

  7. Evaluation of Heat Induced Methane Release from Methane Hydrates

    Science.gov (United States)

    Leeman, J.; Elwood-Madden, M.; Phelps, T. J.; Rawn, C. J.

    2010-12-01

    Clathrates, or gas hydrates, structurally are guest gas molecules populating a cavity in a cage of water molecules. Gas hydrates naturally occur on Earth under low temperature and moderate pressure environments including continental shelf, deep ocean, and permafrost sediments. Large quantities of methane are trapped in hydrates, providing significant near-surface reserves of carbon and energy. Thermodynamics predicts that hydrate deposits may be destabilized by reducing the pressure in the system or raising the temperature. However, the rate of methane release due to varying environmental conditions remains relatively unconstrained and complicated by natural feedback effects of clathrate dissociation. In this study, hydrate dissociation in sediment due to localized increases in temperature was monitored and observed at the mesoscale (>20L) in a laboratory environment. Experiments were conducted in the Seafloor Process Simulator (SPS) at Oak Ridge National Laboratory (ORNL) to simulate heat induced dissociation. The SPS, containing a column of Ottawa sand saturated with water containing 25mg/L Sno-Max to aid nucleation, was pressurized and cooled well into the hydrate stability field. A fiber optic distributed sensing system (DSS) was embedded at four depths in the sediment column. This allowed the temperature strain value (a proxy for temperature) of the system to be measured with high spatial resolution to monitor the clathrate formation/dissociation processes. A heat exchanger embedded in the sediment was heated using hot recirculated ethylene glycol and the temperature drop across the exchanger was measured. These experiments indicate a significant and sustained amount of heat is required to release methane gas from hydrate-bearing sediments. Heat was consumed by hydrate dissociated in a growing sphere around the heat exchanger until steady state was reached. At steady state all heat energy entering the system was consumed in maintaining the temperature profile

  8. Mildly Acidic pH Triggers an Irreversible Conformational Change in the Fusion Domain of Herpes Simplex Virus 1 Glycoprotein B and Inactivation of Viral Entry.

    Science.gov (United States)

    Weed, Darin J; Pritchard, Suzanne M; Gonzalez, Floricel; Aguilar, Hector C; Nicola, Anthony V

    2017-03-01

    Herpes simplex virus (HSV) entry into a subset of cells requires endocytosis and endosomal low pH. Preexposure of isolated virions to mildly acidic pH of 5 to 6 partially inactivates HSV infectivity in an irreversible manner. Acid inactivation is a hallmark of viruses that enter via low-pH pathways; this occurs by pretriggering conformational changes essential for fusion. The target and mechanism(s) of low-pH inactivation of HSV are unclear. Here, low-pH-treated HSV-1 was defective in fusion activity and yet retained normal levels of attachment to cell surface heparan sulfate and binding to nectin-1 receptor. Low-pH-triggered conformational changes in gB reported to date are reversible, despite irreversible low-pH inactivation. gB conformational changes and their reversibility were measured by antigenic analysis with a panel of monoclonal antibodies and by detecting changes in oligomeric conformation. Three-hour treatment of HSV-1 virions with pH 5 or multiple sequential treatments at pH 5 followed by neutral pH caused an irreversible >2.5 log infectivity reduction. While changes in several gB antigenic sites were reversible, alteration of the H126 epitope was irreversible. gB oligomeric conformational change remained reversible under all conditions tested. Altogether, our results reveal that oligomeric alterations and fusion domain changes represent distinct conformational changes in gB, and the latter correlates with irreversible low-pH inactivation of HSV. We propose that conformational change in the gB fusion domain is important for activation of membrane fusion during viral entry and that in the absence of a host target membrane, this change results in irreversible inactivation of virions.IMPORTANCE HSV-1 is an important pathogen with a high seroprevalence throughout the human population. HSV infects cells via multiple pathways, including a low-pH route into epithelial cells, the primary portal into the host. HSV is inactivated by low-pH preexposure, and gB, a

  9. Electrostatics effects on Ca(2+) binding and conformational changes in EF-hand domains: Functional implications for EF-hand proteins.

    Science.gov (United States)

    Ababou, Abdessamad; Zaleska, Mariola

    2015-12-01

    Mutations of Gln41 and Lys75 with nonpolar residues in the N-terminal domain of calmodulin (N-Cam) revealed the importance of solvation energetics in conformational change of Ca(2+) sensor EF-hand domains. While in general these domains have polar residues at these corresponding positions yet the extent of their conformational response to Ca(2+) binding and their Ca(2+) binding affinity can be different from N-Cam. Consequently, here we address the charge state of the polar residues at these positions. The results show that the charge state of these polar residues can affect substantially the conformational change and the Ca(2+) binding affinity of our N-Cam variants. Since all the variants kept their conformational activity in the presence of Ca(2+) suggests that the differences observed among them mainly originate from the difference in their molecular dynamics. Hence we propose that the molecular dynamics of Ca(2+) sensor EF-hand domains is a key factor in the multifunctional aspect of EF-hand proteins.

  10. Imaging of conformational changes of proteins with a new environment-sensitive fluorescent probe designed for site-specific labeling of recombinant proteins in live cells.

    Science.gov (United States)

    Nakanishi, J; Nakajima, T; Sato, M; Ozawa, T; Tohda, K; Umezawa, Y

    2001-07-01

    We demonstrate herein a new method for imaging conformational changes of proteins in live cells using a new synthetic environment-sensitive fluorescent probe, 9-amino-6,8-bis(1,3,2-dithioarsolan-2-yl)-5H-benzo[a]phenoxazin-5-one. This fluorescent probe can be attached to recombinant proteins containing four cysteine residues at the i, i + 1, i + 4, and i + 5 positions of an alpha-helix. The specific binding of the fluorescent probe to this 4Cys motif enables fluorescent labeling inside cells by its extracellular administration. The high sensitivity of the fluorophore to its environment enables monitoring of the conformational changes of the proteins in live cells as changes in its fluorescence intensity. The present method was applied to calmodulin (CaM), a Ca2+-binding protein that was well-known to expose hydrophobic domains, depending on the Ca2+ concentration. A recombinant CaM fused at its C-terminal with a helical peptide containing a 4Cys motif was labeled with the fluorescent probe inside live cells. The fluorescence intensity changed reversibly depending on the intracellular Ca2+ concentration, which reflected the conformational change of the recombinant CaM in the live cells.

  11. Changes in the conformation of 5S rRNA cause alterations in principal functions of the ribosomal nanomachine.

    Science.gov (United States)

    Kouvela, Ekaterini C; Gerbanas, George V; Xaplanteri, Maria A; Petropoulos, Alexandros D; Dinos, George P; Kalpaxis, Dimitrios L

    2007-01-01

    5S rRNA is an integral component of the large ribosomal subunit in virtually all living organisms. Polyamine binding to 5S rRNA was investigated by cross-linking of N1-azidobenzamidino (ABA)-spermine to naked 5S rRNA or 50S ribosomal subunits and whole ribosomes from Escherichia coli cells. ABA-spermine cross-linking sites were kinetically measured and their positions in 5S rRNA were localized by primer extension analysis. Helices III and V, and loops A, C, D and E in naked 5S rRNA were found to be preferred polyamine binding sites. When 50S ribosomal subunits or poly(U)-programmed 70S ribosomes bearing tRNA(Phe) at the E-site and AcPhe-tRNA at the P-site were targeted, the susceptibility of 5S rRNA to ABA-spermine was greatly reduced. Regardless of 5S rRNA assembly status, binding of spermine induced significant changes in the 5S rRNA conformation; loop A adopted an apparent 'loosening' of its structure, while loops C, D, E and helices III and V achieved a more compact folding. Poly(U)-programmed 70S ribosomes possessing 5S rRNA cross-linked with spermine were more efficient than control ribosomes in tRNA binding, peptidyl transferase activity and translocation. Our results support the notion that 5S rRNA serves as a signal transducer between regions of 23S rRNA responsible for principal ribosomal functions.

  12. Augmentation of protein-derived acetic acid production by heat-alkaline-induced changes in protein structure and conformation.

    Science.gov (United States)

    Wang, Xu; Li, Yanbo; Liu, Junxin; Ren, Nan-Qi; Qu, Jiuhui

    2016-01-01

    Waste-derived acetic acid (HAc) is an attractive feedstock for microbe-mediated biofuel production. However, fermentative conversion of HAc from waste-activated sludge (WAS) has low yield because of the high concentration of proteins not readily utilizable by microorganisms without prior hydrolysis. We investigated a combined technology for HAc augmentation during sludge protein fermentation. The maximal HAc yield increased over two-fold, reaching 0.502 ± 0.021 g/g protein (0.36 ± 0.01 g COD/g COD, ∼52% of the total volatile fatty acids) when synthetic sludge protein was heated at 120 °C for 30 min, treated at pH 12 for 24 h, and fermented at pH 9 for 72 h. Comprehensive analysis illustrated that the heat-alkaline pretreatment significantly induced protein fragmentation, simultaneously increasing the efficiency of protein biohydrolysis (from 35.5% to 85.9%) by inducing conformational changes indicative of protein unfolding. Consequently, the native α-helix content was decreased from 67.3% to 32.5% by conversion to an unordered shape, whose content increased from 27.5% to 45.5%; disulfide bonds were cleaved, whereas the main S-S stretching pattern was altered from gauche-gauche-gauche to gauche-gauche-trans, consequently causing increased protein susceptibility to proteolytic hydrolysis (76.3% vs. 47.0%). Economic analysis indicated that anaerobic fermentation with appropriate heat-alkaline pretreatment is a cost-effective approach for waste conversion to energy sources such as HAc.

  13. Conformal Infinity

    Directory of Open Access Journals (Sweden)

    Frauendiener Jörg

    2000-08-01

    Full Text Available The notion of conformal infinity has a long history within the research in Einstein's theory of gravity. Today, ``conformal infinity'' is related with almost all other branches of research in general relativity, from quantisation procedures to abstract mathematical issues to numerical applications. This review article attempts to show how this concept gradually and inevitably evolved out of physical issues, namely the need to understand gravitational radiation and isolated systems within the theory of gravitation and how it lends itself very naturally to solve radiation problems in numerical relativity. The fundamental concept of null-infinity is introduced. Friedrich's regular conformal field equations are presented and various initial value problems for them are discussed. Finally, it is shown that the conformal field equations provide a very powerful method within numerical relativity to study global problems such as gravitational wave propagation and detection.

  14. Conformal Infinity

    Science.gov (United States)

    Frauendiener, Jörg

    2004-12-01

    The notion of conformal infinity has a long history within the research in Einstein's theory of gravity. Today, "conformal infinity" is related to almost all other branches of research in general relativity, from quantisation procedures to abstract mathematical issues to numerical applications. This review article attempts to show how this concept gradually and inevitably evolved from physical issues, namely the need to understand gravitational radiation and isolated systems within the theory of gravitation, and how it lends itself very naturally to the solution of radiation problems in numerical relativity. The fundamental concept of null-infinity is introduced. Friedrich's regular conformal field equations are presented and various initial value problems for them are discussed. Finally, it is shown that the conformal field equations provide a very powerful method within numerical relativity to study global problems such as gravitational wave propagation and detection.

  15. General Conformity

    Science.gov (United States)

    The General Conformity requirements ensure that the actions taken by federal agencies in nonattainment and maintenance areas do not interfere with a state’s plans to meet national standards for air quality.

  16. Conformal Infinity

    Directory of Open Access Journals (Sweden)

    Frauendiener Jörg

    2004-01-01

    Full Text Available The notion of conformal infinity has a long history within the research in Einstein's theory of gravity. Today, 'conformal infinity' is related to almost all other branches of research in general relativity, from quantisation procedures to abstract mathematical issues to numerical applications. This review article attempts to show how this concept gradually and inevitably evolved from physical issues, namely the need to understand gravitational radiation and isolated systems within the theory of gravitation, and how it lends itself very naturally to the solution of radiation problems in numerical relativity. The fundamental concept of null-infinity is introduced. Friedrich's regular conformal field equations are presented and various initial value problems for them are discussed. Finally, it is shown that the conformal field equations provide a very powerful method within numerical relativity to study global problems such as gravitational wave propagation and detection.

  17. Enzymatic hydrolysis of heat-induced aggregates of whey protein isolate.

    Science.gov (United States)

    O'Loughlin, I B; Murray, B A; Kelly, P M; FitzGerald, R J; Brodkorb, A

    2012-05-16

    The effects of heat-induced denaturation and subsequent aggregation of whey protein isolate (WPI) solutions on the rate of enzymatic hydrolysis was investigated. Both heated (60 °C, 15 min; 65 °C, 5 and 15 min; 70 °C, 5 and 15 min, 75 °C, 5 and 15 min; 80 °C, 10 min) and unheated WPI solutions (100 g L(-1) protein) were incubated with a commercial proteolytic enzyme preparation, Corolase PP, until they reached a target degree of hydrolysis (DH) of 5%. WPI solutions on heating were characterized by large aggregate formation, higher viscosity, and surface hydrophobicity and hydrolyzed more rapidly (P whey proteins exhibited differences in their susceptibility to hydrolysis. Both viscosity and surface hydrophobicity along with insolubility declined as hydrolysis progressed. However, microstructural changes observed by light and confocal laser scanning microscopy (CLSM) provided insights to suggest that aggregate size and porosity may be complementary to denaturation in promoting faster enzymatic hydrolysis. This could be clearly observed in the course of aggregate disintegration, gel network breakdown, and improved solution clarification.

  18. Heat-induced destabilization of oil-in-water emulsions formed from hydrolyzed whey protein.

    Science.gov (United States)

    Euston, S R; Finnigan, S R; Hirst, R L

    2001-11-01

    The emulsifying ability, heat stability, and coalescence stability of oil-in-water emulsions prepared with whey protein of varied degrees of hydrolysis (DH), and at varied protein contents, was studied. Whey protein hydrolysates (WPH) with a DH of 4% and 10% had poorer emulsifying ability than non-hydrolyzed whey protein concentrate (WPC), but were more heat stable. Increasing DH between 10 and 27% improved emulsifying ability and further improved the heat stability of the emulsion droplets. Increasing DH from 27 to 35% led to a big decrease in both emulsifying ability and heat stability. The quiescent coalescence stability of WPH emulsions was relatively good up to a DH of 27%. Above DH 27% emulsions become highly unstable. It appears that two mechanisms of instability are at work here. At low DH heat-induced denaturation and aggregation occur. In the DH range of 4-20% heat stability increases as protein globular structure is disrupted. At a DH greater than 27% we see a change from a hydrolysis-induced increase in heat-stability to coalescence instability, with a resultant large increase in emulsion breakdown during heating.

  19. Modification of solubility and heat-induced gelation of amaranth 11S globulin by protein engineering.

    Science.gov (United States)

    Carrazco-Peña, Laura; Osuna-Castro, Juan A; De León-Rodríguez, Antonio; Maruyama, Nobuyuki; Toro-Vazquez, Jorge F; Morales-Rueda, Juan A; Barba de la Rosa, Ana P

    2013-04-10

    The primary structure of amaranth 11S globulin (Ah11S) was engineered with the aim to improve its functional properties. Four continuous methionines were inserted in variable region V, obtaining the Ah11Sr+4M construction. Changes on protein structure and surface characteristics were analyzed in silico. Solubility and heat-induced gelation of recombinant amaranth 11S proglobulin (Ah11Sr and Ah11Sr+4M) were compared with the native protein (Ah11Sn) purified from amaranth seed flour. The Ah11Sr+4 M showed the highest surface hydrophobicity, but as consequence the solubility was reduced. At low ionic strength (μ = 0.2) and acidic pH (proteins Ah11Sr and Ah11Sr+4 M had the highest and lowest solubility values, respectively. All globulins samples formed gels at 90 °C and low ionic strength, but Ah11Sn produced the weakest and Ah11Sr the strongest gels. Differential scanning calorimetry analysis under gel forming conditions revealed only exothermic transitions for all amaranth 11S globulins analyzed. In conclusion, the 3D structure analysis has revealed interesting molecular features that could explain the thermal resistance and gel forming ability of amaranth 11S globulins. The incorporation of four continuous methionines in amaranth increased the hydrophobicity, and self-supporting gels formed had intermediate hardness between Ah11Sn and Ah11Sr. These functional properties could be used in the food industry for the development of new products based on amaranth proteins.

  20. The Murid Herpesvirus-4 gL regulates an entry-associated conformation change in gH.

    Directory of Open Access Journals (Sweden)

    Laurent Gillet

    Full Text Available The glycoprotein H (gH/gL heterodimer is crucial for herpesvirus membrane fusion. Yet how it functions is not well understood. The Murid Herpesvirus-4 gH, like that of other herpesviruses, adopts its normal virion conformation by associating with gL. However, gH switched back to a gL-independent conformation after virion endocytosis. This switch coincided with a conformation switch in gB and with capsid release. Virions lacking gL constitutively expressed the down-stream form of gH, prematurely switched gB to its down-stream form, and showed premature capsid release with poor infectivity. These data argue that gL plays a key role in regulating a gH and gB functional switch from cell binding to membrane fusion.

  1. Calcium binding promotes prion protein fragment 90-231 conformational change toward a membrane destabilizing and cytotoxic structure.

    Directory of Open Access Journals (Sweden)

    Sacha Sorrentino

    Full Text Available The pathological form of prion protein (PrP(Sc, as other amyloidogenic proteins, causes a marked increase of membrane permeability. PrP(Sc extracted from infected Syrian hamster brains induces a considerable change in membrane ionic conductance, although the contribution of this interaction to the molecular mechanism of neurodegeneration process is still controversial. We previously showed that the human PrP fragment 90-231 (hPrP₉₀₋₂₃₁ increases ionic conductance across artificial lipid bilayer, in a calcium-dependent manner, producing an alteration similar to that observed for PrP(Sc. In the present study we demonstrate that hPrP₉₀₋₂₃₁, pre-incubated with 10 mM Ca⁺⁺ and then re-suspended in physiological external solution increases not only membrane conductance but neurotoxicity as well. Furthermore we show the existence of a direct link between these two effects as demonstrated by a highly statistically significant correlation in several experimental conditions. A similar correlation between increased membrane conductance and cell degeneration has been observed assaying hPrP₉₀₋₂₃₁ bearing pathogenic mutations (D202N and E200K. We also report that Ca⁺⁺ binding to hPrP₉₀₋₂₃₁ induces a conformational change based on an alteration of secondary structure characterized by loss of alpha-helix content causing hydrophobic amino acid exposure and proteinase K resistance. These features, either acquired after controlled thermal denaturation or induced by D202N and E200K mutations were previously identified as responsible for hPrP₉₀₋₂₃₁ cytotoxicity. Finally, by in silico structural analysis, we propose that Ca⁺⁺ binding to hPrP₉₀₋₂₃₁ modifies amino acid orientation, in the same way induced by E200K mutation, thus suggesting a pathway for the structural alterations responsible of PrP neurotoxicity.

  2. Calcium binding promotes prion protein fragment 90-231 conformational change toward a membrane destabilizing and cytotoxic structure.

    Science.gov (United States)

    Sorrentino, Sacha; Bucciarelli, Tonino; Corsaro, Alessandro; Tosatto, Alessio; Thellung, Stefano; Villa, Valentina; Schininà, M Eugenia; Maras, Bruno; Galeno, Roberta; Scotti, Luca; Creati, Francesco; Marrone, Alessandro; Re, Nazzareno; Aceto, Antonio; Florio, Tullio; Mazzanti, Michele

    2012-01-01

    The pathological form of prion protein (PrP(Sc)), as other amyloidogenic proteins, causes a marked increase of membrane permeability. PrP(Sc) extracted from infected Syrian hamster brains induces a considerable change in membrane ionic conductance, although the contribution of this interaction to the molecular mechanism of neurodegeneration process is still controversial. We previously showed that the human PrP fragment 90-231 (hPrP₉₀₋₂₃₁) increases ionic conductance across artificial lipid bilayer, in a calcium-dependent manner, producing an alteration similar to that observed for PrP(Sc). In the present study we demonstrate that hPrP₉₀₋₂₃₁, pre-incubated with 10 mM Ca⁺⁺ and then re-suspended in physiological external solution increases not only membrane conductance but neurotoxicity as well. Furthermore we show the existence of a direct link between these two effects as demonstrated by a highly statistically significant correlation in several experimental conditions. A similar correlation between increased membrane conductance and cell degeneration has been observed assaying hPrP₉₀₋₂₃₁ bearing pathogenic mutations (D202N and E200K). We also report that Ca⁺⁺ binding to hPrP₉₀₋₂₃₁ induces a conformational change based on an alteration of secondary structure characterized by loss of alpha-helix content causing hydrophobic amino acid exposure and proteinase K resistance. These features, either acquired after controlled thermal denaturation or induced by D202N and E200K mutations were previously identified as responsible for hPrP₉₀₋₂₃₁ cytotoxicity. Finally, by in silico structural analysis, we propose that Ca⁺⁺ binding to hPrP₉₀₋₂₃₁ modifies amino acid orientation, in the same way induced by E200K mutation, thus suggesting a pathway for the structural alterations responsible of PrP neurotoxicity.

  3. A chemometric analysis of ligand-induced changes in intrinsic fluorescence of folate binding protein indicates a link between altered conformational structure and physico-chemical characteristics

    DEFF Research Database (Denmark)

    Bruun, Susanne W; Holm, Jan; Hansen, Steen Ingemann

    2009-01-01

    Ligand binding alters the conformational structure and physico-chemical characteristics of bovine folate binding protein (FBP). For the purpose of achieving further information we analyzed ligand (folate and methotrexate)-induced changes in the fluorescence landscape of FBP. Fluorescence excitation...... and emission two-dimensional (2D) spectra were recorded over a wide range of wavelengths on a Perkin-Elmer LS 55 spectrofluorometer at varying pH in different buffers, and the resulting three-dimensional data were subjected to a chemometric analysis, parallel factor analysis (PARAFAC). The most important...... of folate accords fairly well with the disappearance of strongly hydrophobic tryptophan residues from the solvent-exposed surface of FBP. The PARAFAC has thus proven useful to establish a hitherto unexplained link between parallel changes in conformational structure and physico-chemical characteristics...

  4. Palm kernel cake extract exerts hepatoprotective activity in heat-induced oxidative stress in chicken hepatocytes.

    Science.gov (United States)

    Oskoueian, Ehsan; Abdullah, Norhani; Idrus, Zulkifli; Ebrahimi, Mahdi; Goh, Yong Meng; Shakeri, Majid; Oskoueian, Armin

    2014-10-02

    Palm kernel cake (PKC), the most abundant by-product of oil palm industry is believed to contain bioactive compounds with hepatoprotective potential. These compounds may serve as hepatoprotective agents which could help the poultry industry to alleviate adverse effects of heat stress on liver function in chickens. This study was performed to evaluate the hepatoprotective potential of PKC extract in heat-induced oxidative stress in chicken hepatocytes. The nature of the active metabolites and elucidation of the possible mechanism involved were also investigated. The PKC extract possessed free radical scavenging activity with values significantly (p Heat-induced oxidative stress in chicken hepatocyte impaired the total protein, lipid peroxidation and antioxidant enzymes activity significantly (p heat-induced hepatocytes with PKC extract (125 μg/ml) and silymarin as positive control increased these values significantly (p stress biomarkers including TNF-like, IFN-γ and IL-1β genes; NF-κB, COX-2, iNOS and Hsp70 proteins expression upon heat stress in chicken hepatocytes. The PKC extract and silymarin were able to alleviate the expression of all of these biomarkers in heat-induced chicken hepatocytes. The gas chromatography-mass spectrometry analysis of PKC extract showed the presence of fatty acids, phenolic compounds, sugar derivatives and other organic compounds such as furfural which could be responsible for the observed hepatoprotective activity. Palm kernel cake extract could be a potential agent to protect hepatocytes function under heat induced oxidative stress.

  5. A Conformational Change of C Fragment of Tetanus Neurotoxin Reduces Its Ganglioside-Binding Activity but Does Not Destroy Its Immunogenicity ▿

    Science.gov (United States)

    Yu, Rui; Yi, Shaoqiong; Yu, Changming; Fang, Ting; Liu, Shuling; Yu, Ting; Song, Xiaohong; Fu, Ling; Hou, Lihua; Chen, Wei

    2011-01-01

    The C fragment of tetanus neurotoxin (TeNT-Hc) with different conformations was observed due to the four cysteine residues within it which could form different intramolecular disulfide bonds. In this study, we prepared and compared three types of monomeric TeNT-Hc with different conformational components: free sulfhydryls (50 kDa), bound sulfhydryls (44 kDa), and a mixture of the two conformational proteins (half 50 kDa and half 44 kDa). TeNT-Hc with bound sulfhydryls reduced its binding activity to ganglioside GT1b and neuronal PC-12 cells compared to what was seen for TeNT-Hc with free sulfhydryls. However, there was no significant difference among their immunogenicities in mice, including induction of antitetanus toxoid IgG titers, antibody types, and protective capacities against tetanus neurotoxin challenge. Our results showed that the conformational changes of TeNT-Hc resulting from disulfide bond formation reduced its ganglioside-binding activity but did not destroy its immunogenicity, and the protein still retained continuous B cell and T cell epitopes; that is, the presence of the ganglioside-binding site within TeNT-Hc may be not essential for the induction of a fully protective antitetanus response. TeNT-Hc with bound sulfhydryls may be developed into an ideal human vaccine with a lower potential for side effects. PMID:21813664

  6. Conformational change induced by electron transfer in a monolayer of cytochrome P450 reductase adsorbed at the Au(110)-phosphate buffer interface.

    Science.gov (United States)

    Weightman, P; Smith, C I; Convery, J H; Harrison, P; Khara, B; Scrutton, N S

    2013-09-01

    The reflection anisotropy spectroscopy profiles of a variant of cytochrome P450 reductase adsorbed at the Au(110)-phosphate buffer interface depend on the sequence of potentials applied to the Au(110) electrode. It is suggested that this dependence arises from changes in the orientation of the isoalloxazine ring structures in the protein with respect to the Au(110) surface. This offers a method of monitoring conformational change in this protein by measuring variations in the reflection anisotropy spectrum arising from changes in the redox potential.

  7. Application of Near-Infrared and Fourier Transform Infrared Spectroscopy in the Characterization of Ligand-Induced Conformation Changes in Folate Binding Protein Purified from Bovine Milk

    DEFF Research Database (Denmark)

    Bruun, Susanne Wrang; Holm, Jan; Hansen, Steen Ingemann

    2006-01-01

    Fourier transform infrared (FT-IR) and near-infrared (NIR) spectroscopy have been applied to detect structural alterations in folate binding protein (FBP) induced by ligation in different buffer types. The amide I region pointed to a beta-sheet to alpha-helix transition upon ligation in acetate...... indicated similar changes at the two pH values. Therefore, we suggest that FT-IR and NIR spectroscopy may complement each other, such that the two techniques in combination may give information on all three types of protein conformational changes. While the secondary structure changes are revealed by FT...

  8. Switchable proline derivatives: tuning the conformational stability of the collagen triple helix by pH changes.

    Science.gov (United States)

    Siebler, Christiane; Erdmann, Roman S; Wennemers, Helma

    2014-09-22

    (4S)-Aminoproline is introduced as a pH-sensitive probe for tuning the conformational properties of peptides and proteins. The pH-triggered flip of the ring puckering and the formation/release of a transannular H bond were used to switch the formation of collagen triple helices on and off reversibly.

  9. Crystallographic observation of pH-induced conformational changes in the Amyelois transitella pheromone-binding protein AtraPBP1.

    Directory of Open Access Journals (Sweden)

    Eric di Luccio

    Full Text Available The navel orangeworm, Amyelois transitella is a major agricultural pest causing large losses in a variety of tree crops. Control of this insect pest may be achieved by interfering with olfactory pathways to block detection of female-produced sex pheromones and consequently, disrupt mating. The first component of this pathway is the pheromone-binding protein AtraPBP1, which recognizes the pheromone and presents it to the odorant receptor housed in a sensory neuron of the male antennae. Release of the ligand depends on a pH-induced conformational change associated with the acidity of the membrane surface. To characterize this conformational change and to understand how pheromones bind, we have determined the high resolution crystal structures of AtraPBP1 in complex with two main constituents of the sex pheromone, i.e., (11Z,13Z-hexadecadienal and (11Z,13Z-hexadecadienol. Comparison with the structure of the unliganded form demonstrates a large ∼90° movement of the C-terminal helix which is observed in other pheromone- or odorant-binding proteins accompanied by an unpredicted 37° displacement of the N-terminal helix. Molecular dynamic trajectories suggest that the conformational change of the α1 helix facilitates the movement of the C-terminal helix.

  10. Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Camila Ramos; Meza, Andreia Navarro [Laboratorio Nacional de Biociencias (LNBio), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil); Hoffmam, Zaira Bruna; Silva, Junio Cota; Alvarez, Thabata Maria; Ruller, Roberto [Laboratorio Nacional de Ciencia e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil); Giesel, Guilherme Menegon; Verli, Hugo [Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Squina, Fabio Marcio [Laboratorio Nacional de Ciencia e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil); Prade, Rolf Alexander [Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK (United States); Murakami, Mario Tyago, E-mail: mario.murakami@lnbio.org.br [Laboratorio Nacional de Biociencias (LNBio), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil)

    2010-12-10

    Research highlights: {yields} The hyperthermostable xylanase 10B from Thermotoga petrophila RKU-1 produces exclusively xylobiose at the optimum temperature. {yields} Circular dichroism spectroscopy suggests a coupling effect of temperature-induced structural changes with its enzymatic behavior. {yields} Crystallographic and molecular dynamics studies indicate that conformational changes in the product release area modulate the enzyme action mode. -- Abstract: Endo-xylanases play a key role in the depolymerization of xylan and recently, they have attracted much attention owing to their potential applications on biofuels and paper industries. In this work, we have investigated the molecular basis for the action mode of xylanases 10B at high temperatures using biochemical, biophysical and crystallographic methods. The crystal structure of xylanase 10B from hyperthermophilic bacterium Thermotoga petrophila RKU-1 (TpXyl10B) has been solved in the native state and in complex with xylobiose. The complex crystal structure showed a classical binding mode shared among other xylanases, which encompasses the -1 and -2 subsites. Interestingly, TpXyl10B displayed a temperature-dependent action mode producing xylobiose and xylotriose at 20 {sup o}C, and exclusively xylobiose at 90 {sup o}C as assessed by capillary zone electrophoresis. Moreover, circular dichroism spectroscopy suggested a coupling effect of temperature-induced structural changes with this particular enzymatic behavior. Molecular dynamics simulations supported the CD analysis suggesting that an open conformational state adopted by the catalytic loop (Trp297-Lys326) provokes significant modifications in the product release area (+1,+2 and +3 subsites), which drives the enzymatic activity to the specific release of xylobiose at high temperatures.

  11. Conformational changes to deamidated wheat gliadins and b-casein upon adsorption to oil-water emulsion interfaces

    DEFF Research Database (Denmark)

    Wong, Benjamin T.; Zhai, Jiali; Hoffmann, Søren Vrønning

    2012-01-01

    The conformation of deamidated gliadins and b-casein in solution and adsorbed at the interface of oil-inwater emulsions was studied using synchrotron radiation circular dichroism (SRCD) and front-facefluorescence spectroscopy. Deamidation led to partial unfolding of gliadins in solution. The a....... In contrast, b-casein adopted a more ordered structure upon adsorption to these two oil/water interfaces, the a-helix content increased from 5.5% (in solution) to 20% and 22.5% respectively after adsorption to tricaprin/water and hexadecane/water interfaces. Both deamidated gliadins and b-casein have...... to oil/water interfaces. The hydrophobicity of the oil phase also has an impact on the conformation of each protein upon adsorption to the oil/water interfaces e systematic trends were observed between oil phase polarity from: i) tryptophan fluorescence emission maxima, and ii) the ahelix content...

  12. The zeaxanthin-independent and zeaxanthin-dependent qE components of nonphotochemical quenching involve common conformational changes within the photosystem II antenna in Arabidopsis.

    Science.gov (United States)

    Johnson, Matthew P; Pérez-Bueno, María L; Zia, Ahmad; Horton, Peter; Ruban, Alexander V

    2009-02-01

    The light-harvesting antenna of higher plant photosystem II (LHCII) has the intrinsic capacity to dissipate excess light energy as heat in a process termed nonphotochemical quenching (NPQ). Recent studies suggest that zeaxanthin and lutein both contribute to the rapidly relaxing component of NPQ, qE, possibly acting in the minor monomeric antenna complexes and the major trimeric LHCII, respectively. To distinguish whether zeaxanthin and lutein act independently as quenchers at separate sites, or alternatively whether zeaxanthin fulfills an allosteric role regulating lutein-mediated quenching, the kinetics of qE and the qE-related conformational changes (DeltaA535) were compared in Arabidopsis (Arabidopsis thaliana) mutant/antisense plants with altered contents of minor antenna (kolhcb6, aslhcb4), trimeric LHCII (aslhcb2), lutein (lut2, lut2npq1, lut2npq2), and zeaxanthin (npq1, npq2). The kinetics of the two components of NPQ induction arising from zeaxanthin-independent and zeaxanthin-dependent qE were both sensitive to changes in the protein composition of the photosystem II antenna. The replacement of lutein by zeaxanthin or violaxanthin in the internal Lhcb protein-binding sites affected the kinetics and relative amplitude of each component as well as the absolute chlorophyll fluorescence lifetime. Both components of qE were characterized by a conformational change leading to nearly identical absorption changes in the Soret region that indicated the involvement of the LHCII lutein 1 domain. Based on these observations, we suggest that both components of qE arise from a common quenching mechanism based upon a conformational change within the photosystem II antenna, optimized by Lhcb subunit-subunit interactions and tuned by the synergistic effects of external and internally bound xanthophylls.

  13. Heat-Induced Gel Formation by Soy Proteins at Neutral pH

    NARCIS (Netherlands)

    Renkema, J.M.S.; Vliet, van T.

    2002-01-01

    Heat-induced gel formation by soy protein isolate at pH 7 is discussed. Different heating and cooling rates, heating times, and heating temperatures were used to elucidate the various processes that occur and to study the relative role of covalent and noncovalent protein interactions therein. Gel fo

  14. Heat-induced whey protein isolate fibrils: Conversion, hydrolysis, and disulphide bond formation

    NARCIS (Netherlands)

    Bolder, S.G.; Vasbinder, A.; Sagis, L.M.C.; Linden, van der E.

    2007-01-01

    Fibril formation of individual pure whey proteins and whey protein isolate (WPI) was studied. The heat-induced conversion of WPI monomers into fibrils at pH 2 and low ionic strength increased with heating time and protein concentration. Previous studies, using a precipitation method, size-exclusion

  15. Conformational changes in IpaD from Shigella flexneri upon binding bile salts provide insight into the second step of type III secretion.

    Science.gov (United States)

    Dickenson, Nicholas E; Zhang, Lingling; Epler, Chelsea R; Adam, Philip R; Picking, Wendy L; Picking, William D

    2011-01-18

    Shigella flexneri uses its type III secretion apparatus (TTSA) to inject host-altering proteins into targeted eukaryotic cells. The TTSA is composed of a basal body and an exposed needle with invasion plasmid antigen D (IpaD) forming a tip complex that controls secretion. The bile salt deoxycholate (DOC) stimulates recruitment of the translocator protein IpaB into the maturing TTSA needle tip complex. This process appears to be triggered by a direct interaction between DOC and IpaD. Fluorescence spectroscopy and NMR spectroscopy are used here to confirm the DOC-IpaD interaction and to reveal that IpaD conformational changes upon DOC binding trigger the appearance of IpaB at the needle tip. Förster resonance energy transfer between specific sites on IpaD was used here to identify changes in distances between IpaD domains as a result of DOC binding. To further explore the effects of DOC binding on IpaD structure, NMR chemical shift mapping was employed. The environments of residues within the proposed DOC binding site and additional residues within the "distal" globular domain were perturbed upon DOC binding, further indicating that conformational changes occur within IpaD upon DOC binding. These events are proposed to be responsible for the recruitment of IpaB at the TTSA needle tip. Mutation analyses combined with additional spectroscopic analyses confirm that conformational changes in IpaD induced by DOC binding contribute to the recruitment of IpaB to the S. flexneri TTSA needle tip. These findings lay the foundation for determining how environmental factors promote TTSA needle tip maturation prior to host cell contact.

  16. A spectroscopic and computational investigation of the conformational structural changes induced by hydrogen bonding networks in the glycidol-water complex.

    Science.gov (United States)

    Conrad, A R; Teumelsan, N H; Wang, P E; Tubergen, M J

    2010-01-14

    Rotational spectra were recorded in natural abundance for the (13)C isotopomers of two conformers of glycidol. Moments of inertia from the (13)C isotopomers were used to calculate the substitution coordinates and C-C bond lengths of two glycidol monomer conformations. The structures of seven different conformational minima were found from ab initio (MP2/6-311++G(d,p)) optimizations of glycidol-water. The rotational spectrum of glycidol-water was recorded using microwave spectroscopy, and the rotational constants were determined to be A = 3902.331 (11) MHz, B = 2763.176 (3) MHz, and C = 1966.863 (3) MHz. Rotational spectra were also recorded for glycidol-H(2)(18)O, glycidol-D(b)OH, and glycidol-d(O)-D(2)O. The rotational spectra were assigned to the lowest-energy ab initio structure, and the structure was improved by fitting to the experimental moments of inertia. The best-fit structure shows evidence for structural changes in glycidol to accommodate formation of the intermolecular hydrogen bonding network: the O-C-C-O torsional angle in glycidol was found to increase from 40.8 degrees for the monomer to 49.9 degrees in the water complex.

  17. The interaction of human serum albumin with selected lanthanide and actinide ions: Binding affinities, protein unfolding and conformational changes.

    Science.gov (United States)

    Ali, Manjoor; Kumar, Amit; Kumar, Mukesh; Pandey, Badri N

    2016-04-01

    Human serum albumin (HSA), the most abundant soluble protein in blood plays critical roles in transportation of biomolecules and maintenance of osmotic pressure. In view of increasing applications of lanthanides- and actinides-based materials in nuclear energy, space, industries and medical applications, the risk of exposure with these metal ions is a growing concern for human health. In present study, binding interaction of actinides/lanthanides [thorium: Th(IV), uranium: U(VI), lanthanum: La(III), cerium: Ce(III) and (IV)] with HSA and its structural consequences have been investigated. Ultraviolet-visible, Fourier transform-infrared, Raman, Fluorescence and Circular dichroism spectroscopic techniques were applied to study the site of metal ions interaction, binding affinity determination and the effect of metal ions on protein unfolding and HSA conformation. Results showed that these metal ions interacted with carbonyl (CO..:)/amide(N..-H) groups and induced exposure of aromatic residues of HSA. The fluorescence analysis indicated that the actinide binding altered the microenvironment around Trp214 in the subdomain IIA. Binding affinity of U(VI) to HSA was slightly higher than that of Th(IV). Actinides and Ce(IV) altered the secondary conformation of HSA with a significant decrease of α-helix and an increase of β-sheet, turn and random coil structures, indicating a partial unfolding of HSA. A correlation was observed between metal ion's ability to alter HSA conformation and protein unfolding. Both cationic effects and coordination ability of metal ions seemed to determine the consequences of their interaction with HSA. Present study improves our understanding about the protein interaction of these heavy ions and their impact on its secondary structure. In addition, binding characteristics may have important implications for the development of rational antidote for the medical management of health effects of actinides and lanthanides.

  18. Conformational Transitions

    Science.gov (United States)

    Czerminski, Ryszard; Roitberg, Adrian; Choi, Chyung; Ulitsky, Alexander; Elber, Ron

    1991-10-01

    Two computational approaches to study plausible conformations of biological molecules and the transitions between them are presented and discussed. The first approach is a new search algorithm which enhances the sampling of alternative conformers using a mean field approximation. It is argued and demonstrated that the mean field approximation has a small effect on the location of the minima. The method is a combination of the LES protocol (Locally Enhanced Sampling) and simulated annealing. The LES method was used in the past to study the diffusion pathways of ligands from buried active sites in myoglobin and leghemoglobin to the exterior of the protein. The present formulation of LES and its implementation in a Molecular Dynamics program is described. An application for side chain placement in a tetrapeptide is presented. The computational effort associated with conformational searches using LES grows only linearly with the number of degrees of freedom, whereas in the exact case the computational effort grows exponentially. Such saving is of course associated with a mean field approximation. The second branch of studies pertains to the calculation of reaction paths in large and flexible biological systems. An extensive mapping of minima and barriers for two different tetrapeptides is calculated from the known minima and barriers of alanine tetrapeptide which we calculated recently.1 The tetrapeptides are useful models for the formation of secondary structure elements since they are the shortest possible polymers of this type which can still form a complete helical turn. The tetrapeptides are isobutyryl-val(χ1=60)-ala-ala and isobutyryl-val(χ1=-60)-ala-ala. Properties of the hundreds of minima and of the hundreds intervening barriers are discussed. Estimates for thermal transition times between the many conformers (and times to explore the complete phase space) are calculated and compared. It is suggested that the most significant effect of the side chain size is

  19. Tunable electron heating induced giant magnetoresistance in the high mobility GaAs/AlGaAs 2D electron system

    Science.gov (United States)

    Wang, Zhuo; Samaraweera, R. L.; Reichl, C.; Wegscheider, W.; Mani, R. G.

    2016-12-01

    Electron-heating induced by a tunable, supplementary dc-current (Idc) helps to vary the observed magnetoresistance in the high mobility GaAs/AlGaAs 2D electron system. The magnetoresistance at B = 0.3 T is shown to progressively change from positive to negative with increasing Idc, yielding negative giant-magnetoresistance at the lowest temperature and highest Idc. A two-term Drude model successfully fits the data at all Idc and T. The results indicate that carrier heating modifies a conductivity correction σ1, which undergoes sign reversal from positive to negative with increasing Idc, and this is responsible for the observed crossover from positive- to negative- magnetoresistance, respectively, at the highest B.

  20. Conformational changes in human serum albumin studied by fluorescence and absorption spectroscopy. Distance measurements as a function of pH and fatty acids

    DEFF Research Database (Denmark)

    Honoré, B; Pedersen, A O

    1989-01-01

    pH- and fatty acid-induced conformational changes in human serum albumin were investigated by fluorescence-energy transfer, determining the distance between Trp-214 and bound bilirubin at 25 degrees C. This distance changes significantly with the pH, being 2.52 +/- 0.01 nm at pH 6, 2.31 +/- 0.04 ...... chromophores. The contraction of the protein carrying long-chain saturated fatty acids is even more pronounced at pH 9. Udgivelsesdato: 1989-Feb-15......pH- and fatty acid-induced conformational changes in human serum albumin were investigated by fluorescence-energy transfer, determining the distance between Trp-214 and bound bilirubin at 25 degrees C. This distance changes significantly with the pH, being 2.52 +/- 0.01 nm at pH 6, 2.31 +/- 0.04 nm...... at pH 9, 2.13 +/- 0.07 nm at pH 11.0 and 2.77 nm at pH 11.9. The influence of different fatty acids on the distance was also determined. At pH 7.4 medium-chain fatty acids seem to increase this distance, whereas long-chain fatty acids, at low concentrations, decrease the distance between the two...

  1. Monitoring Protein Conformation Changes as an Activating Step for Protein Interactions with Cross-linking/MS Analysis. / Chen, Zhuo; Rasmussen, Morten; Tahir, Salman; Clark, C.A.C; Barlow, Paul; Rappsilber, Juri

    DEFF Research Database (Denmark)

    Rasmussen, Morten

    Monitoring protein conformation changes as an activating step for protein interactions with cross-linking/MS analysis. Chen, Zhou; Rasmussen, Morten; Tahir, Salman; Clark, C.A.C; Barlow, Paul; Rappsilber, Juri.   Introduction Protein interactions often require conformational changes in proteins....... Chemical cross-linking of proteins coupled with mass spectrometric analysis is emerging as a versatile tool for determining low-resolution three-dimensional structures of proteins. We show in this study that this technique is also able to resolve protein conformation changes, investigating the transition......-linked peptides and manual validation were performed using in-house software. The structural information determined by validated cross-links was compared against C3 and C3b crystal structure using Pymol.   Preliminary results We portray conformation changes from C3 to C3b through observing different group...

  2. Molecular Dynamics Simulation of Tau Peptides for the Investigation of Conformational Changes Induced by Specific Phosphorylation Patterns.

    Science.gov (United States)

    Gandhi, Neha S; Kukic, Predrag; Lippens, Guy; Mancera, Ricardo L

    2017-01-01

    The Tau protein plays an important role due to its biomolecular interactions in neurodegenerative diseases. The lack of stable structure and various posttranslational modifications such as phosphorylation at various sites in the Tau protein pose a challenge for many experimental methods that are traditionally used to study protein folding and aggregation. Atomistic molecular dynamics (MD) simulations can help around deciphering relationship between phosphorylation and various intermediate and stable conformations of the Tau protein which occur on longer timescales. This chapter outlines protocols for the preparation, execution, and analysis of all-atom MD simulations of a 21-amino acid-long phosphorylated Tau peptide with the aim of generating biologically relevant structural and dynamic information. The simulations are done in explicit solvent and starting from nearly extended configurations of the peptide. The scaled MD method implemented in AMBER14 was chosen to achieve enhanced conformational sampling in addition to a conventional MD approach, thereby allowing the characterization of folding for such an intrinsically disordered peptide at 293 K. Emphasis is placed on the analysis of the simulation trajectories to establish correlations with NMR data (i.e., chemical shifts and NOEs). Finally, in-depth discussions are provided for commonly encountered problems.

  3. Cholesterol-induced conformational changes in the sterol-sensing domain of the Scap protein suggest feedback mechanism to control cholesterol synthesis.

    Science.gov (United States)

    Gao, Yansong; Zhou, Yulian; Goldstein, Joseph L; Brown, Michael S; Radhakrishnan, Arun

    2017-05-26

    Scap is a polytopic protein of endoplasmic reticulum (ER) membranes that transports sterol regulatory element-binding proteins to the Golgi complex for proteolytic activation. Cholesterol accumulation in ER membranes prevents Scap transport and decreases cholesterol synthesis. Previously, we provided evidence that cholesterol inhibition is initiated when cholesterol binds to loop 1 of Scap, which projects into the ER lumen. Within cells, this binding causes loop 1 to dissociate from loop 7, another luminal Scap loop. However, we have been unable to demonstrate this dissociation when we added cholesterol to isolated complexes of loops 1 and 7. We therefore speculated that the dissociation requires a conformational change in the intervening polytopic sequence separating loops 1 and 7. Here we demonstrate such a change using a protease protection assay in sealed membrane vesicles. In the absence of cholesterol, trypsin or proteinase K cleaved cytosolic loop 4, generating a protected fragment that we visualized with a monoclonal antibody against loop 1. When cholesterol was added to these membranes, cleavage in loop 4 was abolished. Because loop 4 is part of the so-called sterol-sensing domain separating loops 1 and 7, these results support the hypothesis that cholesterol binding to loop 1 alters the conformation of the sterol-sensing domain. They also suggest that this conformational change helps transmit the cholesterol signal from loop 1 to loop 7, thereby allowing separation of the loops and facilitating the feedback inhibition of cholesterol synthesis. These insights suggest a new structural model for cholesterol-mediated regulation of Scap activity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Secondary structure of prothymosin alpha evidenced for conformational transitions induced by changes in temperature and concentration of n-dodecyltrimethylammonium bromide.

    Science.gov (United States)

    Pombo, C; Suarez, M J; Nogueira, M; Czarnecki, J; Ruso, J M; Sarmiento, F; Prieto, G

    2001-08-01

    Conformational changes of prothymosin alpha (ProTalpha) induced by changes in temperature and concentration of the denaturant n-dodecyltrimethylammonium bromide (C12TAB) were studied by difference spectroscopy. The conformational transition of ProTalpha by C12TAB was followed as a function of denaturant concentration by absorbance measurements at 230 nm and the data were analyzed to obtain the Gibbs energy of the transition in water (deltaG0(w)) and in a hydrophobic environment (deltaG0(hc)) for saturated protein-surfactant complexes. The value of deltaG0(w) was 6.38 kJ mol(-1) and that for deltaG0(hc), which is not affected by temperature, was -18.62 kJ mol(-1). Changes of absorbance at 230 nm of ProTalpha with temperature can be assumed to resemble a transition in the secondary structure. The parameters characterizing the thermodynamics of unfolding, melting temperature (Tm), enthalpy (deltaHm), entropy (deltaSm) and heat capacity (deltaCp) were determined. The values obtained for Tm, deltaHm, and deltaSm are smaller that those found for other globular proteins; deltaCp was found to be much smaller. These results suggest that ProTalpha exhibits some type of secondary structure under these conditions (10 mM glycine buffer, pH 2.4).

  5. Dual changes in conformation and optical properties of fluorophores within a metal-organic framework during framework construction and associated sensing event.

    Science.gov (United States)

    Cho, Won; Lee, Hee Jung; Choi, Goeun; Choi, Sora; Oh, Moonhyun

    2014-09-03

    Microsized chemosensor particle (CPP-16, CPP means coordination polymer particle), which is made from a metal-organic framework (MOF), is synthesized using pyrene-functionalized organic building block. This building block contains three important parts, a framework construction part, a Cu(2+) detection part, and a fluorophore part. PXRD studies have revealed that CPP-16 has a 3D cubic structure of MOF-5. During both MOF formation and sensing event, fluorophores within CPP-16 undergo dual changes in conformation and optical properties. After MOF construction, pyrene moieties experience an unusual complete conversion from monomer to excimer form. This conversion takes place due to a confinement effect induced by space limitations within the MOF structure. The selective sensing ability of CPP-16 on Cu(2+) over many other metal ions is verified by emission spectra and is also visually identified by fluorescence microscopy images. Specific interaction of Cu(2+) with binding sites within CPP-16 causes a second conformational change of the fluorophores, where they change from stacked excimer (CPP-16) to quenched excimer states (CPP-16·Cu(2+)).

  6. Water drives peptide conformational transitions

    CERN Document Server

    Nerukh, Dmitry

    2011-01-01

    Transitions between metastable conformations of a dipeptide are investigated using classical molecular dynamics simulation with explicit water molecules. The distribution of the surrounding water at different moments before the transitions and the dynamical correlations of water with the peptide's configurational motions indicate that water is the main driving force of the conformational changes.

  7. Structural modifications leading to changes in supramolecular aggregation of thiazolo[3, 2-]pyrimidines: Insights into their conformational features

    Indian Academy of Sciences (India)

    H Nagarajaiah; Noor Shahina Begum

    2014-09-01

    The compounds, 7-methyl-3,5-diphenyl-5-thiazolo[3,2-a]pyrimidine-6-carboxylic acid ethyl ester (1), 3-amino-2-cyano-7-methyl-5-phenyl-5-thiazolo[3,2-]pyrimidine-6-carboxylic acid methyl ester (2), 2-dimethylaminomethylene-7-methyl-3-oxo-5-phenyl-2,3-dihydro-5-thiazolo[3,2-]pyrimidine-6-carboxylic acid ethyl ester (3), 2-(3-cyano-benzylidene)-5-(4-hydroxy-phenyl)-7-methyl-3-oxo-2,3-dihydro-5-thiazolo[3,2-]pyrimidine-6-carboxylic acid methyl ester; with ,-dimethyl-formamide (4) and 3-ethoxycarbonylmethyl-5-(4-hydroxy-3-methoxy-phenyl)-7-methyl-5-thiazolo[3,2-]pyrimidine-6-carboxylic acid methyl ester (5) have been synthesized and their structures evaluated crystallographically. Compound 1 crystallizes in the space group $\\bar{ı}$ with Z=8, with four molecules in the asymmetric unit. Compound 2 also crystallizes in the space group $\\bar{ı}$ with Z=4 wherein asymmetric unit accommodates two molecules. Compound 3 belongs to 21/ with Z=4, compound 4 crystallizes in bc21 with Z= 4 and compound 5 belongs to $\\bar{ı}$ with Z=2. In all the above compounds, the aryl ring positioned at C5 of thiazolopyrimidine ring is almost perpendicular. In the case of compounds with substituted phenyl ring, aryl group-up conformation predominates. However, for compounds with unsubstituted phenyl ring, aryl group-down conformation is adopted. By varying the substituents at positions C2, C3, C6 and on the aryl at C5 in the main molecular scaffold of (1-5), we have observed significant differences in the intermolecular interaction patterns. The packing features of the compounds are controlled by C-H…O, C-H…N, N-H…N O-H…N, C-H$\\ldots$ and $\\ldots$ weak interactions.

  8. Insulin degrading enzyme induces a conformational change in varicella-zoster virus gE, and enhances virus infectivity and stability.

    Directory of Open Access Journals (Sweden)

    Qingxue Li

    Full Text Available Varicella-zoster virus (VZV glycoprotein E (gE is essential for virus infectivity and binds to a cellular receptor, insulin-degrading enzyme (IDE, through its unique amino terminal extracellular domain. Previous work has shown IDE plays an important role in VZV infection and virus cell-to-cell spread, which is the sole route for VZV spread in vitro. Here we report that a recombinant soluble IDE (rIDE enhances VZV infectivity at an early step of infection associated with an increase in virus internalization, and increases cell-to-cell spread. VZV mutants lacking the IDE binding domain of gE were impaired for syncytia formation and membrane fusion. Pre-treatment of cell-free VZV with rIDE markedly enhanced the stability of the virus over a range of conditions. rIDE interacted with gE to elicit a conformational change in gE and rendered it more susceptible to proteolysis. Co-incubation of rIDE with gE modified the size of gE. We propose that the conformational change in gE elicited by IDE enhances infectivity and stability of the virus and leads to increased fusogenicity during VZV infection. The ability of rIDE to enhance infectivity of cell-free VZV over a wide range of incubation times and temperatures suggests that rIDE may be useful for increasing the stability of varicella or zoster vaccines.

  9. Probing the interaction of ferrocene containing hyperbranched poly-ester with model plasma protein: Effect on the interaction mechanism and conformational change

    Energy Technology Data Exchange (ETDEWEB)

    Xiao, Fengjuan, E-mail: xfj66@126.com; Gu, Muqing; Liang, Ye; Li, Lanlan; Yu, Xiaolei; Wu, Xiangfeng

    2014-05-01

    Interaction mechanism and conformational change of model plasma protein-bovine serum albumin (BSA) induced by ferrocenyl-functionalized hyperbranched polyester (HBPE-Fc) were investigated using cyclicvoltammetry (CV), differential pulsed voltammetry (DPV), fluorescence, UV–vis absorption spectrometry and circular dichroism (CD). Some complicated interactions occurred between BSA and HBPE-Fc and the new redox centers appeared in the BSA/HBPE-Fc complex that changed and hindered the electron transfer of Fe/Fe{sup 2+}. Fluorescence quenching data showed that the fluorescence of BSA was statically quenched by HBPE-Fc, which implied that ground state complex formed between BSA and HBPE-Fc. van der Waals force and hydrogen bond played major roles in the interaction of HBPE-Fc with BSA. The binding constant Ka for HBPE-Fc–protein interaction is in the order of 10{sup 6} at room temperature indicates that there is a strong interaction between HBPE-Fc and BSA. Synchronous, three-dimensional fluorescence and CD studies indicated that the interaction of BSA with HBPE-Fc induced conformational changes in BSA with overall decrease in the α-helical structure and increase in β-pleated sheet structure. The molecular model of the interaction between HBPE-Fc and BSA was also presented according to the results in this study. - Highlights: • A novel ferrocenyl-functionalized hyperbranched polymer (HBPE-Fc) with potential anticancer effects. • New redox centers appear in the BSA/HBPE-Fc complex that changed and hindered the electron transfer of Fe/Fe{sup 2+}. • BSA fluorescence was statically quenched by HBPE-Fc. • BSA/HBPE-Fc ground state complex was mainly formed by the hydrogen bonds and van der Waals force. • HBPE-Fc induced conformational changes in BSA with overall decrease in the α-helical structure and increase in β-pleated sheet structure. • The molecular model of the interaction was presented according to the results in this study.

  10. Palm kernel cake extract exerts hepatoprotective activity in heat-induced oxidative stress in chicken hepatocytes

    OpenAIRE

    Oskoueian, Ehsan; Abdullah, Norhani; Idrus, Zulkifli; Ebrahimi, Mahdi; Goh, Yong Meng; SHAKERI, Majid; Oskoueian, Armin

    2014-01-01

    Background Palm kernel cake (PKC), the most abundant by-product of oil palm industry is believed to contain bioactive compounds with hepatoprotective potential. These compounds may serve as hepatoprotective agents which could help the poultry industry to alleviate adverse effects of heat stress on liver function in chickens. Methods This study was performed to evaluate the hepatoprotective potential of PKC extract in heat-induced oxidative stress in chicken hepatocytes. The nature of the acti...

  11. Denaturant-Dependent Conformational Changes in a [beta]-Trefoil Protein: Global and Residue-Specific Aspects of an Equilibrium Denaturation Process

    Energy Technology Data Exchange (ETDEWEB)

    Latypov, Ramil F.; Liu, Dingjiang; Jacob, Jaby; Harvey, Timothy S.; Bondarenko, Pavel V.; Kleemann, Gerd R.; Brems, David N.; Raibekas, Andrei A.; (Amgen)

    2010-01-12

    Conformational properties of the folded and unfolded ensembles of human interleukin-1 receptor antagonist (IL-1ra) are strongly denaturant-dependent as evidenced by high-resolution two-dimensional nuclear magnetic resonance (NMR), limited proteolysis, and small-angle X-ray scattering (SAXS). The folded ensemble was characterized in detail in the presence of different urea concentrations by 1H-15N HSQC NMR. The {beta}-trefoil fold characteristic of native IL-1ra was preserved until the unfolding transition region beginning at 4 M urea. At the same time, a subset of native resonances disappeared gradually starting at low denaturant concentrations, indicating noncooperative changes in the folded state. Additional evidence of structural perturbations came from the chemical shift analysis, nonuniform and bell-shaped peak intensity profiles, and limited proteolysis. In particular, the following nearby regions of the tertiary structure became progressively destabilized with increasing urea concentrations: the {beta}-hairpin interface of trefoils 1 and 2 and the H2a-H2 helical region. These regions underwent small-scale perturbations within the native baseline region in the absence of populated molten globule-like states. Similar regions were affected by elevated temperatures known to induce irreversible aggregation of IL-1ra. Further evidence of structural transitions invoking near-native conformations came from an optical spectroscopy analysis of its single-tryptophan variant W17A. The increase in the radius of gyration was associated with a single equilibrium unfolding transition in the case of two different denaturants, urea and guanidine hydrochloride (GuHCl). However, the compactness of urea- and GuHCl-unfolded molecules was comparable only at high denaturant concentrations and deviated under less denaturing conditions. Our results identified the role of conformational flexibility in IL-1ra aggregation and shed light on the nature of structural transitions within the

  12. Conformational Changes in Orotidine 5’-Monophosphate Decarboxylase: A Structure-Based Explanation for How the 5’-Phosphate Group Activates the Enzyme†

    Science.gov (United States)

    Desai, Bijoy J.; Wood, McKay; Fedorov, Alexander A.; Fedorov, Elena V.; Goryanova, Bogdana; Amyes, Tina L.; Richard, John P.; Almo, Steven C.; Gerlt, John A.

    2012-01-01

    The binding of a ligand to orotidine 5’-monophosphate decarboxylase (OMPDC) is accompanied by a conformational change from an open, inactive conformation (Eo) to a closed, active conformation (Ec). As the substrate traverses the reaction coordinate to form the stabilized vinyl carbanion/carbene intermediate, interactions are enforced that destabilize the carboxylate group of the substrate as well as stabilize the intermediate (in the Ec•S‡ complex). Focusing on the OMPDC from Methanothermobacter thermautotrophicus, the “remote” 5’-phosphate group of the substrate activates the enzyme 2.4 × 108-fold; the activation is equivalently described by an intrinsic binding energy (IBE) of 11.4 kcal/mol. We studied residues in the activation that 1) directly contact the 5’-phosphate group; 2) participate in a hydrophobic cluster near the base of the active site loop that sequesters the bound substrate from solvent; and 3) form hydrogen-bonding interactions across the interface between the “mobile” and “fixed” half-barrel domains of the (β/α8-barrel structure. Our data support a model in which the IBE provided by the 5’-phosphate group is used to enable interactions both near the N-terminus of the active site loop and across the domain interface that stabilize both the Ec•S and Ec•S‡ complexes relative to the Eo•S complex. The conclusion that the IBE of the 5’-phosphate group provides stabilization of both the Ec•S and Ec•S‡ complexes, not just the Ec•S‡ complex, is central to understanding the structural origins of enzymatic catalysis as well as the requirements for the de novo design of enzymes that catalyze novel reactions. PMID:23030629

  13. Conformation and structural changes of diblock copolymers with octopus-like micelle formation in the presence of external stimuli

    Science.gov (United States)

    Dammertz, K.; Saier, A. M.; Marti, O.; Amirkhani, M.

    2014-04-01

    External stimuli such as vapours and electric fields can be used to manipulate the formation of AB-diblock copolymers on surfaces. We study the conformational variation of PS-b-PMMA (polystyrene-block-poly(methyl methacrylate)), PS and PMMA adsorbed on mica and their response to saturated water or chloroform atmospheres. Using specimens with only partial polymer coverage, new unanticipated effects were observed. Water vapour, a non-solvent for all three polymers, was found to cause high surface mobility. In contrast, chloroform vapour (a solvent for all three polymers) proved to be less efficient. Furthermore, the influence of an additional applied electric field was investigated. A dc field oriented parallel to the sample surface induces the formation of polymer islands which assemble into wormlike chains. Moreover, PS-b-PMMA forms octopus-like micelles (OLMs) on mica. Under the external stimuli mentioned above, the wormlike formations of OLMs are able to align in the direction of the external electric field. In the absence of an electric field, the OLMs disaggregate and exhibit phase separated structures under chloroform vapour.

  14. Exciton coupling effects and conformational change of perhexyloligosilanes with optically active methyl(1-naphthyl)phenylsilyl terminals.

    Science.gov (United States)

    Oh, Hyun-Shik; Park, Lee-Soon; Kawakami, Yusuke

    2003-08-01

    Perhexyloligosilanes (R,R)-(+)-MeNpPhSi*(Hex(2)Si)(n)Si*PhNpMe (n = 2; (R,R)-(+)-4a, n = 4; (R,R)-(+)-6a, n = 6; (R,R)-(+)-8a) with chiral methyl(1-naphthyl)phenylsilyl terminals were synthesized and characterized. The absorption wavelengths lambda(max) by (1)L(a,Ph) transition of phenyl chromophore conjugated with oligosilane units in (R,R)-(+)-4a - (R,R)-(+)-8a show bathochromic shift of about 3-4 nm compared with those of the alpha,omega-phenyl substituted perhexyloligosilanes Ph(Hex(2)Si)(m)Ph (m = 4; 4b, m = 6; 6b, m = 8; 8b) having the same silicon chain length. Longer chain length induces the separated lambda(max) of (1)L(a,Ph) from (1)B(b,Np) of naphthyl chromophore with positive exciton chiralities. In (R,R)-(+)-8a, although the extremum wavelengths lambda(ext) of exciton coupling between (1)B(b,Np) and (1)L(a,Ph) are separated by about 80 nm, the compound retains the positive exciton chirality, which provides definite information on the absolute configuration of terminal chiral silicon atoms. Bulky terminal substituents and lowering the temperature affect the conformation of the main chain, inducing extended silicon backbone structure.

  15. Towards Al3+-Induced Manganese-Containing Superoxide Dismutase Inactivation and Conformational Changes: An Integrating Study with Docking Simulations

    Directory of Open Access Journals (Sweden)

    Jiang-Liu Yang

    2011-01-01

    Full Text Available Superoxide dismutase (SOD, EC 1.15.1.1 plays an important antioxidant defense role in skins exposed to oxygen. We studied the inhibitory effects of Al3+ on the activity and conformation of manganese-containing SOD (Mn-SOD. Mn-SOD was significantly inactivated by Al3+ in a dose-dependent manner. The kinetic studies showed that Al3+ inactivated Mn-SOD follows the first-order reaction. Al3+ increased the degree of secondary structure of Mn-SOD and also disrupted the tertiary structure of Mn-SOD, which directly resulted in enzyme inactivation. We further simulated the docking between Mn-SOD and Al3+ (binding energy for Dock 6.3: −14.07 kcal/mol and suggested that ASP152 and GLU157 residues were predicted to interact with Al3+, which are not located in the Mn-contained active site. Our results provide insight into the inactivation of Mn-SOD during unfolding in the presence of Al3+ and allow us to describe a ligand binding via inhibition kinetics combined with the computational prediction.

  16. The quality of high pressure-induced and heat-induced yuzu marmalade

    Science.gov (United States)

    Kuwada, Hiroko; Jibu, Yuri; Teramoto, Ai; Fuchigami, Michiko

    2010-12-01

    Yuzu is a typical Japanese citrus with a desirable smell. The objectives of this study are to establish a process for pressure-induced marmalade (without both heating or the addition of pectin) and compare it with heat-induced marmalade. Sliced peel (flavedo) was soaked in 2% citric acid solution (pH 2.0). Albedo, endocarp and juice sacs were homogenized with 0.3% citric acid solution (pH 2.5). After soaking for 24 h, these were mixed and 50% or 60% sucrose of the total weight was added, then pressurized at 500 MPa or boiled (process A). Process B: all processing was done at pH 2.7. Peel of high pressure-induced marmalade maintained a natural color. Flavedo in heat-induced marmalade was softer than that of pressure-induced marmalade. There was no difference in viscosity between heat-induced and high pressure-induced marmalade. High pressure-induced marmalade with 50% sugar was preferred by a sensory test because fresh flavor and color were maintained.

  17. Effects of Pressure, Temperature, Treatment Time, and Storage on Rheological, Textural, and Structural Properties of Heat-Induced Chickpea Gels

    Science.gov (United States)

    Alvarez, María Dolores; Fuentes, Raúl; Canet, Wenceslao

    2015-01-01

    Pressure-induced gelatinization of chickpea flour (CF) was studied in combination with subsequent temperature-induced gelatinization. CF slurries (with 1:5 flour-to-water ratio) and CF in powder form were treated with high hydrostatic pressure (HHP), temperature (T), and treatment time (t) at three levels (200, 400, 600 MPa; 10, 25, 50 °C; 5, 15, 25 min). In order to investigate the effect of storage (S), half of the HHP-treated CF slurries were immediately analyzed for changes in oscillatory rheological properties under isothermal heating at 75 °C for 15 min followed by cooling to 25 °C. The other half of the HHP-treated CF slurries were refrigerated (at 4 °C) for one week and subsequently analyzed for changes in oscillatory properties under the same heating conditions as the unrefrigerated samples. HHP-treated CF in powder form was analyzed for changes in textural properties of heat-induced CF gels under isothermal heating at 90 °C for 5 min and subsequent cooling to 25 °C. Structural changes during gelatinization were investigated using microscopy. Pressure had a more significant effect on rheological and textural properties, followed by T and treatment t (in that order). Gel aging in HHP-treated CF slurries during storage was supported by rheological measurements. PMID:28231191

  18. Effects of Pressure, Temperature, Treatment Time, and Storage on Rheological, Textural, and Structural Properties of Heat-Induced Chickpea Gels

    Directory of Open Access Journals (Sweden)

    María Dolores Alvarez

    2015-04-01

    Full Text Available Pressure-induced gelatinization of chickpea flour (CF was studied in combination with subsequent temperature-induced gelatinization. CF slurries (with 1:5 flour-to-water ratio and CF in powder form were treated with high hydrostatic pressure (HHP, temperature (T, and treatment time (t at three levels (200, 400, 600 MPa; 10, 25, 50 °C; 5, 15, 25 min. In order to investigate the effect of storage (S, half of the HHP-treated CF slurries were immediately analyzed for changes in oscillatory rheological properties under isothermal heating at 75 °C for 15 min followed by cooling to 25 °C. The other half of the HHP-treated CF slurries were refrigerated (at 4 °C for one week and subsequently analyzed for changes in oscillatory properties under the same heating conditions as the unrefrigerated samples. HHP-treated CF in powder form was analyzed for changes in textural properties of heat-induced CF gels under isothermal heating at 90 °C for 5 min and subsequent cooling to 25 °C. Structural changes during gelatinization were investigated using microscopy. Pressure had a more significant effect on rheological and textural properties, followed by T and treatment t (in that order. Gel aging in HHP-treated CF slurries during storage was supported by rheological measurements.

  19. The Cytosolic DNA Sensor cGAS Forms an Oligomeric Complex with DNA and Undergoes Switch-like Conformational Changes in the Activation Loop

    Directory of Open Access Journals (Sweden)

    Xu Zhang

    2014-02-01

    Full Text Available The presence of DNA in the cytoplasm is a danger signal that triggers immune and inflammatory responses. Cytosolic DNA binds to and activates cyclic GMP-AMP (cGAMP synthase (cGAS, which produces the second messenger cGAMP. cGAMP binds to the adaptor protein STING and activates a signaling cascade that leads to the production of type I interferons and other cytokines. Here, we report the crystal structures of human cGAS in its apo form, representing its autoinhibited conformation as well as in its cGAMP- and sulfate-bound forms. These structures reveal switch-like conformational changes of an activation loop that result in the rearrangement of the catalytic site. The structure of DNA-bound cGAS reveals a complex composed of dimeric cGAS bound to two molecules of DNA. Functional analyses of cGAS mutants demonstrate that both the protein-protein interface and the two DNA binding surfaces are critical for cGAS activation. These results provide insights into the mechanism of DNA sensing by cGAS.

  20. The cytosolic DNA sensor cGAS forms an oligomeric complex with DNA and undergoes switch-like conformational changes in the activation loop.

    Science.gov (United States)

    Zhang, Xu; Wu, Jiaxi; Du, Fenghe; Xu, Hui; Sun, Lijun; Chen, Zhe; Brautigam, Chad A; Zhang, Xuewu; Chen, Zhijian J

    2014-02-13

    The presence of DNA in the cytoplasm is a danger signal that triggers immune and inflammatory responses. Cytosolic DNA binds to and activates cyclic GMP-AMP (cGAMP) synthase (cGAS), which produces the second messenger cGAMP. cGAMP binds to the adaptor protein STING and activates a signaling cascade that leads to the production of type I interferons and other cytokines. Here, we report the crystal structures of human cGAS in its apo form, representing its autoinhibited conformation as well as in its cGAMP- and sulfate-bound forms. These structures reveal switch-like conformational changes of an activation loop that result in the rearrangement of the catalytic site. The structure of DNA-bound cGAS reveals a complex composed of dimeric cGAS bound to two molecules of DNA. Functional analyses of cGAS mutants demonstrate that both the protein-protein interface and the two DNA binding surfaces are critical for cGAS activation. These results provide insights into the mechanism of DNA sensing by cGAS. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  1. The Cytosolic DNA Sensor cGAS Forms An Oligomeric Complex with DNA and Undergoes Switch-like Conformational Changes in the Activation Loop

    Science.gov (United States)

    Zhang, Xu; Wu, Jiaxi; Du, Fenghe; Xu, Hui; Sun, Lijun; Chen, Zhe; Brautigam, Chad A.; Zhang, Xuewu; Chen, Zhijian J.

    2014-01-01

    The presence of DNA in the cytoplasm is a danger signal that triggers immune and inflammatory responses. Cytosolic DNA binds to and activates cyclic GMP-AMP (cGAMP) synthase (cGAS), which produces the second messenger cGAMP. cGAMP binds to the adaptor protein STING and activates a signaling cascade that leads to the production of type-I interferons and other cytokines. Here we report the crystal structures of human cGAS in its apo form, representing its auto-inhibited conformation, as well as cGAMP-bound and sulfate-bound forms. These structures reveal switch-like conformational changes of an activation loop that result in the rearrangement of the catalytic site. The structure of DNA-bound cGAS reveals a complex composed of dimeric cGAS bound to two molecules of DNA. Functional analyses of cGAS mutants demonstrate that both the protein-protein interface and the two DNA binding surfaces are critical for cGAS activation. These results provide new insights into the mechanism of DNA sensing by cGAS. PMID:24462292

  2. Crystal structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase from the hyperthermophile Thermotoga maritima for insights into the coordination of conformational changes and an inhibitor binding.

    Science.gov (United States)

    Takenoya, Mihoko; Ohtaki, Akashi; Noguchi, Keiichi; Endo, Kiwamu; Sasaki, Yasuyuki; Ohsawa, Kanju; Yajima, Shunsuke; Yohda, Masafumi

    2010-06-01

    Isopentenyl diphosphate is a precursor of various isoprenoids and is produced by the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in plastids of plants, protozoa and many eubacteria. A key enzyme in the MEP pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), has been shown to be the target of fosmidomycin, which works as an antimalarial, antibacterial and herbicidal compound. In this paper, we report studies of kinetics and the crystal structures of the thermostable DXR from the hyperthermophile Thermotoga maritima. Unlike the mesophilic DXRs, Thermotoga DXR (tDXR) showed activity only with Mg(2+) at its growth temperature. We solved the crystal structures of tDXR with and without fosmidomycin. The structure without fosmidomycin but unexpectedly bound with 2-methyl-2,4-pentanediol (MPD), revealing a new extra space available for potential drug design. This structure adopted the closed form by rigid domain rotation but without the flexible loop over the active site, which was considered as a novel conformation. Further, the conserved Asp residue responsible for cation binding seemed to play an important role in adjusting the position of fosmidomycin. Taken together, our kinetic and the crystal structures illustrate the binding mode of fosmidomycin that leads to its slow, tight binding according to the conformational changes of DXR.

  3. Continuous wave W- and D-Band EPR spectroscopy offer “sweet-spots” for characterizing conformational changes and dynamics in intrinsically disordered proteins

    Energy Technology Data Exchange (ETDEWEB)

    Casey, Thomas M.; Liu, Zhanglong; Esquiaqui, Jackie M.; Pirman, Natasha L.; Milshteyn, Eugene; Fanucci, Gail E., E-mail: fanucci@chem.ufl.edu

    2014-07-18

    Highlights: • W- and D-Band line shapes are sensitive to motions in the 0.1–2 ns time regime. • These frequencies effectively report on conformational dynamics of IDPs. • W-band spectra reflecting helical formation in IA{sub 3} is experimentally demonstrated. - Abstract: Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for characterizing conformational sampling and dynamics in biological macromolecules. Here we demonstrate that nitroxide spectra collected at frequencies higher than X-band (∼9.5 GHz) have sensitivity to the timescale of motion sampled by highly dynamic intrinsically disordered proteins (IDPs). The 68 amino acid protein IA{sub 3}, was spin-labeled at two distinct sites and a comparison of X-band, Q-band (35 GHz) and W-band (95 GHz) spectra are shown for this protein as it undergoes the helical transition chemically induced by tri-fluoroethanol. Experimental spectra at W-band showed pronounced line shape dispersion corresponding to a change in correlation time from ∼0.3 ns (unstructured) to ∼0.6 ns (α-helical) as indicated by comparison with simulations. Experimental and simulated spectra at X- and Q-bands showed minimal dispersion over this range, illustrating the utility of SDSL EPR at higher frequencies for characterizing structural transitions and dynamics in IDPs.

  4. Convergent evolution of heat-inducibility during subfunctionalization of the Hsp70 gene family.

    Science.gov (United States)

    Krenek, Sascha; Schlegel, Martin; Berendonk, Thomas U

    2013-02-21

    Heat-shock proteins of the 70 kDa family (Hsp70s) are essential chaperones required for key cellular functions. In eukaryotes, four subfamilies can be distinguished according to their function and localisation in different cellular compartments: cytosol, endoplasmic reticulum, mitochondria and chloroplasts. Generally, multiple cytosol-type Hsp70s can be found in metazoans that show either constitutive expression and/or stress-inducibility, arguing for the evolution of different tasks and functions. Information about the hsp70 copy number and diversity in microbial eukaryotes is, however, scarce, and detailed knowledge about the differential gene expression in most protists is lacking. Therefore, we have characterised the Hsp70 gene family of Paramecium caudatum to gain insight into the evolution and differential heat stress response of the distinct family members in protists and to investigate the diversification of eukaryotic hsp70s focusing on the evolution of heat-inducibility. Eleven putative hsp70 genes could be detected in P. caudatum comprising homologs of three major Hsp70-subfamilies. Phylogenetic analyses revealed five evolutionarily distinct Hsp70-groups, each with a closer relationship to orthologous sequences of Paramecium tetraurelia than to another P. caudatum Hsp70-group. These highly diverse, paralogous groups resulted from duplications preceding Paramecium speciation, underwent divergent evolution and were subject to purifying selection. Heat-shock treatments were performed to test for differential expression patterns among the five Hsp70-groups as well as for a functional conservation within Paramecium. These treatments induced exceptionally high mRNA up-regulations in one cytosolic group with a low basal expression, indicative for the major heat inducible hsp70s. All other groups showed comparatively high basal expression levels and moderate heat-inducibility, signifying constitutively expressed genes. Comparative EST analyses for P. tetraurelia

  5. A Single Mutation at the Sheet Switch Region Results in Conformational Changes Favoring 6 Light-Chain Fibrillogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Hernández-Santoyo, A.; Del Pozo Yauner, L; Fuentes-Silva, D; Ortiz, E; Rudiño-Piñera, E; Sánchez-López, R; Horjales, E; Becerril, B; Rodríguez-Romero, A

    2010-01-01

    Systemic amyloid light-chain (LC) amyloidosis is a disease process characterized by the pathological deposition of monoclonal LCs in tissue. All LC subtypes are capable of fibril formation although {lambda} chains, particularly those belonging to the {lambda}6 type, are overrepresented. Here, we report the thermodynamic and in vitro fibrillogenic properties of several mutants of the {lambda}6 protein 6aJL2 in which Pro7 and/or His8 was substituted by Ser or Pro. The H8P and H8S mutants were almost as stable as the wild-type protein and were poorly fibrillogenic. In contrast, the P7S mutation decreased the thermodynamic stability of 6aJL2 and greatly enhanced its capacity to form amyloid-like fibrils in vitro. The crystal structure of the P7S mutant showed that the substitution induced both local and long-distance effects, such as the rearrangement of the VL (variable region of the light chain)-VL interface. This mutant crystallized in two orthorhombic polymorphs, P2{sub 1}2{sub 1}2{sub 1} and C222{sub 1}. In the latter, a monomer that was not arranged in the typical Bence-Jones dimer was observed for the first time. Crystal-packing analysis of the C222{sub 1} lattice showed the establishment of intermolecular {beta}-{beta} interactions that involved the N-terminus and {beta}-strand B and that these could be relevant in the mechanism of LC fibril formation. Our results strongly suggest that Pro7 is a key residue in the conformation of the N-terminal sheet switch motif and, through long-distance interactions, is also critically involved in the contacts that stabilized the VL interface in {lambda}6 LCs.

  6. Conformational heterogeneity and propagation of structural changes in the LOV2/Jalpha domain from Avena sativa phototropin 1 as recorded by temperature-dependent FTIR spectroscopy.

    Science.gov (United States)

    Alexandre, Maxime T A; van Grondelle, Rienk; Hellingwerf, Klaas J; Kennis, John T M

    2009-07-01

    Phototropins control phototropism, chloroplast movement, stomatal opening, and leaf expansion in plants. Phototropin 1 (phot1) is composed of a kinase domain linked to two blue light-sensing domains, LOV2 and LOV1, which bind flavin mononucleotide. Disruption of the interaction between the LOV2 domain and a helical segment named Jalpha, joining LOV to the kinase domain, induces the subsequent kinase activity of phototropin 1 and further-downstream signal transduction. Here we study the effects of temperature and hydration on the light-triggered signal propagation in the phot1 LOV2 domain of Avena sativa (AsLOV2/Jalpha), using Fourier transform infrared spectroscopy to unravel part of the molecular mechanism of phototropin 1. We report that AsLOV2/Jalpha shows an intense signal in the amide I and II regions, arising mainly from beta-sheet changes and the unbinding of the Jalpha helix from the Per-ARNT-Sim core and its subsequent partial unfolding. Importantly, these structural changes only occur under conditions of full hydration and at temperatures above 280 K. We characterized a newly isolated low-hydration intermediate that shows a downshift of high-frequency amide I signals and that possibly corresponds to loop tightening, without large beta-sheet or Jalpha structural changes. In addition, we report a heterogeneity in AsLOV2/Jalpha involving two different C(4)=O conformer populations, coexisting in the dark state and characterized by C(4)=O carbonyl frequencies at 1712 cm(-1) and 1694 cm(-1) that are attributable to a single H-bond and two H-bonds at this site, respectively. Such conformers display slightly shifted absorption spectra and cause a splitting of the 475-nm band in the ultraviolet/visible spectra of LOV domains at low temperature.

  7. Supplement data for conformation changes by some binding - ConfC | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available ed, and the Z score of distance is computed.The information about a regions of st...h residues are calculated, and the Z score of distance is computed. Finally, the regions of structural chang

  8. Molecular Dynamics Simulations of Ligand-Induced Flap Conformational Changes in Cathepsin-D-A Comparative Study.

    Science.gov (United States)

    Arodola, Olayide A; Soliman, Mahmoud E S

    2016-11-01

    The flap region in aspartic proteases is a unique structural feature to this class of enzymes, and found to have a profound impact on protein overall structure, function, and dynamics. Understanding the structure and dynamic behavior of the flap regions is crucial in the design of selective inhibitors against aspartic proteases. Cathepsin-D, an aspartic protease enzyme, has been implicated in a long list of degenerative diseases as well as breast cancer progression. Presented herein, for the first time, is a comprehensive description of the conformational flap dynamics of cathepsin-D using a comparative 50 ns "multiple" molecular dynamics simulations. Diverse collective metrics were proposed to accurately define flap dynamics. These are distance d1 between the flap tips residues (Gly79 and Met301); dihedral angle ϕ; in addition to TriCα angles Gly79-Asp33-Asp223, θ1 , and Gly79-Asp223-Met301, θ2 . The maximum distance attained throughout the simulation was 17.42 and 11.47 Å for apo and bound cathepsin-D, respectively, while the minimum distance observed was 8.75 and 6.32 Å for apo and bound cathepsin-D, respectively. The movement of the flap as well as the twist of the active pocket can properly be explained by measuring the angle, θ1 , between Gly79-Asp33-Met301 and correlating it with the distance Cα of the flap tip residues. The asymmetrical opening of the binding cavity was best described by the large shift of -6.26° to +20.94° in the dihedral angle, ϕ, corresponding to the full opening of the flap at a range of 31-33 ns. A wide-range of post-dynamic analyses was also applied in this report to supplement our findings. We believe that this report would augment current efforts in designing potent structure-based inhibitors against cathepsin-D in the treatment of breast cancer and other degenerative diseases. J. Cell. Biochem. 117: 2643-2657, 2016. © 2016 Wiley Periodicals, Inc.

  9. Medicare determinations and income-related monthly adjustment amounts to Medicare Part B premiums; conforming changes to regulations. Interim final rule with request for comments.

    Science.gov (United States)

    2013-09-18

    We are modifying our regulations regarding Medicare Part B income-related monthly adjustment amounts (IRMAA) in order to conform to changes made to the Social Security Act (Act) by the Affordable Care Act. This rule freezes the modified adjusted gross income threshold and ranges from 2011 through 2019 and removes the requirement that beneficiaries consent to our release of Internal Revenue Service (IRS) information to the U.S. Department of Health and Human Services (HHS) for the purpose of adjudicating any appeal of an IRMAA to the Part B premium subsidy. We are also removing provisions that phased in IRMAA between 2007 and 2009 and updating a citation to reflect the transfer of authority for hearing appeals under Title XVIII of the Act from the Social Security Administration to HHS.

  10. Conformational changes of the bacterial type I ATP-binding cassette importer HisQMP2 at distinct steps of the catalytic cycle.

    Science.gov (United States)

    Heuveling, Johanna; Frochaux, Violette; Ziomkowska, Joanna; Wawrzinek, Robert; Wessig, Pablo; Herrmann, Andreas; Schneider, Erwin

    2014-01-01

    Prokaryotic solute binding protein-dependent ATP-binding cassette import systems are divided into type I and type II and mechanistic differences in the transport process going along with this classification are under intensive investigation. Little is known about the conformational dynamics during the catalytic cycle especially concerning the transmembrane domains. The type I transporter for positively charged amino acids from Salmonella enterica serovar Typhimurium (LAO-HisQMP2) was studied by limited proteolysis in detergent solution in the absence and presence of co-factors including ATP, ADP, LAO/arginine, and Mg(2+) ions. Stable peptide fragments could be obtained and differentially susceptible cleavage sites were determined by mass spectrometry as Lys-258 in the nucleotide-binding subunit, HisP, and Arg-217/Arg-218 in the transmembrane subunit, HisQ. In contrast, transmembrane subunit HisM was gradually degraded but no stable fragment could be detected. HisP and HisQ were equally resistant under pre- and post-hydrolysis conditions in the presence of arginine-loaded solute-binding protein LAO and ATP/ADP. Some protection was also observed with LAO/arginine alone, thus reflecting binding to the transporter in the apo-state and transmembrane signaling. Comparable digestion patterns were obtained with the transporter reconstituted into proteoliposomes and nanodiscs. Fluorescence lifetime spectroscopy confirmed the change of HisQ(R218) to a more apolar microenvironment upon ATP binding and hydrolysis. Limited proteolysis was subsequently used as a tool to study the consequences of mutations on the transport cycle. Together, our data suggest similar conformational changes during the transport cycle as described for the maltose ABC transporter of Escherichia coli, despite distinct structural differences between both systems.

  11. Polyethylene damage and deformation on fixed-bearing, non-conforming unicondylar knee replacements corresponding to progressive changes in alignment and fixation.

    Science.gov (United States)

    Harman, Melinda K; Schmitt, Sabine; Rössing, Sven; Banks, Scott A; Sharf, Hans-Peter; Viceconti, Marco; Hodge, W Andrew

    2010-07-01

    Deviations from nominal alignment of unicondylar knee replacements impact knee biomechanics, including the load and stress distribution at the articular contact surfaces. This study characterizes relationships between the biomechanical environment, distinguished by progressive changes in alignment and fixation, and articular damage and deformation in a consecutive series of retrieved unicondylar knee replacements. Twenty seven fixed-bearing, non-conforming unicondylar knee replacements of one design were retrieved after 2 to 13 years of in vivo function. The in vivo biomechanical environment was characterized by grading component migration measured from full-length radiographs and grading component fixation based on intraoperative manual palpation. Articular damage patterns and linear deformation on the polyethylene inserts were measured using optical photogrammetry and contact point digitization. Articular damage patterns and surface deformation on the explanted polyethylene inserts corresponded to progressive changes in component alignment and fixation. Component migration produced higher deformation rates, whereas loosening contributed to larger damage areas but lower deformation rates. Migration and loosening of the femoral component, but not the tibial component, were factors contributing to large regions of abrasion concentrated on the articular periphery. Classifying component migration and fixation at revision proved useful for distinguishing common biomechanical conditions associated with the varied polyethylene damage patterns and linear deformation for this fixed-bearing, non-conforming design. Pre-clinical evaluations of unicondylar knee replacements that are capable of reproducing variations in clinical alignment and predicting the observed wear mechanisms are necessary to better understand the impact of knee biomechanics and design on unicondylar knee replacement longevity. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  12. Handheld Chem/Biosensor Using Extreme Conformational Changes in Designed Binding Proteins to Enhance Surface Plasmon Resonance (SPR)

    Science.gov (United States)

    2016-04-01

    detection system for chemical and biological toxins . Surface Plasmon Resonance (SPR), protein design, protein engineering, supercharged protein ...chemical and biological toxins . Keywords: Surface Plasmon Resonance (SPR), protein design, protein engineering, supercharged protein , metamaterials...even this small index change, should be capable of detecting larger target molecules, such as proteins or even viral or bacterial pathogens, which

  13. Effects of the I559P gp41 change on the conformation and function of the human immunodeficiency virus (HIV-1 membrane envelope glycoprotein trimer.

    Directory of Open Access Journals (Sweden)

    Nirmin Alsahafi

    Full Text Available The mature human immunodeficiency virus (HIV-1 envelope glycoprotein (Env trimer is produced by proteolytic cleavage of a precursor and consists of three gp120 exterior and three gp41 transmembrane subunits. The metastable Env complex is induced to undergo conformational changes required for virus entry by the binding of gp120 to the receptors, CD4 and CCR5/CXCR4. An isoleucine-to-proline change (I559P in the gp41 ectodomain has been used to stabilize soluble forms of HIV-1 Env trimers for structural characterization and for use as immunogens. In the native membrane-anchored HIV-1BG505 Env, the I559P change modestly decreased proteolytic maturation, increased the non-covalent association of gp120 with the Env trimer, and resulted in an Env conformation distinctly different from that of the wild-type HIV-1BG505 Env. Compared with the wild-type Env, the I559P Env was recognized inefficiently by polyclonal sera from HIV-1-infected individuals, by several gp41-directed antibodies, by some antibodies against the CD4-binding site of gp120, and by antibodies that preferentially recognize the CD4-bound Env. Some of the gp120-associated antigenic differences between the wild-type HIV-1BG505 Env and the I559P mutant were compensated by the SOS disulfide bond between gp120 and gp41, which has been used to stabilize cleaved soluble Env trimers. Nonetheless, regardless of the presence of the SOS changes, Envs with proline 559 were recognized less efficiently than Envs with isoleucine 559 by the VRC01 neutralizing antibody, which binds the CD4-binding site of gp120, and the PGT151 neutralizing antibody, which binds a hybrid gp120-gp41 epitope. The I559P change completely eliminated the ability of the HIV-1BG505 Env to mediate cell-cell fusion and virus entry, and abolished the capacity of the SOS Env to support virus infection in the presence of a reducing agent. These results suggest that differences exist between the quaternary structures of functional Env

  14. Prediction of conformational changes by single mutation in the hepatitis B virus surface antigen (HBsAg identified in HBsAg-negative blood donors

    Directory of Open Access Journals (Sweden)

    Roni Martono

    2010-11-01

    Full Text Available Abstract Background Selection of hepatitis B virus (HBV by host immunity has been suggested to give rise to variants with amino acid substitutions at or around the 'a' determinant of the surface antigen (HBsAg, the main target of antibody neutralization and diagnostic assays. However, there have never been successful attempts to provide evidence for this hypothesis, partly because the 3 D structure of HBsAg molecules has not been determined. Tertiary structure prediction of HBsAg solely from its primary amino acid sequence may reveal the molecular energetic of the mutated proteins. We carried out this preliminary study to analyze the predicted HBsAg conformation changes of HBV variants isolated from Indonesian blood donors undetectable by HBsAg assays and its significance, compared to other previously-reported variants that were associated with diagnostic failure. Results Three HBV variants (T123A, M133L and T143M and a wild type sequence were analyzed together with frequently emerged variants T123N, M133I, M133T, M133V, and T143L. Based on the Jameson-Wolf algorithm for calculating antigenic index, the first two amino acid substitutions resulted in slight changes in the antigenicity of the 'a' determinant, while all four of the comparative variants showed relatively more significant changes. In the pattern T143M, changes in antigenic index were more significant, both in its coverage and magnitude, even when compared to variant T143L. These data were also partially supported by the tertiary structure prediction, in which the pattern T143M showed larger shift in the HBsAg second loop structure compared to the others. Conclusions Single amino acid substitutions within or near the 'a' determinant of HBsAg may alter antigenicity properties of variant HBsAg, which can be shown by both its antigenic index and predicted 3 D conformation. Findings in this study emphasize the significance of variant T143M, the prevalent isolate with highest degree of

  15. Conformal transformations and conformal invariance in gravitation

    CERN Document Server

    Dabrowski, Mariusz P; Blaschke, David B

    2008-01-01

    Conformal transformations are frequently used tools in order to study relations between various theories of gravity and Einstein relativity. Because of that, in this paper we discuss the rules of conformal transformations for geometric quantities in general relativity. In particular, we discuss the conformal transformations of the matter energy-momentum tensor. We thoroughly discuss the latter and show the subtlety of the conservation law (i.e., the geometrical Bianchi identity) imposed in one of the conformal frames in reference to the other. The subtlety refers to the fact that conformal transformation ``creates'' an extra matter term composed of the conformal factor which enters the conservation law. In an extreme case of the flat original spacetime the matter is ``created'' due to work done by the conformal transformation to bend the spacetime which was originally flat. We also discuss how to construct the conformally invariant gravity which, in the simplest version, is a special case of the Brans-Dicke t...

  16. MRI temperature map reconstruction directly from k-space with compensation for heating-induced geometric distortions

    Science.gov (United States)

    Gaur, Pooja; Grissom, William A.

    2017-03-01

    Proton resonance frequency (PRF) change is used to measure tissue heating, but also distorts the image and causes geometric distortions in temperature estimates in the same manner as other chemical shift distortions if left uncompensated. We propose an algorithm that produces PRF temperature maps free of these distortions by fitting a signal model directly to acquired k-space data that accounts for PRF-induced phase both up to and during the readout. We also introduce a faster method compatible with Cartesian data that corrects distortions from image-domain temperature maps. Gel heating experiments show the proposed CS compensation algorithms correct magnitude image artifacts and hotspot distortions. Without CS compensation, thermal dose values are overestimated in spiral data, and are spatially offset in 2DFT and EPI data. Compensating for heat-induced CS distortions improves the accuracy of temperature change and thermal dose measurements, and can have a significant positive impact on clinical and research applications of PRF-shift thermometry.

  17. Role of Heavy Meromyosin in Heat-Induced Gelation in Low Ionic Strength Solution Containing L-Histidine.

    Science.gov (United States)

    Hayakawa, Toru; Yoshida, Yuri; Yasui, Masanori; Ito, Toshiaki; Wakamatsu, Jun-ichi; Hattori, Akihito; Nishimura, Takanori

    2015-08-01

    The gelation of myosin has a very important role in meat products. We have already shown that myosin in low ionic strength solution containing L-histidine forms a transparent gel after heating. To clarify the mechanism of this unique gelation, we investigated the changes in the nature of myosin subfragments during heating in solutions with low and high ionic strengths with and without L-histidine. The hydrophobicity of myosin and heavy meromyosin (HMM) in low ionic strength solution containing L-histidine was lower than in high ionic strength solution. The SH contents of myosin and HMM in low ionic strength solution containing l-histidine did not change during the heating process, whereas in high ionic strength solution they decreased slightly. The heat-induced globular masses of HMM in low ionic strength solution containing L-histidine were smaller than those in high ionic strength solution. These findings suggested that the polymerization of HMM molecules by heating was suppressed in low ionic strength solution containing L-histidine, resulting in formation of the unique gel. © 2015 Institute of Food Technologists®

  18. On the mechanism of biological methane formation: structural evidence for conformational changes in methyl-coenzyme M reductase upon substrate binding.

    Science.gov (United States)

    Grabarse, W; Mahlert, F; Duin, E C; Goubeaud, M; Shima, S; Thauer, R K; Lamzin, V; Ermler, U

    2001-05-25

    Methyl-coenzyme M reductase (MCR) catalyzes the final reaction of the energy conserving pathway of methanogenic archaea in which methylcoenzyme M and coenzyme B are converted to methane and the heterodisulfide CoM-S-S-CoB. It operates under strictly anaerobic conditions and contains the nickel porphinoid F430 which is present in the nickel (I) oxidation state in the active enzyme. The known crystal structures of the inactive nickel (II) enzyme in complex with coenzyme M and coenzyme B (MCR-ox1-silent) and in complex with the heterodisulfide CoM-S-S-CoB (MCR-silent) were now refined at 1.16 A and 1.8 A resolution, respectively. The atomic resolution structure of MCR-ox1-silent describes the exact geometry of the cofactor F430, of the active site residues and of the modified amino acid residues. Moreover, the observation of 18 Mg2+ and 9 Na+ ions at the protein surface of the 300 kDa enzyme specifies typical constituents of binding sites for either ion. The MCR-silent and MCR-ox1-silent structures differed in the occupancy of bound water molecules near the active site indicating that a water chain is involved in the replenishment of the active site with water molecules. The structure of the novel enzyme state MCR-red1-silent at 1.8 A resolution revealed an active site only partially occupied by coenzyme M and coenzyme B. Increased flexibility and distinct alternate conformations were observed near the active site and the substrate channel. The electron density of the MCR-red1-silent state aerobically co-crystallized with coenzyme M displayed a fully occupied coenzyme M-binding site with no alternate conformations. Therefore, the structure was very similar to the MCR-ox1-silent state. As a consequence, the binding of coenzyme M induced specific conformational changes that postulate a molecular mechanism by which the enzyme ensures that methylcoenzyme M enters the substrate channel prior to coenzyme B as required by the active-site geometry. The three different

  19. Physical changes on the radiation and heat induced poly(Vinyl alcohol) fibers

    Energy Technology Data Exchange (ETDEWEB)

    Nho, Young Chang; Park, Kyung Ran; Park, Jong Seok; Lim, Youn Mook; Kang, Phil Hyun [Research Division for Industry and Environment, Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2016-11-15

    Poly(vinyl alcohol) (PVA) has high tensile strength and flexibility as well as oxygen barrier properties. The water, which acts as a plasticizer, reduces the tensile strength of PVA and swells PVA as well. It needs to have high water-resistant depending on its applications, especially for the use as tire cords. The aim of this work was to investigate the effect of the annealing temperature and radiation on the Gel content and tensile strength of PVA fbers. For this purpose, gel fraction and tensile strength were determined and analyzed. Annealing was performed in the temperature range of 120-180℃ for 10 min. PVA fbers were irradiated by using a conventional electron beam irradiation at a radiation dose of 50⁓200 kGy, at a dose of 10 kGy/pass at room temperature (acceleration voltage of 1 MeV). Annealing markedly increased the Gel content and the tensile strength of PVA fbers. However irradiation lowered the Gel content and the tensile strength of PVA fbers because they were degraded easily by radiation.

  20. Predictive response surface model for heat-induced rheological changes and aggregation of whey protein concentrate.

    Science.gov (United States)

    Alvarez, Pedro A; Emond, Charles; Gomaa, Ahmed; Remondetto, Gabriel E; Subirade, Muriel

    2015-02-01

    Whey proteins are now far more than a by-product of cheese processing. In the last 2 decades, food manufacturers have developed them as ingredients, with the dairy industry remaining as a major user. For many applications, whey proteins are modified (denatured) to alter their structure and functional properties. The objective of this research was to study the influence of 85 to 100 °C, with protein concentration of 8% to 12%, and treatment times of 5 to 30 min, while measuring rheological properties (storage modulus, loss modulus, and complex viscosity) and aggregation (intermolecular beta-sheet formation) in dispersions of whey protein concentrate (WPC). A Box-Behnken Response Surface Methodology modeled the heat denaturation of liquid sweet WPC at 3 variables and 3 levels. The model revealed a very significant fit for viscoelastic properties, and a lesser fit for protein aggregation, at temperatures not previously studied. An exponential increase of rheological parameters was governed by protein concentration and temperature, while a modest linear relationship of aggregation was governed by temperature. Models such as these can serve as valuable guides to the ingredient and dairy industries to develop target products, as whey is a major ingredient in many functional foods.

  1. Extensive dry heating-induced changes in physicochemical and immunological properties of whey proteins

    NARCIS (Netherlands)

    Liu, Fahui

    2016-01-01

    Baked milk products, e.g. milk-protein containing muffins or baked cheese, can be tolerated by most cow’s milk allergic subjects. These products were also reported to contribute to the development of immune tolerance in allergic subjects. The main objective of this thesis was to investigate th

  2. Nucleotide binding triggers a conformational change of the CBS module of the magnesium transporter CNNM2 from a twisted towards a flat structure.

    Science.gov (United States)

    Corral-Rodríguez, María Ángeles; Stuiver, Marchel; Abascal-Palacios, Guillermo; Diercks, Tammo; Oyenarte, Iker; Ereño-Orbea, June; de Opakua, Alain Ibáñez; Blanco, Francisco J; Encinar, José Antonio; Spiwok, Vojtêch; Terashima, Hiroyuki; Accardi, Alessio; Müller, Dominik; Martínez-Cruz, Luis Alfonso

    2014-11-15

    Recent studies suggest CNNM2 (cyclin M2) to be part of the long-sought basolateral Mg2+ extruder at the renal distal convoluted tubule, or its regulator. In the present study, we explore structural features and ligand-binding capacities of the Bateman module of CNNM2 (residues 429-584), an intracellular domain structurally equivalent to the region involved in Mg2+ handling by the bacterial Mg2+ transporter MgtE, and AMP binding by the Mg2+ efflux protein CorC. Additionally, we studied the structural impact of the pathogenic mutation T568I located in this region. Our crystal structures reveal that nucleotides such as AMP, ADP or ATP bind at only one of the two cavities present in CNNM2429-584. Mg2+ favours ATP binding by alleviating the otherwise negative charge repulsion existing between acidic residues and the polyphosphate group of ATP. In crystals CNNM2429-584 forms parallel dimers, commonly referred to as CBS (cystathionine β-synthase) modules. Interestingly, nucleotide binding triggers a conformational change in the CBS module from a twisted towards a flat disc-like structure that mostly affects the structural elements connecting the Bateman module with the transmembrane region. We furthermore show that the T568I mutation, which causes dominant hypomagnesaemia, mimics the structural effect induced by nucleotide binding. The results of the present study suggest that the T568I mutation exerts its pathogenic effect in humans by constraining the conformational equilibrium of the CBS module of CNNM2, which becomes 'locked' in its flat form.

  3. Acidic pH triggers conformational changes at the NH2-terminal propeptide of the precursor of pulmonary surfactant protein B to form a coiled coil structure.

    Science.gov (United States)

    Bañares-Hidalgo, A; Pérez-Gil, J; Estrada, P

    2014-07-01

    Pulmonary surfactant protein SP-B is synthesized as a larger precursor, proSP-B. We report that a recombinant form of human SP-BN forms a coiled coil structure at acidic pH. The protonation of a residue with pK=4.8±0.06 is the responsible of conformational changes detected by circular dichroism and intrinsic fluorescence emission. Sedimentation velocity analysis showed protein oligomerisation at any pH condition, with an enrichment of the species compatible with a tetramer at acidic pH. Low 2,2,2,-trifluoroethanol concentration promoted β-sheet structures in SP-BN, which bind Thioflavin T, at acidic pH, whereas it promoted coiled coil structures at neutral pH. The amino acid stretch predicted to form β-sheet parallel association in SP-BN overlaps with the sequence predicted by several programs to form coiled coil structure. A synthetic peptide ((60)W-E(85)) designed from the sequence of the amino acid stretch of SP-BN predicted to form coiled coil structure showed random coil conformation at neutral pH but concentration-dependent helical structure at acidic pH. Sedimentation velocity analysis of the peptide indicated monomeric state at neutral pH (s20, w=0.55S; Mr~3kDa) and peptide association (s20, w=1.735S; Mr=~14kDa) at acidic pH, with sedimentation equilibrium fitting to a Monomer-Nmer-Mmer model with N=6 and M=4 (Mr=14692Da). We propose that protein oligomerisation through coiled-coil motifs could then be a general feature in the assembly of functional units in saposin-like proteins in general and in the organization of SP-B in a functional surfactant, in particular. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Monoclonal antibody against brain calmodulin-dependent protein kinase type II detects putative conformational changes induced by Ca/sup 2 +/-calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    LeVine, H. III; Su, J.L.; Sahyoun, N.E.

    1988-08-23

    A mouse monoclonal IgG1 antibody has been generated against the soluble form of the calmodulin-dependent protein kinase type II. This antibody recognizes both the soluble and cytoskeletal forms of the enzyme, requiring Ca/sup 2 +/ for the interaction. Other divalent cations such as Zn/sup 2 +/, Mn/sup 2 +/, Cd/sup 2 +/, Co/sup 2 +/, and Ni/sup 2 +/ will substitute for Ca/sup 2 +/, while Mg/sup 2 +/ and Ba/sup 2 +/ will not. The antibody reacts with both the ..cap alpha..- and ..beta..-subunits on Western blots in a similar Ca/sup 2 +/-dependent fashion but with a lower sensitivity. The affinity of the antibody for the kinase is 0.13 nM determined by displacement of /sup 125/I Bolton-Hunter-labeled kinase with unlabeled enzyme. Calmodulin and antibody reciprocally potentiate each other's interaction with the enzyme. This is illustrated both by direct binding studies and by a decrease of the K/sub m app/ for calmodulin and an increase in the V/sub max/ for the autophosphorylation reaction of the enzyme. The antibody thus appears to recognize and stabilize a conformation of the kinase which favors calmodulin binding although it does not itself activate the kinase in the absence of calmodulin. Since the M/sub r/ 30,000 catalytic fragment of the kinase is not immunoreactive, either the antibody combining site of the kinase must be present in the noncatalytic portion of the protein along with the calmodulin binding site or proteolysis interferes with the putative Ca/sup 2 +/-dependent conformational change. Thus, monoclonal antibodies can be useful tools in elucidating the mechanism by which Ca/sup 2 +/ and calmodulin act on the kinase molecule.

  5. Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET of Dihydropyridine Receptor (DHPR β1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes.

    Directory of Open Access Journals (Sweden)

    Dipankar Bhattacharya

    Full Text Available The dihydropyridine receptor (DHPR β1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1 complex is still debatable. We used fluorescence resonance energy transfer (FRET to probe proximity relationships within the β1a subunit in cultured skeletal myotubes lacking or expressing RyR1. The fluorescein biarsenical reagent FlAsH was used as the FRET acceptor, which exhibits fluorescence upon binding to specific tetracysteine motifs, and enhanced cyan fluorescent protein (CFP was used as the FRET donor. Ten β1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β1a with either carboxyl or amino terminal fused CFP. FRET efficiency was largest when CCPGCC was positioned next to CFP, and significant intramolecular FRET was observed for all constructs suggesting that in situ the β1a subunit has a relatively compact conformation in which the carboxyl and amino termini are not extended. Comparison of the FRET efficiency in wild type to that in dyspedic (lacking RyR1 myotubes revealed that in only one construct (H458 CCPGCC β1a -CFP FRET efficiency was specifically altered by the presence of RyR1. The present study reveals that the C-terminal of the β1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β1a and RyR1 in resting myotubes.

  6. Kinetic flow dichroism study of conformational changes in supercoiled DNA induced by ethidium bromide and noncovalent and covalent binding of benz[a]pyrene diol epoxide.

    Science.gov (United States)

    Yoshida, H; Swenberg, C E; Geacintov, N E

    1987-03-10

    The dynamic conformational changes due to the noncovalent intercalative binding of ethidium bromide and racemic trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), and the covalent binding of BPDE to supercoiled phi X174 DNA, have been studied by gel electrophoresis and a novel application of a kinetic flow linear dichroism technique. The magnitude of the linear dichroism (delta A) of the DNA oriented in the flow gradient is sensitive to the hydrodynamic shape of the DNA molecule which is affected by the binding of the drug or the carcinogen BPDE. While the linear dichroism of ethidium bromide supercoiled DNA is time independent, the delta A spectra of BPDE-DNA reaction mixtures vary on time scales of minutes, which correspond to the reaction rate constant of BPDE to form 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene hydrolysis products and covalent DNA adducts. The rapid noncovalent intercalation of BPDE causes an initial large increase in delta A (up to 250%, corresponding to the dichroism observed with relaxed circular DNA), followed by a slower decrease in the linear dichroism signal. This decrease in delta A is attributed to the removal of intercalated diol epoxide molecules and the resulting reversible increase in the number of superhelical turns. The kinetic flow dichroism spectra indicate that the noncovalent BPDE-DNA complexes are intercalative in nature, while the covalent adducts are characterized by a very different conformation in which the long axes of the pyrenyl residues are oriented at a large angle with respect to the average orientation of the planes of the DNA bases.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Conformal isoparametric hypersurfaces with two distinct conformal principal curvatures in conformal space

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The conformal geometry of regular hypersurfaces in the conformal space is studied.We classify all the conformal isoparametric hypersurfaces with two distinct conformal principal curvatures in the conformal space up to conformal equivalence.

  8. Spectral characterization of the binding and conformational changes of serum albumins upon interaction with an anticancer drug, anastrozole

    Science.gov (United States)

    Punith, Reeta; Seetharamappa, J.

    2012-06-01

    The present study employed different optical spectroscopic techniques viz., fluorescence, FTIR, circular dichroism (CD) and UV-vis absorption spectroscopy to investigate the mechanism of interaction of an anticancer drug, anastrozole (AZ) with transport proteins viz., bovine serum albumin (BSA) and human serum albumin (HSA). The drug, AZ quenched the intrinsic fluorescence of protein and the analysis of results revealed the presence of dynamic quenching mechanism. The binding characteristics of drug-protein were computed. The thermodynamic parameters, enthalpy change (ΔH°) and entropy change (ΔS°) were calculated to be +92.99 kJ/mol and +159.18 J/mol/K for AZ-BSA and, +99.43 kJ/mol and +159.19 J/mol/K for AZ-HSA, respectively. These results indicated that the hydrophobic forces stabilized the interaction between the drug and protein. CD, FTIR, absorption, synchronous and 3D fluorescence results indicated that the binding of AZ to protein induced structural perturbation in both serum albumins. The distance, r between the drug and protein was calculated based on the theory of Förster's resonance energy transfer and found to be 5.9 and 6.24 nm, respectively for AZ-BSA and AZ-HSA.

  9. Study of structural and conformational change in cytochrome, C through molecular dynamic simulation in presence of gold nanoparticles

    Science.gov (United States)

    Moudgil, Lovika; Singh, Baljinder; Kaura, Aman; Singh, Gurinder; Tripathi, S. K.; Saini, G. S. S.

    2017-05-01

    Proteins are the most abundant organic molecules in living system having diverse structures and various functions than the other classes of macromolecules. We have done Molecular Dynamics (MD) simulation of the Cytochrome,C (Cyt,c) protein found in plants, animals and many unicellular animals in the presence of gold nanoparticles (Au NPs). MD results helped to recognize the amino acids that play important role to make the interaction possible between protein and gold surface. In the present study we have examined the structural change of protein in the presence of gold surface and its adsorption on the surface through MD simulations with the help of Gold-Protein (GolP) force field. Results were further analyzed to understand the protein interaction up to molecular level.

  10. Decipher the mechanisms of protein conformational changes induced by nucleotide binding through free-energy landscape analysis: ATP binding to Hsp70.

    Directory of Open Access Journals (Sweden)

    Adrien Nicolaï

    Full Text Available ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD of Hsp70 propagates a signal to its substrate-binding domain (SBD. Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in

  11. Heat-inducible Cre-lox system for marker excision in transgenic rice

    Indian Academy of Sciences (India)

    Abhilasha Khattri; Soumen Nandy; Vibha Srivastava

    2011-03-01

    The present study assessed the efficacy of a heat-inducible cre gene for conditional removal of the marker gene from a rice genome via Cre-lox recombination. A cre gene controlled by the soybean heat-shock promoter was introduced into the rice genome along with the recombination target (lox) construct. Cre-mediated recombination was expected to remove the marker gene and activate the promoter-less GUS gene. Six transgenic lines displayed well-regulated heat-inducible Cre activity in the callus. However, only one line that contained a single copy of the cre gene maintained this property in the regenerated plants and their progeny. Marker-free progeny were obtained from the plant that was heat-treated at the seedling stage, indicating the inheritance of the recombination `footprint’. The presence of the `footprint’ was verified by polymerase chain reaction and Southern analysis. Therefore, the cre gene controlled by the soybean heat-shock promoter is an effective tool for conditional removal of the marker gene in rice.

  12. Homologous recombination preferentially repairs heat-induced DNA double-strand breaks in mammalian cells.

    Science.gov (United States)

    Takahashi, Akihisa; Mori, Eiichiro; Nakagawa, Yosuke; Kajihara, Atsuhisa; Kirita, Tadaaki; Pittman, Douglas L; Hasegawa, Masatoshi; Ohnishi, Takeo

    2016-11-13

    Heat shock induces DNA double-strand breaks (DSBs), but the precise mechanism of repairing heat-induced damage is unclear. Here, we investigated the DNA repair pathways involved in cell death induced by heat shock. B02, a specific inhibitor of human RAD51 (homologous recombination; HR), and NU7026, a specific inhibitor of DNA-PK (non-homologous end-joining; NHEJ), were used for survival assays of human cancer cell lines with different p53-gene status. Mouse embryonic fibroblasts (MEFs) lacking Lig4 (NHEJ) and/or Rad54 (HR) were used for survival assays and a phosphorylated histone H2AX at Ser139 (γH2AX) assay. MEFs lacking Rad51d (HR) were used for survival assays. SPD8 cells were used to measure HR frequency after heat shock. Human cancer cells were more sensitive to heat shock in the presence of B02 despite their p53-gene status, and the effect of B02 on heat sensitivity was specific to the G2 phase. Rad54-deficient MEFs were sensitive to heat shock and showed prolonged γH2AX signals following heat shock. Rad51d-deficient MEFs were also sensitive to heat shock. Moreover, heat shock-stimulated cells had increased HR. The HR pathway plays an important role in the survival of mammalian cells against death induced by heat shock via the repair of heat-induced DNA DSBs.

  13. Conformational changes induced by the A21G Flemish mutation in the amyloid precursor protein lead to increased Aβ production.

    Science.gov (United States)

    Tang, Tzu-Chun; Hu, Yi; Kienlen-Campard, Pascal; El Haylani, Laetitia; Decock, Marie; Van Hees, Joanne; Fu, Ziao; Octave, Jean-Noel; Constantinescu, Stefan N; Smith, Steven O

    2014-03-04

    Proteolysis of the β C-terminal fragment (β-CTF) of the amyloid precursor protein generates the Aβ peptides associated with Alzheimer's disease. Familial mutations in the β-CTF, such as the A21G Flemish mutation, can increase Aβ secretion. We establish how the Flemish mutation alters the structure of C55, the first 55 residues of the β-CTF, using FTIR and solid-state NMR spectroscopy. We show that the A21G mutation reduces β sheet structure of C55 from Leu17 to Ala21, an inhibitory region near the site of the mutation, and increases α-helical structure from Gly25 to Gly29, in a region near the membrane surface and thought to interact with cholesterol. Cholesterol also increases Aβ peptide secretion, and we show that the incorporation of cholesterol into model membranes enhances the structural changes induced by the Flemish mutant, suggesting a common link between familial mutations and the cellular environment.

  14. Structure of the active N-terminal domain of Ezrin. Conformational and mobility changes identify keystone interactions.

    Science.gov (United States)

    Smith, William James; Nassar, Nicolas; Bretscher, Anthony; Cerione, Richard A; Karplus, P Andrew

    2003-02-14

    Ezrin is a member of the ERM (ezrin, radixin, moesin) family of proteins that cross-link the actin cytoskeleton to the plasma membrane and also may function in signaling cascades that regulate the assembly of actin stress fibers. Here, we report a crystal structure for the free (activated) FERM domain (residues 2-297) of recombinant human ezrin at 2.3 A resolution. Structural comparison among the dormant moesin FERM domain structure and the three known active FERM domain structures (radixin, moesin, and now ezrin) allows the clear definition of regions that undergo structural changes during activation. The key regions affected are residues 135-150 and 155-180 in lobe F2 and residues 210-214 and 235-267 in lobe F3. Furthermore, we show that a large increase in the mobilities of lobes F2 and F3 accompanies activation, suggesting that their integrity is compromised. This leads us to propose a new concept that we refer to as keystone interactions. Keystone interactions occur when one protein (or protein part) contributes residues that allow another protein to complete folding, meaning that it becomes an integral part of the structure and would rarely dissociate. Such interactions are well suited for long-lived cytoskeletal protein interactions. The keystone interactions concept leads us to predict two specific docking sites within lobes F2 and F3 that are likely to bind target proteins.

  15. Substrate preference of the HIF-prolyl hydroxylase-2 (PHD2) and substrate-induced conformational change.

    Science.gov (United States)

    Pektas, Serap; Knapp, Michael J

    2013-09-01

    HIF prolyl-4-hydroxylase 2 (PHD2) is a non-heme Fe, 2-oxoglutarate (2OG) dependent dioxygenase that regulates the hypoxia inducible transcription factor (HIF) by hydroxylating two conserved prolyl residues in N-terminal oxygen degradation domain (NODD) and C-terminal oxygen degradation domain (CODD) of HIF-1α. Prior studies have suggested that the substrate preference of PHD2 arises from binding contacts with the β2β3 loop of PHD2. In this study we tested the substrate selectivity of PHD2 by kinetic competition assays, varied ionic strength, and global protein flexibility using amide H/D exchange (HDX). Our results revealed that PHD2 preferred CODD by 20-fold over NODD and that electrostatics influenced this effect. Global HDX monitored by mass spectrometry indicated that binding of Fe(II) and 2OG stabilized the overall protein structure but the saturating concentrations of either NODD or CODD caused an identical change in protein flexibility. These observations imply that both substrates stabilize the β2β3 loop to the same extent. Under unsaturated substrate conditions NODD led to a higher HDX rate than CODD due to its lower binding affinity to PHD2. Our results suggest that loop closure is the dominant contributor to substrate selectivity in PHD2.

  16. Bovine serum albumin conformational changes upon adsorption on titania and on hydroxyapatite and their relation with biomineralization.

    Science.gov (United States)

    Serro, A P; Bastos, M; Pessoa, J Costa; Saramago, B

    2004-09-01

    The biocompatibility of implant materials used for substitution of bone tissue depends on its ability to induce the deposition of a hydroxyapatite layer when in contact with body fluids. In previous work, some of the authors found that bovine serum albumin (BSA) promotes calcium phosphate deposition if preadsorbed on hydroxyapatite and retards precipitation if preadsorbed on titania. In the present study, we investigated the adsorption of BSA upon particles of titania and hydroxyapatite in order to understand the different role played by the protein on the mineralization of both biomaterials. The adsorption isotherms were determined and the structural changes induced by adsorption at different surface coverages were investigated by circular dichroism spectroscopy and differential scanning microcalorimetry. At low surface coverages, the adsorbed BSA molecules lost part of their alpha-helix content. However, at high surface coverages, corresponding to the plateau values of the adsorption isotherms, the BSA molecules did not undergo structural rearrangements upon adsorption. In the latter circumstances, the availability of BSA calcium binding sites, which should be responsible for inducing mineralization, depends on the electrostatic interactions between BSA and the sorbent surface. A possible explanation for the different mineralization behavior of hydroxyapatite and titania is advanced.

  17. Konjac flour improved textural and water retention properties of transglutaminase-mediated, heat-induced porcine myofibrillar protein gel: Effect of salt level and transglutaminase incubation.

    Science.gov (United States)

    Chin, Koo B; Go, Mi Y; Xiong, Youling L

    2009-03-01

    Functional properties of heat-induced gels prepared from microbial transglutaminase (TG)-treated porcine myofibrillar protein (MP) containing sodium caseinate with or without konjac flour (KF) under various salt concentrations (0.1, 0.3 and 0.6MNaCl) were evaluated. The mixed MP gels with KF exhibited improved cooking yields at all salt concentrations. TG treatment greatly enhanced gel strength and elasticity (storage modulus, G') at 0.6M NaCl, but not at lower salt concentrations. The combination of KF and TG improved the gel strength at 0.1 and 0.3M NaCl and G' at all salt concentrations, when compared with non-TG controls. Incubation of MP suspensions (sols) with TG promoted the disappearance of myosin heavy chain and the production of polymers. The TG-treated MP mixed gels had a compact structure, compared to those without TG, and the KF incorporation modified the gel matrix and increased its water-holding capacity. Results from differential scanning calorimetry suggested possible interactions of MP with KF, which may explain the changes in the microstructure of the heat-induced gels.

  18. Fourier transform-infrared studies on the effects of salt and drought stress on the chemical composition and pro-tein conformation changes in Arabidopsis leaves

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    We examined the changes of chemical composition and protein conformation in Arabidopsis leaves by Fourier transform-infrared (FT-IR) spectrometry Arabidopsis under 50 mmol/L NaCl salt and -0.5 mPa polyethylene glycols 8000 (PEG 8000) drought stress during the early stages of growth.We primarily analyzed the absorption band areas in the 1,745 cm-1 (ester),1,600-1,700 cm-1 (amide I),and 1,100 cm-1 (carbohydrate) changes under salt stress and drought stress within 24 hours.The results showed that ester content declined at the beginning and then increased steadily during 24 hours of drought stress.But under salt stress,it de-clined steadily,and it was about 40 percent of the control after 24 hours.The protein synthesis increased by 25 percent after one hour of salt stress and then reached about 85 percent of the control after 24 hours.Under drought stress,the protein synthesis de-creased and reached aminimal level at the 4-hr time point;it then recovered to the control level at the 24-hr point.The patterns of the accumulation of carbohydrates in the 1,100 cm-1 band areas resembled that of amide I band changes under drought stress and salt stress.Analyzing the ratio A1,627cm-1/A1,658cm-1 under drought stress revealed that the leaves’ entire protein structure maintained a higher-level ordered form than did those under salt stress.Thus our results indicate the existence of different strategies of the Arabidopsis adaptation to salt stress and drought stress.

  19. Organic compounds characteristics associated with heat-induced increases of water repellency in Australian eucalypt forest soils

    Science.gov (United States)

    Atanassova, Irena; Doerr, Stefan H.

    2010-05-01

    Ground surface heating during wildfires often leads to increased water repellency in soils. The effect of elevated soil temperature on water repellency has been investigated in many laboratory-based studies and temperature thresholds for increases in, and destruction of, water repellency have been established. However, little is known about the changes in organic compounds patterns and their chemical structure that associated with these changes. Here we report on the characterisation of the chemical changes of organic compounds associated with heat-induced increases in water repellency in Eucalypt soils of different repellency levels. Fires are very common in eucalypt forest environments and soils under eucalypt species exhibit one of the most severe repellency levels, providing an ideal study case. Three SE Australian eucalypt forest soils from different locations (two sands and one sandy loam) were heated in the laboratory for 10 min at 300° C. Laboratory heating resulted in extreme repellency in the three soils studied. Heated and unheated control samples were then extracted by accelerated solvent extraction (ASE) with iso-propanol/ammonia mixture (IPA/NH3 95:5). Extraction led to the elimination of any water repellency present both in the original (heated) and the control samples. Organic compounds in the IPA/NH3 solvent were measured in extracts of increasing polarity in order to solubilise the residue. Before heating, the total solvent extracts from the soils with sandy texture were dominated by n-alkanols, terpenoids, C16 acid, C29 alkane, β-sitosterol and polar compounds such as glycerol, monosaccharides and glycosides. Fatty acids with chain length over C20 were detected in the sandy soils, while the soil of heavier texture (sandy loam) lacked longer than C20 fatty acids and had lower concentrations of alkanols (exceeding C26 chain lenght) and alkanes (C29, C31). Alkane patterns were characterized by the predominance of C21 - C31 homologues with a

  20. Non-conformable, partial and conformable transposition

    DEFF Research Database (Denmark)

    König, Thomas; Mäder, Lars Kai

    2013-01-01

    Although member states are obliged to transpose directives into domestic law in a conformable manner and receive considerable time for their transposition activities, we identify three levels of transposition outcomes for EU directives: conformable, partially conformable and non-conformable....... Compared with existing transposition models, which do not distinguish between different transposition outcomes, we examine the factors influencing each transposition process by means of a competing risk analysis. We find that preference-related factors, in particular the disagreement of a member state...... and the Commission regarding a directive’s outcome, play a much more strategic role than has to date acknowledged in the transposition literature. Whereas disagreement of a member state delays conformable transposition, it speeds up non-conformable transposition. Disagreement of the Commission only prolongs...

  1. Study of conformational changes and protein aggregation of bovine serum albumin in presence of Sb(III) and Sb(V).

    Science.gov (United States)

    Verdugo, Marcelo; Ruiz Encinar, Jorge; Costa-Fernández, José Manuel; Menendez-Miranda, Mario; Bouzas-Ramos, Diego; Bravo, Manuel; Quiroz, Waldo

    2017-01-01

    Antimony is a metalloid that affects biological functions in humans due to a mechanism still not understood. There is no doubt that the toxicity and physicochemical properties of Sb are strongly related with its chemical state. In this paper, the interaction between Sb(III) and Sb(V) with bovine serum albumin (BSA) was investigated in vitro by fluorescence spectroscopy, and circular dichroism (CD) under simulated physiological conditions. Moreover, the coupling of the separation technique, asymmetric flow field-flow fractionation, with elemental mass spectrometry to understand the interaction of Sb(V) and Sb(III) with the BSA was also used. Our results showed a different behaviour of Sb(III) vs. Sb(V) regarding their effects on the interaction with the BSA. The effects in terms of protein aggregates and conformational changes were higher in the presence of Sb(III) compared to Sb(V) which may explain the differences in toxicity between both Sb species in vivo. Obtained results demonstrated the protective effect of GSH that modifies the degree of interaction between the Sb species with BSA. Interestingly, in our experiments it was possible to detect an interaction between BSA and Sb species, which may be related with the presence of labile complex between the Sb and a protein for the first time.

  2. Poly(N-isopropylacrylamide) thin films densely grafted onto gold surface: preparation, characterization, and dynamic AFM study of temperature-induced chain conformational changes.

    Science.gov (United States)

    Montagne, Franck; Polesel-Maris, Jérome; Pugin, Raphael; Heinzelmann, Harry

    2009-01-20

    Thermally responsive poly(N-isopropylacrylamide) (PNIPAM) films are attracting considerable attention since they offer the possibility to achieve reversible control over surface wettability and biocompatibility. In this paper, we first report a new and simple method for the grafting under melt of amine-terminated PNIPAM chains onto gold surfaces modified with a self-assembled monolayer (SAM) of reactive thiols. The formation of homogeneous tethered PNIPAM films, whose thickness can be tuned by adjusting polymer molecular weight or SAM reactivity, is evidenced by using the combination of ellipsometry, X-ray photon spectroscopy, infrared spectroscopy (PM-IRRAS), and atomic force microscopy. The calculation of grafting parameters from experimental measurements indicated the synthesis of densely grafted PNIPAM films and allowed us to predict a "brushlike" regime for the chains in good solvent. In a second part, the temperature-induced responsive properties are studied in situ by conducting dynamic AFM measurements using the amplitude modulation technique. Imaging in water environment first revealed the reversible modification of surface morphology below and above the theoretical lower critical solution temperature (LCST) of PNIPAM. Then, the determination of amplitude and phase approach curves at various temperatures provided direct measurement of the evolution of the damping factor, or similarly the dissipated energy, as a function of the probe indentation into the PNIPAM film. Most interestingly, we clearly showed the subtle and progressive thermally induced chain conformational change occurring at the scale of several nanometers around the expected LCST.

  3. Conformational Change of a Tryptophan Residue in BtuF Facilitates Binding and Transport of Cobinamide by the Vitamin B12 Transporter BtuCD-F

    Science.gov (United States)

    Mireku, S. A.; Ruetz, M.; Zhou, T.; Korkhov, V. M.; Kräutler, B.; Locher, K. P.

    2017-01-01

    BtuCD-F is an ABC transporter that mediates cobalamin uptake into Escherichia coli. Early in vivo data suggested that BtuCD-F might also be involved in the uptake of cobinamide, a cobalamin precursor. However, neither was it demonstrated that BtuCD-F indeed transports cobinamide, nor was the structural basis of its recognition known. We synthesized radiolabeled cyano-cobinamide and demonstrated BtuCD-catalyzed in vitro transport, which was ATP- and BtuF-dependent. The crystal structure of cobinamide-bound BtuF revealed a conformational change of a tryptophan residue (W66) in the substrate binding cleft compared to the structure of cobalamin-bound BtuF. High-affinity binding of cobinamide was dependent on W66, because mutation to most other amino acids substantially reduced binding. The structures of three BtuF W66 mutants revealed that tight packing against bound cobinamide was only provided by tryptophan and phenylalanine, in line with the observed binding affinities. In vitro transport rates of cobinamide and cobalamin were not influenced by the substitutions of BtuF W66 under the experimental conditions, indicating that W66 has no critical role in the transport reaction. Our data present the molecular basis of the cobinamide versus cobalamin specificity of BtuCD-F and provide tools for in vitro cobinamide transport and binding assays. PMID:28128319

  4. Crystal structures of a poplar thioredoxin peroxidase that exhibits the structure of glutathione peroxidases: insights into redox-driven conformational changes.

    Science.gov (United States)

    Koh, Cha San; Didierjean, Claude; Navrot, Nicolas; Panjikar, Santosh; Mulliert, Guillermo; Rouhier, Nicolas; Jacquot, Jean-Pierre; Aubry, André; Shawkataly, Omar; Corbier, Catherine

    2007-07-13

    Glutathione peroxidases (GPXs) are a group of enzymes that regulate the levels of reactive oxygen species in cells and tissues, and protect them against oxidative damage. Contrary to most of their counterparts in animal cells, the higher plant GPX homologues identified so far possess cysteine instead of selenocysteine in their active site. Interestingly, the plant GPXs are not dependent on glutathione but rather on thioredoxin as their in vitro electron donor. We have determined the crystal structures of the reduced and oxidized form of Populus trichocarpaxdeltoides GPX5 (PtGPX5), using a selenomethionine derivative. PtGPX5 exhibits an overall structure similar to that of the known animal GPXs. PtGPX5 crystallized in the assumed physiological dimeric form, displaying a pseudo ten-stranded beta sheet core. Comparison of both redox structures indicates that a drastic conformational change is necessary to bring the two distant cysteine residues together to form an intramolecular disulfide bond. In addition, a computer model of a complex of PtGPX5 and its in vitro recycling partner thioredoxin h1 is proposed on the basis of the crystal packing of the oxidized form enzyme. A possible role of PtGPX5 as a heavy-metal sink is also discussed.

  5. Structures of the Bacillus subtilis glutamine synthetase dodecamer reveal large intersubunit catalytic conformational changes linked to a unique feedback inhibition mechanism.

    Science.gov (United States)

    Murray, David S; Chinnam, Nagababu; Tonthat, Nam Ky; Whitfill, Travis; Wray, Lewis V; Fisher, Susan H; Schumacher, Maria A

    2013-12-13

    Glutamine synthetase (GS), which catalyzes the production of glutamine, plays essential roles in nitrogen metabolism. There are two main bacterial GS isoenzymes, GSI-α and GSI-β. GSI-α enzymes, which have not been structurally characterized, are uniquely feedback-inhibited by Gln. To gain insight into GSI-α function, we performed biochemical and cellular studies and obtained structures for all GSI-α catalytic and regulatory states. GSI-α forms a massive 600-kDa dodecameric machine. Unlike other characterized GS, the Bacillus subtilis enzyme undergoes dramatic intersubunit conformational alterations during formation of the transition state. Remarkably, these changes are required for active site construction. Feedback inhibition arises from a hydrogen bond network between Gln, the catalytic glutamate, and the GSI-α-specific residue, Arg(62), from an adjacent subunit. Notably, Arg(62) must be ejected for proper active site reorganization. Consistent with these findings, an R62A mutation abrogates Gln feedback inhibition but does not affect catalysis. Thus, these data reveal a heretofore unseen restructuring of an enzyme active site that is coupled with an isoenzyme-specific regulatory mechanism. This GSI-α-specific regulatory network could be exploited for inhibitor design against Gram-positive pathogens.

  6. Study of the mechanism of protonated histidine-induced conformational changes in the Zika virus dimeric envelope protein using accelerated molecular dynamic simulations.

    Science.gov (United States)

    Sun, Jixue; Li, Yang; Liu, Pi; Lin, Jianping

    2017-06-01

    The Zika virus has drawn worldwide attention because of the epidemic diseases it causes. It is a flavivirus that has an icosahedral protein shell constituted by an envelope glycoprotein (E-protein) and membrane protein (M-protein) in the mature virion. The multistep process of membrane fusion to infect the host cell is pH-induced. To understand the mechanism of the conformational changes in the (E-M)2 protein homodimer embedded in the membrane, two 200-ns accelerated dynamic simulations were performed under different pH conditions. The low pH condition weakens the interactions and correlations in both E-protein monomers and in the E-M heterodimer. The highly conserved residues, His249, His288, His323 and His446, are protonated under low pH conditions and play key roles in driving the fusion process. The analysis and discussion in this study may provide some insight into the molecular mechanism of Zika virus infection. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. The repeat domain of the type III effector protein PthA shows a TPR-like structure and undergoes conformational changes upon DNA interaction.

    Science.gov (United States)

    Murakami, Mário Tyago; Sforça, Mauricio Luis; Neves, Jorge Luiz; Paiva, Joice Helena; Domingues, Mariane Noronha; Pereira, André Luiz Araujo; Zeri, Ana Carolina de Mattos; Benedetti, Celso Eduardo

    2010-12-01

    Many plant pathogenic bacteria rely on effector proteins to suppress defense and manipulate host cell mechanisms to cause disease. The effector protein PthA modulates the host transcriptome to promote citrus canker. PthA possesses unusual protein architecture with an internal region encompassing variable numbers of near-identical tandem repeats of 34 amino acids termed the repeat domain. This domain mediates protein-protein and protein-DNA interactions, and two polymorphic residues in each repeat unit determine DNA specificity. To gain insights into how the repeat domain promotes protein-protein and protein-DNA contacts, we have solved the structure of a peptide corresponding to 1.5 units of the PthA repeat domain by nuclear magnetic resonance (NMR) and carried out small-angle X-ray scattering (SAXS) and spectroscopic studies on the entire 15.5-repeat domain of PthA2 (RD2). Consistent with secondary structure predictions and circular dichroism data, the NMR structure of the 1.5-repeat peptide reveals three α-helices connected by two turns that fold into a tetratricopeptide repeat (TPR)-like domain. The NMR structure corroborates the theoretical TPR superhelix predicted for RD2, which is also in agreement with the elongated shape of RD2 determined by SAXS. Furthermore, RD2 undergoes conformational changes in a pH-dependent manner and upon DNA interaction, and shows sequence similarities to pentatricopeptide repeat (PPR), a nucleic acid-binding motif structurally related to TPR. The results point to a model in which the RD2 structure changes its compactness as it embraces the DNA with the polymorphic diresidues facing the interior of the superhelix oriented toward the nucleotide bases.

  8. Effect of CdTe quantum dots size on the conformational changes of human serum albumin: results of spectroscopy and isothermal titration calorimetry.

    Science.gov (United States)

    Yang, Bingjun; Liu, Rutao; Hao, Xiaopeng; Wu, Yongzhong; Du, Jie

    2013-10-01

    Quantum dots (QDs) are recognized as some of the most promising candidates for future applications in biomedicine. However, concerns about their safety have delayed their widespread application. Human serum albumin (HSA) is the main protein component of the circulatory system. It is important to explore the interaction of QDs with HSA for the potential in vivo application of QDs. Herein, using spectroscopy and isothermal titration calorimetry (ITC), the effect of glutathione-capped CdTe quantum dots of different sizes on the HSA was investigated. After correction for the inner filter effect, the fluorescence emission spectra and synchronous fluorescence spectra showed that the microenvironment of aromatic acid residues in the protein was slightly changed when the glutathione (GSH)-cadmium telluride (CdTe) QDs was added, and GSH-CdTe QDs with larger particle size exhibited a much higher effect on HSA than the small particles. Although a ground-state complex between HSA and GSH-CdTe QDs was formed, the UV-vis absorption and circular dichroism spectroscopic results did not find appreciable conformational changes of HSA. ITC has been used for the first time to characterize the binding of QDs with HSA. The ITC results revealed that the binding was a thermodynamically spontaneous process mainly driven by hydrophobic interactions, and the binding constant tended to increase as the GSH-CdTe QDs size increased. These findings are helpful in understanding the bioactivities of QDs in vivo and can be used to assist in the design of biocompatible and stable QDs.

  9. Changes in Pulmonary Function After Three-Dimensional Conformal Radiotherapy, Intensity-Modulated Radiotherapy, or Proton Beam Therapy for Non-Small-Cell Lung Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Lopez Guerra, Jose L. [Department of Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Department of Radiation Oncology, Hospitales Universitarios Virgen del Rocio, Seville (Spain); Department of Medicine, Universitat Autonoma de Barcelona, Barcelona (Spain); Gomez, Daniel R., E-mail: dgomez@mdanderson.org [Department of Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Zhuang Yan; Levy, Lawrence B. [Department of Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Eapen, George [Department of Pulmonary Medicine, University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Liu, Hongmei; Mohan, Radhe; Komaki, Ritsuko; Cox, James D.; Liao Zhongxing [Department of Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas (United States)

    2012-07-15

    Purpose: To investigate the extent of change in pulmonary function over time after definitive radiotherapy for non-small-cell lung cancer (NSCLC) with modern techniques and to identify predictors of changes in pulmonary function according to patient, tumor, and treatment characteristics. Patients and Methods: We analyzed 250 patients who had received {>=}60 Gy radio(chemo)therapy for primary NSCLC in 1998-2010 and had undergone pulmonary function tests before and within 1 year after treatment. Ninety-three patients were treated with three-dimensional conformal radiotherapy, 97 with intensity-modulated radiotherapy, and 60 with proton beam therapy. Postradiation pulmonary function test values were evaluated among individual patients compared with the same patient's preradiation value at the following time intervals: 0-4 (T1), 5-8 (T2), and 9-12 (T3) months. Results: Lung diffusing capacity for carbon monoxide (DLCO) was reduced in the majority of patients along the three time periods after radiation, whereas the forced expiratory volume in 1 s per unit of vital capacity (FEV1/VC) showed an increase and decrease after radiation in a similar percentage of patients. There were baseline differences (stage, radiotherapy dose, concurrent chemotherapy) among the radiation technology groups. On multivariate analysis, the following features were associated with larger posttreatment declines in DLCO: pretreatment DLCO, gross tumor volume, lung and heart dosimetric data, and total radiation dose. Only pretreatment DLCO was associated with larger posttreatment declines in FEV1/VC. Conclusions: Lung diffusing capacity for carbon monoxide is reduced in the majority of patients after radiotherapy with modern techniques. Multiple factors, including gross tumor volume, preradiation lung function, and dosimetric parameters, are associated with the DLCO decline. Prospective studies are needed to better understand whether new radiation technology, such as proton beam therapy or

  10. A generalized recipe to construct elementary or multi-step reaction paths via a stochastic formulation: Application to the conformational change in noble gas clusters

    Energy Technology Data Exchange (ETDEWEB)

    Talukder, Srijeeta; Sen, Shrabani [Department of Chemistry, University of Calcutta, 92 A P C Road, Kolkata 700 009 (India); Sharma, Rahul [Department of Chemistry, St. Xavier’s College, 30 Mother Teresa Sarani, Kolkata 700 016 (India); Banik, Suman K., E-mail: skbanik@bic.boseinst.ernet.in [Department of Chemistry, Bose Institute, 93/1 A P C Road, Kolkata 700 009 (India); Chaudhury, Pinaki, E-mail: pinakc@rediffmail.com [Department of Chemistry, University of Calcutta, 92 A P C Road, Kolkata 700 009 (India)

    2014-03-18

    Highlights: • We demonstrate a general strategy to map out reaction paths irrespective of the number of kinetic steps involved. • The objective function proposed does not need the information of gradient norm and eigenvalue of Hessian matrix explicitly. • A stochastic optimizer Simulated Annealing is used in searching reaction path. • The strategy is applied in mapping out the path for conformational changes in pure Ar clusters and Ar{sub N}Xe mixed clusters. - abstract: In this paper we demonstrate a general strategy to map out reaction paths irrespective of the number of kinetic steps required to bring about the change. i.e., whether the transformation takes place in a single step or in multiple steps with the appearance of intermediates. The objective function proposed is unique and works equally well for a concerted or a multiple step pathway. As the objective function proposed does not explicitly involves the calculation of the gradient of the potential energy function or the eigenvalues of the Hessian Matrix during the iterative process, the calculation is computationally economical. To map out the reaction path, we cast the entire problem as one of optimization and the solution is done with the use of the stochastic optimizer Simulated Annealing. The formalism is tested on Argon clusters (Ar{sub N}) and Argon clusters singly doped with Xenon (Ar{sub N-1}Xe). The size of the systems for which the method is applied ranges from N=7-25, where N is the total number of atoms in the cluster. We also test the results obtained by us by comparing with an established gradient only method. Moreover to demonstrate that our strategy can overcome the standard problems of drag method, we apply our strategy to a two dimensional LEPS + harmonic oscillator Potential to locate the TS, in which standard drag method has been seen to encounter problems.

  11. Hot Conformal Gauge Theories

    CERN Document Server

    Mojaza, Matin; Sannino, Francesco

    2010-01-01

    We compute the nonzero temperature free energy up to the order g^6 \\ln(1/g) in the coupling constant for vector like SU(N) gauge theories featuring matter transforming according to different representations of the underlying gauge group. The number of matter fields, i.e. flavors, is arranged in such a way that the theory develops a perturbative stable infrared fixed point at zero temperature. Due to large distance conformality we trade the coupling constant with its fixed point value and define a reduced free energy which depends only on the number of flavors, colors and matter representation. We show that the reduced free energy changes sign, at the second, fifth and sixth order in the coupling, when decreasing the number of flavors from the upper end of the conformal window. If the change in sign is interpreted as signal of an instability of the system then we infer a critical number of flavors. Surprisingly this number, if computed to the order g^2, agrees with previous predictions for the lower boundary o...

  12. Dynamic Conformational Change Regulates the Protein-DNA Recognition: An Investigation on Binding of a Y-Family Polymerase to Its Target DNA

    Science.gov (United States)

    Chu, Xiakun; Liu, Fei; Maxwell, Brian A.; Wang, Yong; Suo, Zucai; Wang, Haijun; Han, Wei; Wang, Jin

    2014-01-01

    Protein-DNA recognition is a central biological process that governs the life of cells. A protein will often undergo a conformational transition to form the functional complex with its target DNA. The protein conformational dynamics are expected to contribute to the stability and specificity of DNA recognition and therefore may control the functional activity of the protein-DNA complex. Understanding how the conformational dynamics influences the protein-DNA recognition is still challenging. Here, we developed a two-basin structure-based model to explore functional dynamics in Sulfolobus solfataricus DNA Y-family polymerase IV (DPO4) during its binding to DNA. With explicit consideration of non-specific and specific interactions between DPO4 and DNA, we found that DPO4-DNA recognition is comprised of first 3D diffusion, then a short-range adjustment sliding on DNA and finally specific binding. Interestingly, we found that DPO4 is under a conformational equilibrium between multiple states during the binding process and the distributions of the conformations vary at different binding stages. By modulating the strength of the electrostatic interactions, the flexibility of the linker, and the conformational dynamics in DPO4, we drew a clear picture on how DPO4 dynamically regulates the DNA recognition. We argue that the unique features of flexibility and conformational dynamics in DPO4-DNA recognition have direct implications for low-fidelity translesion DNA synthesis, most of which is found to be accomplished by the Y-family DNA polymerases. Our results help complete the description of the DNA synthesis process for the Y-family polymerases. Furthermore, the methods developed here can be widely applied for future investigations on how various proteins recognize and bind specific DNA substrates. PMID:25188490

  13. Ligand- and subunit-specific conformational changes in the ligand-binding domain and the TM2-TM3 linker of {alpha}1 {beta}2 {gamma}2 GABAA receptors

    DEFF Research Database (Denmark)

    Wang, Qian; Pless, Stephan Alexander; Lynch, Joseph W

    2010-01-01

    changes are essential for gating. Here we used voltage clamp fluorometry to investigate the roles of loops C and F in gating the α1 β2 γ2 GABA(A) receptor. Voltage clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements......Cys-loop receptor ligand binding sites are located at subunit interfaces where they are lined by loops A-C from one subunit and loops D-F from the adjacent subunit. Agonist binding induces large conformational changes in loops C and F. However, it is controversial as to whether these conformational...... from ligand-induced fluorescence changes. Previous attempts to define the roles of loops C and F using this technique have focused on homomeric Cys-loop receptors. However, the problem with studying homomeric receptors is that it is difficult to eliminate the possibility of bound ligands interacting...

  14. Sensing Conformational Changes in DNA upon Ligand Binding Using QCM-D. Polyamine Condensation and Rad51 Extension of DNA Layers

    KAUST Repository

    Sun, Lu

    2014-10-16

    © 2014 American Chemical Society. Biosensors, in which binding of ligands is detected through changes in the optical or electrochemical properties of a DNA layer confined to the sensor surface, are important tools for investigating DNA interactions. Here, we investigate if conformational changes induced in surface-attached DNA molecules upon ligand binding can be monitored by the quartz crystal microbalance with dissipation (QCM-D) technique. DNA duplexes containing 59-184 base pairs were formed on QCM-D crystals by stepwise assembly of synthetic oligonucleotides of designed base sequences. The DNA films were exposed to the cationic polyamines spermidine and spermine, known to condense DNA molecules in bulk experiments, or to the recombination protein Rad51, known to extend the DNA helix. The binding and dissociation of the ligands to the DNA films were monitored in real time by measurements of the shifts in resonance frequency (Δf) and in dissipation (ΔD). The QCM-D data were analyzed using a Voigt-based model for the viscoelastic properties of polymer films in order to evaluate how the ligands affect thickness and shear viscosity of the DNA layer. Binding of spermine shrinks all DNA layers and increases their viscosity in a reversible fashion, and so does spermidine, but to a smaller extent, in agreement with its lower positive charge. SPR was used to measure the amount of bound polyamines, and when combined with QCM-D, the data indicate that the layer condensation leads to a small release of water from the highly hydrated DNA films. The binding of Rad51 increases the effective layer thickness of a 59bp film, more than expected from the know 50% DNA helix extension. The combined results provide guidelines for a QCM-D biosensor based on ligand-induced structural changes in DNA films. The QCM-D approach provides high discrimination between ligands affecting the thickness and the structural properties of the DNA layer differently. The reversibility of the film

  15. Viscous conformal gauge theories

    DEFF Research Database (Denmark)

    Toniato, Arianna; Sannino, Francesco; Rischke, Dirk H.

    2017-01-01

    We present the conformal behavior of the shear viscosity-to-entropy density ratio and the fermion-number diffusion coefficient within the perturbative regime of the conformal window for gauge-fermion theories.......We present the conformal behavior of the shear viscosity-to-entropy density ratio and the fermion-number diffusion coefficient within the perturbative regime of the conformal window for gauge-fermion theories....

  16. Spectroscopic analysis of the role of extractives on heat-induced discoloration of black locust (Robinia pseudoacacia)

    Science.gov (United States)

    Yao Chen; Yongming Fan; Jianmin Gao; Mandla A. Tshabalala; Nicole M. Stark

    2012-01-01

    To investigate the role of extractives on heat-induced discoloration of wood, samples of black locust (Robinia pseudoacacia) wood flour were extracted with various solvents prior to heat-treatment. Analysis of their color parameters and chromophoric structures showed that the chroma value of the unextracted sample decreased while that of the...

  17. Heat-induced denaturation and aggregation of ovalbumin at neutral pH described by irreversible first-order kinetics

    NARCIS (Netherlands)

    Weijers, M.; Barneveld, P.A.; Cohen Stuart, M.A.; Visschers, R.W.

    2003-01-01

    The heat-induced denaturation kinetics of two different sources of ovalbumin at pH 7 was studied by chromatography and differential scanning calorimetry. The kinetics was found to be independent of protein concentration and salt concentration, but was strongly dependent on temperature. For highly

  18. Single-sided and small-scaled grasping of delicate tissues : Effectiveness of indirect heat-induced attachment and detachment

    NARCIS (Netherlands)

    Tukker, Arnoud A.; Knulst, Arjan J.; Maaijwee, Kristel J. M.; Schutte, Sander; Van Zeeburg, Elsbeth J. T.; Van Meurs, Jan C.; Dankelman, Jenny

    2012-01-01

    Introduction: Indirect heat-induced attachment and detachment (iHIAD) is a promising concept for gripping delicate tissues in microsurgery. However, the optimal settings of iHIAD are unknown. This study evaluates the effects of the instrument heating properties and initial contact force on the adhes

  19. IMP improves water-holding capacity, physical and sensory properties of heat-induced gels from porcine meat.

    Science.gov (United States)

    Nakamura, Yukinobu; Migita, Koshiro; Okitani, Akihiro; Matsuishi, Masanori

    2014-05-01

    Water-holding capacity (WHC) of heat-induced pork gels was examined. The heat-induced gels were obtained from meat homogenates prepared by adding nine volumes of 0.3-0.5 mol/L NaCl solutions containing 9-36 mmol/L disodium inosine-5'-monophosphate (IMP) or 9 mmol/L tetrapotassium pyrophosphate (KPP) to minced pork. IMP at 36 mmol/L enhanced the WHC to the same level as attained by KPP. Physical and sensory properties of heat-induced gels were also examined. The heat-induced gels were prepared from porcine meat homogenates containing 0.3 mol/L NaCl and 9-36 mmol/L IMP or 9 mmol/L KPP. IMP at 36 mmol/L enhanced the values of hardness, cohesiveness, gumminess and springiness, measured with a Tensipresser, and several organoleptic scores to the same level as the score attained by KPP. Thus, it is concluded that IMP is expected to be a practical substitute for pyrophosphates to improve the quality of sausages.

  20. Superspace conformal field theory

    Energy Technology Data Exchange (ETDEWEB)

    Quella, Thomas [Koeln Univ. (Germany). Inst. fuer Theoretische Physik; Schomerus, Volker [Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany)

    2013-07-15

    Conformal sigma models and WZW models on coset superspaces provide important examples of logarithmic conformal field theories. They possess many applications to problems in string and condensed matter theory. We review recent results and developments, including the general construction of WZW models on type I supergroups, the classification of conformal sigma models and their embedding into string theory.

  1. Heat-induced protein structure and subfractions in relation to protein degradation kinetics and intestinal availability in dairy cattle.

    Science.gov (United States)

    Doiron, K; Yu, P; McKinnon, J J; Christensen, D A

    2009-07-01

    The objectives of this study were to reveal protein structures of feed tissues affected by heat processing at a cellular level, using the synchrotron-based Fourier transform infrared microspectroscopy as a novel approach, and quantify protein structure in relation to protein digestive kinetics and nutritive value in the rumen and intestine in dairy cattle. The parameters assessed included 1) protein structure alpha-helix to beta-sheet ratio; 2) protein subfractions profiles; 3) protein degradation kinetics and effective degradability; 4) predicted nutrient supply using the intestinally absorbed protein supply (DVE)/degraded protein balance (OEB) system for dairy cattle. In this study, Vimy flaxseed protein was used as a model feed protein and was autoclave-heated at 120 degrees C for 20, 40, and 60 min in treatments T1, T2, and T3, respectively. The results showed that using the synchrotron-based Fourier transform infrared microspectroscopy revealed and identified the heat-induced protein structure changes. Heating at 120 degrees C for 40 and 60 min increased the protein structure alpha-helix to beta-sheet ratio. There were linear effects of heating time on the ratio. The heating also changed chemical profiles, which showed soluble CP decreased upon heating with concomitant increases in nonprotein nitrogen, neutral, and acid detergent insoluble nitrogen. The protein subfractions with the greatest changes were PB1, which showed a dramatic reduction, and PB2, which showed a dramatic increase, demonstrating a decrease in overall protein degradability. In situ results showed a reduction in rumen-degradable protein and in rumen-degradable dry matter without differences between the treatments. Intestinal digestibility, determined using a 3-step in vitro procedure, showed no changes to rumen undegradable protein. Modeling results showed that heating increased total intestinally absorbable protein (feed DVE value) and decreased degraded protein balance (feed OEB value

  2. Heat-induced Protein Structure and Subfractions in Relation to Protein Degradation Kinetics and Intestinal Availability in Dairy Cattle

    Energy Technology Data Exchange (ETDEWEB)

    Doiron, K.; Yu, P; McKinnon, J; Christensen, D

    2009-01-01

    The objectives of this study were to reveal protein structures of feed tissues affected by heat processing at a cellular level, using the synchrotron-based Fourier transform infrared microspectroscopy as a novel approach, and quantify protein structure in relation to protein digestive kinetics and nutritive value in the rumen and intestine in dairy cattle. The parameters assessed included (1) protein structure a-helix to e-sheet ratio; (2) protein subfractions profiles; (3) protein degradation kinetics and effective degradability; (4) predicted nutrient supply using the intestinally absorbed protein supply (DVE)/degraded protein balance (OEB) system for dairy cattle. In this study, Vimy flaxseed protein was used as a model feed protein and was autoclave-heated at 120C for 20, 40, and 60 min in treatments T1, T2, and T3, respectively. The results showed that using the synchrotron-based Fourier transform infrared microspectroscopy revealed and identified the heat-induced protein structure changes. Heating at 120C for 40 and 60 min increased the protein structure a-helix to e-sheet ratio. There were linear effects of heating time on the ratio. The heating also changed chemical profiles, which showed soluble CP decreased upon heating with concomitant increases in nonprotein nitrogen, neutral, and acid detergent insoluble nitrogen. The protein subfractions with the greatest changes were PB1, which showed a dramatic reduction, and PB2, which showed a dramatic increase, demonstrating a decrease in overall protein degradability. In situ results showed a reduction in rumen-degradable protein and in rumen-degradable dry matter without differences between the treatments. Intestinal digestibility, determined using a 3-step in vitro procedure, showed no changes to rumen undegradable protein. Modeling results showed that heating increased total intestinally absorbable protein (feed DVE value) and decreased degraded protein balance (feed OEB value), but there were no differences

  3. Expression of ACO1, ERS1 and ERF1 genes in harvested bananas in relation to heat-induced defense against Colletotrichum musae.

    Science.gov (United States)

    Zhu, Xiangfei; Wang, Aiping; Zhu, Shijiang; Zhang, Lubin

    2011-09-15

    The aim of this study was to investigate the connection between heat-induced ethylene signal changes and enhanced disease resistance. Heat enhanced ripening and elevated MaACO1 expression in naturally ripened bananas (NRB), while it delayed ripening and reduced MaACO1expression in the ethephon-treated bananas (ETB). However, in both cases, heat reduced lesion sizes infected by Colletotrichum musae. This indicates that heat-induced disease resistance in bananas was independent of ripening rate. The expression of MaERS1 gene was inhibited by heat treatment in both NRB and ETB, implying that heat as a physical signal could be sensed by banana fruits through the inhibition of ethylene receptor gene expression. The intensity of MaERF1 transcript signals was elevated in heated bananas, suggesting that the enhanced accumulation of MaERF1 transcript following heat treatment could play an important role in activation of the defense system. In ETB, inhibition of JA biosynthesis by application of IBU down-regulated the expression of MaERF and significantly weakened disease resistance, suggesting involvement of endogenous JA in induction of the gene expression, which was reconfirmed by the fact that exposure to exogenous MeJA following the combination of heat plus IBU treatment restored part of the gene expression. On the other hand, in NRB, application of IBU elevated level of MaERF1 expression at 24h and enhanced disease resistance, suggesting that, when banana was not exposed to ethephon, the expression of MaERF1 gene was not JA dependent, which was verified by the fact that MeJA application did not enhance MaERF1 gene expression. In conclusion, heat-induced disease resistance in harvested bananas could involve down-regulation of MaERS1 expression and up-regulation of MaERF1 expression and JA pathway could be involved in heat activation of the defense system in bananas exposed to ethephon.

  4. The cryo-electron microscopy structure of feline calicivirus bound to junctional adhesion molecule A at 9-angstrom resolution reveals receptor-induced flexibility and two distinct conformational changes in the capsid protein VP1.

    Science.gov (United States)

    Bhella, David; Goodfellow, Ian G

    2011-11-01

    Caliciviridae are small icosahedral positive-sense RNA-containing viruses and include the human noroviruses, a leading cause of infectious acute gastroenteritis and feline calicivirus (FCV), which causes respiratory illness and stomatitis in cats. FCV attachment and entry is mediated by feline junctional adhesion molecule A (fJAM-A), which binds to the outer face of the capsomere, inducing a conformational change in the capsid that may be important for viral uncoating. Here we present the results of our structural investigation of the virus-receptor interaction and ensuing conformational changes. Cryo-electron microscopy and three-dimensional image reconstruction were used to solve the structure of the virus decorated with a soluble fragment of the receptor at subnanometer resolution. In initial reconstructions, the P domains of the capsid protein VP1 and fJAM-A were poorly resolved. Sorting experiments led to improved reconstructions of the FCV-fJAM-A complex both before and after the induced conformational change, as well as in three transition states. These data showed that the P domain becomes flexible following fJAM-A binding, leading to a loss of icosahedral symmetry. Furthermore, two distinct conformational changes were seen; an anticlockwise rotation of up to 15° of the P domain was observed in the AB dimers, while tilting of the P domain away from the icosahedral 2-fold axis was seen in the CC dimers. A list of putative contact residues was calculated by fitting high-resolution coordinates for fJAM-A and VP1 to the reconstructed density maps, highlighting regions in both virus and receptor important for virus attachment and entry.

  5. Ataxia Telangiectasia-Mutated (ATM) kinase activity is regulated by ATP-driven conformational changes in the Mre11/Rad50/Nbs1 (MRN) complex

    NARCIS (Netherlands)

    J.-H. Lee (Ji-Hoon); M.R. Mand (Michael); R.A. Deshpande (Rajashree); E. Kinoshita (Eri); S.-H. Yang (Soo-Hyun); C. Wyman (Claire); T.T. Paull

    2013-01-01

    textabstractThe Ataxia Telangiectasia-Mutated (ATM) protein kinase is recruited to sites of double-strand DNA breaks by the Mre11/Rad50/Nbs1 (MRN) complex, which also facilitates ATM monomerization and activation. MRN exists in at least two distinct conformational states, dependent on ATP binding an

  6. Prediction of heat-induced polymerization of different globular food proteins in mixtures with wheat gluten.

    Science.gov (United States)

    Lambrecht, Marlies A; Rombouts, Ine; De Ketelaere, Bart; Delcour, Jan A

    2017-04-15

    Egg, soy or whey protein co-exists with wheat gluten in different food products. Different protein types impact each other during heat treatment. A positive co-protein effect occurs when heat-induced polymerization of a mixture of proteins is more intense than that of the isolated proteins. The intrinsic protein characteristics of globular proteins which enhance polymerization in mixtures with gluten are unknown. In this report, a model was developed to predict potential co-protein effects in mixtures of gluten and globular proteins during heating at 100°C. A negative co-protein effect with addition of lysozyme, no co-protein effect with soy glycinin or egg yolk and positive co-protein effects with bovine serum albumin, (S-)ovalbumin, egg white, whole egg, defatted egg yolk, wheat albumins and wheat globulins were detected. The level of accessible free sulfhydryl groups and the surface hydrophobicity of unfolded globular proteins were the main characteristics in determining the co-protein effects in gluten mixtures.

  7. Heat shock transcription factors regulate heat induced cell death in a rat histiocytoma

    Indian Academy of Sciences (India)

    Kolla V, P Rasad; Aftab Taiyab; D Jyothi; Usha K Srinivas; Amere S Sreedhar

    2007-04-01

    Heat shock response is associated with the synthesis of heat shock proteins (Hsps) which is strictly regulated by different members of heat shock transcription factors (HSFs). We previously reported that a rat histiocytoma, BC-8 failed to synthesize Hsps when subjected to typical heat shock conditions (42°C, 60 min). The lack of Hsp synthesis in these cells was due to a failure in HSF1 DNA binding activity. In the present study we report that BC-8 tumor cells when subjected to heat shock at higher temperature (43°C, 60 min) or incubation for longer time at 42°C, exhibited necrosis characteristics; however, under mild heat shock (42°C, 30 min) conditions cells showed activation of autophagy. Mild heat shock treatment induced proteolysis of HSF1, and under similar conditions we observed an increase in HSF2 expression followed by its enhanced DNA binding activity. Inhibiting HSF1 proteolysis by reversible proteasome inhibition failed to inhibit heat shock induced autophagy. Compromising HSF2 expression but not HSF1 resulted in the inhibition of autophagy, suggesting HSF2 dependent activation of autophagy. We are reporting for the first time that HSF2 is heat inducible and functions in heat shock induced autophagic cell death in BC-8 tumor cells.

  8. Integrated physiological and hormonal profile of heat-induced thermotolerance in Pinus radiata.

    Science.gov (United States)

    Escandón, Mónica; Cañal, María Jesús; Pascual, Jesús; Pinto, Glória; Correia, Barbara; Amaral, Joana; Meijón, Mónica

    2016-01-01

    Despite great interest, not only from the economic point of view but also in terms of basic science, research on heat stress tolerance in conifers remains scarce. To fill this gap, a time-course experiment using expected temperature increase was performed aiming to identify physiological and biochemical traits that allow the characterization of heat-induced thermotolerance and recovery in Pinus radiata D. Don plants. Several physiological parameters were assessed during heat exposure and after recovery, and multiple phytohormones-abscisic acid (ABA), indole-3-acetic acid (IAA), cytokinins (CKs), gibberellins, jasmonic acid, salicylic acid (SA) and brassinosteroids-were quantified by ultra-performance liquid chromatography-mass spectrometry from unique sample. Furthermore, tissue specific stress-signaling was monitored by IAA and ABA immunolocalization. Multivariate statistical analysis of the data enabled clustering of the shorter- and longer-term effects of heat stress exposure. Two sequential physiological responses were identified: an immediate and a delayed response, essentially determined by specific phytohormones, proline, malondialdehyde and total soluble sugar patterns. Results showed that ABA and SA play a crucial role in the first stage of response to heat stress, probably due to the plant's urgent need to regulate stomatal closure and counteract the increase in oxidative membrane damage demonstrated in shorter-term exposures. However, in longer exposures and recovery, proline, total sugars, IAA and CKs seem to be more relevant. This integrated approach pinpointed some basic mechanisms of P. radiata physiological responses underlying thermotolerance processes and after recovery.

  9. Heating-Induced Evaporation of Nine Different Secondary Organic Aerosol Types.

    Science.gov (United States)

    Kolesar, Katheryn R; Li, Ziyue; Wilson, Kevin R; Cappa, Christopher D

    2015-10-20

    The volatility of the compounds comprising organic aerosol (OA) determines their distribution between the gas and particle phases. However, there is a disconnect between volatility distributions as typically derived from secondary OA (SOA) growth experiments and the effective particle volatility as probed in evaporation experiments. Specifically, the evaporation experiments indicate an overall much less volatile SOA. This raises questions regarding the use of traditional volatility distributions in the simulation and prediction of atmospheric SOA concentrations. Here, we present results from measurements of thermally induced evaporation of SOA for nine different SOA types (i.e., distinct volatile organic compound and oxidant pairs) encompassing both anthropogenic and biogenic compounds and O3 and OH to examine the extent to which the low effective volatility of SOA is a general phenomenon or specific to a subset of SOA types. The observed extents of evaporation with temperature were similar for all the SOA types and indicative of a low effective volatility. Furthermore, minimal variations in the composition of all the SOA types upon heating-induced evaporation were observed. These results suggest that oligomer decomposition likely plays a major role in controlling SOA evaporation, and since the SOA formation time scale in these measurements was less than a minute, the oligomer-forming reactions must be similarly rapid. Overall, these results emphasize the importance of accounting for the role of condensed phase reactions in altering the composition of SOA when assessing particle volatility.

  10. Heat-induced structure formation in metal films generated by single ultrashort laser pulses

    Science.gov (United States)

    Koch, Jürgen; Unger, Claudia; Chichkov, Boris N.

    2012-03-01

    Ultrashort pulsed lasers are increasingly used in micromachining applications. Their short pulse lengths lead to well defined thresholds for the onset of material ablation and to the formation of only very small heat affected zones, which can be practically neglected in the majority of cases. Structure sizes down to the sub-micron range are possible in almost all materials - including heat sensitive materials. Ultrashort pulse laser ablation - even though called "cold ablation" - in fact is a heat driven process. Ablation takes place after a strong and fast temperature increase carrying away most of the heat with the ablated particles. This type of heat convection is not possible when reducing the laser fluence slightly below the ablation threshold. In this case temperature decreases slower giving rise to heat-induced material deformations and melt dynamics. After cooling down protruding structures can remain - ablation-free laser surface structuring is possible. Structure formation is boosted on thin metal films and offers best reproducibility and broadest processing windows for metals with high ductility and weak electron phonon coupling strength. All approaches to understand the process formation are currently based only on images of the final structures. The pump-probe imaging investigations presented here lead to a better process understanding.

  11. Insights into the Molecular Events That Regulate Heat-Induced Chilling Tolerance in Citrus Fruits

    Science.gov (United States)

    Lafuente, María T.; Establés-Ortíz, Beatriz; González-Candelas, Luis

    2017-01-01

    Low non-freezing temperature may cause chilling injury (CI), which is responsible for external quality deterioration in many chilling-sensitive horticultural crops. Exposure of chilling-sensitive citrus cultivars to non-lethal high-temperature conditioning may increase their chilling tolerance. Very little information is available about the molecular events involved in such tolerance. In this work, the molecular events associated with the low temperature tolerance induced by heating Fortune mandarin, which is very sensitive to chilling, for 3 days at 37°C prior to cold storage is presented. A transcriptomic analysis reveals that heat-conditioning has an important impact favoring the repression of genes in cold-stored fruit, and that long-term heat-induced chilling tolerance is an active process that requires activation of transcription factors involved in transcription initiation and of the WRKY family. The analysis also shows that chilling favors degradation processes, which affect lipids and proteins, and that the protective effect of the heat-conditioning treatment is more likely to be related to the repression of the genes involved in lipid degradation than to the modification of fatty acids unsaturation, which affects membrane permeability. Another major factor associated with the beneficial effect of the heat treatment on reducing CI is the regulation of stress-related proteins. Many of the genes that encoded such proteins are involved in secondary metabolism and in oxidative stress-related processes. PMID:28694818

  12. Heat-induced-radiolabeling and click chemistry: A powerful combination for generating multifunctional nanomaterials

    Science.gov (United States)

    Wilks, Moses Q.; El Fakhri, Georges; Normandin, Marc D.; Kaittanis, Charalambos; Josephson, Lee

    2017-01-01

    A key advantage of nanomaterials for biomedical applications is their ability to feature multiple small reporter groups (multimodality), or combinations of reporter groups and therapeutic agents (multifunctionality), while being targeted to cell surface receptors. Here a facile combination of techniques for the syntheses of multimodal, targeted nanoparticles (NPs) is presented, whereby heat-induced-radiolabeling (HIR) labels NPs with radiometals and so-called click chemistry is used to attach bioactive groups to the NP surface. Click-reactive alkyne or azide groups were first attached to the nonradioactive clinical Feraheme (FH) NPs. Resulting “Alkyne-FH” and “Azide-FH” intermediates, like the parent NP, tolerated 89Zr labeling by the HIR method previously described. Subsequently, biomolecules were quickly conjugated to the radioactive NPs by either copper-catalyzed or copper-free click reactions with high efficiency. Synthesis of the Alkyne-FH or Azide-FH intermediates, followed by HIR and then by click reactions for biomolecule attachment, provides a simple and potentially general path for the synthesis of multimodal, multifunctional, and targeted NPs for biomedical applications. PMID:28225818

  13. A site-directed spin-labeling study of ligand-induced conformational change in the ferric enterobactin receptor, FepA.

    Science.gov (United States)

    Liu, J; Rutz, J M; Klebba, P E; Feix, J B

    1994-11-15

    the CW saturation properties of MTSL bound to these sites, but did influence their accessibility to O2. These results provide consistent evidence for a ligand-specific conformational change in the surface peptides of FepA upon the binding of ferric enterobactin.

  14. Transitive conformal holonomy groups

    CERN Document Server

    Alt, Jesse

    2011-01-01

    For $(M,[g])$ a conformal manifold of signature $(p,q)$ and dimension at least three, the conformal holonomy group $\\mathrm{Hol}(M,[g]) \\subset O(p+1,q+1)$ is an invariant induced by the canonical Cartan geometry of $(M,[g])$. We give a description of all possible connected conformal holonomy groups which act transitively on the M\\"obius sphere $S^{p,q}$, the homogeneous model space for conformal structures of signature $(p,q)$. The main part of this description is a list of all such groups which also act irreducibly on $\\R^{p+1,q+1}$. For the rest, we show that they must be compact and act decomposably on $\\R^{p+1,q+1}$, in particular, by known facts about conformal holonomy the conformal class $[g]$ must contain a metric which is locally isometric to a so-called special Einstein product.

  15. Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change.

    Science.gov (United States)

    Hetrick, Byron; Han, Min Suk; Helgeson, Luke A; Nolen, Brad J

    2013-05-23

    Actin-related protein 2/3 (Arp2/3) complex is a seven-subunit assembly that nucleates branched actin filaments. Small molecule inhibitors CK-666 and CK-869 bind to Arp2/3 complex and inhibit nucleation, but their modes of action are unknown. Here, we use biochemical and structural methods to determine the mechanism of each inhibitor. Our data indicate that CK-666 stabilizes the inactive state of the complex, blocking movement of the Arp2 and Arp3 subunits into the activated filament-like (short pitch) conformation, while CK-869 binds to a serendipitous pocket on Arp3 and allosterically destabilizes the short pitch Arp3-Arp2 interface. These results provide key insights into the relationship between conformation and activity in Arp2/3 complex and will be critical for interpreting the influence of the inhibitors on actin filament networks in vivo.

  16. Simultaneous application of microbial transglutaminase and high hydrostatic pressure to improve heat induced gelation of pork plasma.

    Science.gov (United States)

    Fort, N; Lanier, T C; Amato, P M; Carretero, C; Saguer, E

    2008-11-01

    The effects of treating porcine plasma with microbial tranglutaminase (MTGase) under high hydrostatic pressure (HHP) were studied as a means of improving its gel-forming properties when subsequently heated at pH 5.5, near the pH of meats. Plasma containing varying levels of commercial MTGase was pressurized (400MPa, room temperature, pH 7) for different times, and adjusted to pH 5.5 prior to heating to induce gelation. MTGase-treatment under HHP led to greater enhancement of heat-induced plasma gel properties as compared to control samples. The greatest improvements were achieved by pressurising plasma with 43.3U MTGase/g protein for 30min, thereby achieving recoveries of 49% and 63% in fracture force (gel strength) and fracture distance (gel deformability) of the subsequently heat-induced gels, respectively, relative to gel properties obtained by heating untreated plasma at physiological conditions (pH 7.5).

  17. Using IR spectroscopy and multivariate curve resolution to elucidate mechanism of heat-induced decomposition of an organic complex

    DEFF Research Database (Denmark)

    Karpushkin, Evgeny; Gvozdik, Nataliya; Kucheryavskiy, Sergey V.

    and discuss the results of TGA-DSC-IR study of heat-induced decomposition of tris(acetylacetonato)manganese(III) complex. Using this process as an example, we have indicated the complications of the evolved gas analysis and demonstrated that they can be partially overcome taking advantage of multivariate......Many practically important processes occur upon elevated temperatures, and the induced chemical transformations are accompanied by gases evolution: heat-induced degradation of plastics, conversion of acrylic fibers into carbon ones, and fossil fuels pyrolysis – to name just a few. Generally...... curve resolution methods. Moreover, we have shown that the conventional methods based on TGA and IR analysis of the solid decomposition products produce incomplete data that is not enough for reliable elucidation of the decomposition mechanism. Finally, we have de- veloped a workflow to extract...

  18. Reversible heat-induced dissociation of β2-microglobulin amyloid fibrils.

    Science.gov (United States)

    Kardos, József; Micsonai, András; Pál-Gábor, Henriett; Petrik, Éva; Gráf, László; Kovács, János; Lee, Young-Ho; Naiki, Hironobu; Goto, Yuji

    2011-04-19

    Recent progress in the field of amyloid research indicates that the classical view of amyloid fibrils, being irreversibly formed highly stable structures resistant to perturbating conditions and proteolytic digestion, is getting more complex. We studied the thermal stability and heat-induced depolymerization of amyloid fibrils of β(2)-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis. We found that freshly polymerized β2m fibrils at 0.1-0.3 mg/mL concentration completely dissociated to monomers upon 10 min incubation at 99 °C. Fibril depolymerization was followed by thioflavin-T fluorescence and circular dichroism spectroscopy at various temperatures. Dissociation of β2m fibrils was found to be a reversible and dynamic process reaching equilibrium between fibrils and monomers within minutes. Repolymerization experiments revealed that the number of extendable fibril ends increased significantly upon incubation at elevated temperatures suggesting that the mechanism of fibril unfolding involves two distinct processes: (1) dissociation of monomers from the fibril ends and (2) the breakage of fibrils. The breakage of fibrils may be an important in vivo factor multiplying the number of fibril nuclei and thus affecting the onset and progress of disease. We investigated the effects of some additives and different factors on the stability of amyloid fibrils. Sample aging increased the thermal stability of β2m fibril solution. 0.5 mM SDS completely prevented β2m fibrils from dissociation up to the applied highest temperature of 99 °C. The generality of our findings was proved on fibrils of K3 peptide and α-synuclein. Our simple method may also be beneficial for screening and developing amyloid-active compounds for therapeutic purposes.

  19. Heat-induced whey protein gels: protein-protein interactions and functional properties.

    Science.gov (United States)

    Havea, Palatasa; Watkinson, Philip; Kuhn-Sherlock, Barbara

    2009-02-25

    Heat-induced gelation (80 degrees C for 30 min or 85 degrees C for 60 min) of whey protein concentrate (WPC) solutions was studied using small deformation dynamic rheology, small and large deformation compression, and polyacrylamide gel electrophoresis (PAGE). The WPC solutions (15% w/w, pH 6.9) were prepared by dispersing WPC powder in water (control), 1% (w/w) sodium dodecyl sulfate (SDS) solution, and N-ethylmaleimide (NEM) solution at a protein/NEM molar ratio of 1:1 or in 10 mM dithiothreitol (DTT) solution. PAGE analyses showed that the heat treatment of control solutions contained both disulfide and non-covalent linkages between denatured protein molecules. Only disulfide linkages were formed in heated SDS-WPC solutions, whereas only non-covalent linkages were formed in DTT-WPC and NEM-WPC solutions during heating. In heated NEM-WPC solutions, the pre-existing disulfide linkages remained unaltered. Small deformation rheology measurements showed that the storage modulus (G') values, compared with those of the control WPC gels (approximately 14000 Pa), were 3 times less for the SDS-WPC gels (approximately 4000 Pa), double for the NEM-WPC gels (approximately 24000 Pa), and even higher for the DTT-WPC gels (approximately 30000 Pa). Compression tests suggested that the rubberiness (fracture strain) of the WPC gels increased as the degree of disulfide linkages within the gels increased, whereas the stiffness (modulus) of the gels increased as the degree of non-covalent associations among the denatured protein molecules increased.

  20. Formation of tRNA granules in the nucleus of heat-induced human cells

    Energy Technology Data Exchange (ETDEWEB)

    Miyagawa, Ryu [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Department of Biological Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654 (Japan); Mizuno, Rie [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Watanabe, Kazunori, E-mail: watanabe@ric.u-tokyo.ac.jp [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Ijiri, Kenichi [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032 (Japan); Department of Biological Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654 (Japan)

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer tRNAs are tranlocated into the nucleus in heat-induced HeLa cells. Black-Right-Pointing-Pointer tRNAs form the unique granules in the nucleus. Black-Right-Pointing-Pointer tRNA ganules overlap with nuclear stress granules. -- Abstract: The stress response, which can trigger various physiological phenomena, is important for living organisms. For instance, a number of stress-induced granules such as P-body and stress granule have been identified. These granules are formed in the cytoplasm under stress conditions and are associated with translational inhibition and mRNA decay. In the nucleus, there is a focus named nuclear stress body (nSB) that distinguishes these structures from cytoplasmic stress granules. Many splicing factors and long non-coding RNA species localize in nSBs as a result of stress. Indeed, tRNAs respond to several kinds of stress such as heat, oxidation or starvation. Although nuclear accumulation of tRNAs occurs in starved Saccharomyces cerevisiae, this phenomenon is not found in mammalian cells. We observed that initiator tRNA{sup Met} (Meti) is actively translocated into the nucleus of human cells under heat stress. During this study, we identified unique granules of Meti that overlapped with nSBs. Similarly, elongator tRNA{sup Met} was translocated into the nucleus and formed granules during heat stress. Formation of tRNA granules is closely related to the translocation ratio. Then, all tRNAs may form the specific granules.

  1. Successive pH- and heat-induced homogenous liquid-liquid extraction.

    Science.gov (United States)

    Farajzadeh, Mir Ali; Feriduni, Behruz

    2016-08-12

    A simple and efficient analytical method known as pH- and heat-induced homogenous liquid-liquid extraction combined with high-performance liquid chromatography has been successfully developed for the extraction and determination of neonicotinoid pesticides in aqueous samples. In this method, a few mL of a water-miscible basic extraction solvent is mixed with a high volume of an aqueous phase containing the analytes and passed through a tube which a portion of the tube is filled with sodium carbonate as a separating agent. By passing the solution, salt is dissolved and the fine droplets of the extraction solvent are formed. The produced droplets go up through the remained solution and collect as a separated layer. In the following, the collected organic phase is removed and placed into a micro tube. Then it is heated in a water bath to form two phases. Several experimental parameters that influence extraction efficiency such as type and volume of extraction solvent, type of phase separation agent, temperature, and extraction time were investigated. Under the optimum conditions, the extraction recoveries and enrichment factors ranged between 51 and 81% and 680 and 1080, respectively. Calibration curves showed a high-level of linearity for all target analytes with coefficients of determination ranging between 0.997 and 0.999. The repeatability of the proposed method expressed as relative standard deviation varied between 3 and 5% (n=6, C=50μgL(-1)), and the detection limits were in the range of 0.52-1.0μgL(-1). Finally, the performance of the method was evaluated by analyzing the selected pesticides in different fruit juice and vegetable samples.

  2. DNA extraction from archival formalin-fixed, paraffin-embedded tissues: heat-induced retrieval in alkaline solution.

    Science.gov (United States)

    Shi, Shan-Rong; Datar, Ram; Liu, Cheng; Wu, Lin; Zhang, Zina; Cote, Richard J; Taylor, Clive R

    2004-09-01

    Based on the antigen retrieval principle, our previous study has demonstrated that heating archival formalin-fixed, paraffin-embedded (FFPE) tissues at a higher temperature and at higher pH value of the retrieval solution may achieve higher efficiency of extracted DNA, when compared to the traditional enzyme digestion method. Along this line of heat-induced retrieval, this further study is focused on development of a simpler and more effective heat-induced DNA retrieval technique by testing various retrieval solutions. Three major experiments using a high temperature heating method to extract DNA from FFPE human lymphoid and other tissue sections were performed to compare: (1) different concentrations of alkaline solution (NaOH or KOH, pH 11.5-12) versus Britton and Robinson type of buffer solution (BR buffer) of pH 12 that was the only retrieval solution tested in our previous study; (2) several chemical solutions (SDS, Tween 20, and GITC of various concentrations) versus BR buffer or alkaline solution; and (3) alkaline solution mixed with chemicals versus BR buffer or single alkaline solution. Efficiency of DNA extraction was evaluated by measuring yields using spectrophotometry, electrophoretic pattern, semiquantitation of tissue dissolution, PCR amplification, and kinetic thermocycling-PCR methods. Results showed that boiling tissue sections in 0.1 M NaOH or KOH or its complex retrieval solutions produced higher yields and better quality of DNA compared to BR buffer or chemical solutions alone. The conclusion was that boiling FFPE tissue sections in 0.1 M alkaline solution is a simpler and more effective heat-induced retrieval protocol for DNA extraction. Combination with some chemicals (detergents) may further significantly improve efficiency of the heat-induced retrieval technique.

  3. Toward Consistent Terminology for Cyclohexane Conformers in Introductory Organic Chemistry

    Science.gov (United States)

    Nelson, Donna J.; Brammer, Christopher N.

    2011-01-01

    Recommended changes in use of cyclohexane conformers and their nomenclature will remedy inconsistencies in cyclohexane conformers and their nomenclature that exist across currently used organic chemistry textbooks. These inconsistencies prompted this logical analysis and the resulting recommendations. Recommended conformer names are "chair",…

  4. The effect of conformal symmetry on charged wormholes

    CERN Document Server

    Kuhfittig, Peter K F

    2016-01-01

    This paper discusses the effect that conformal symmetry can have on a charged wormhole. The analysis yields a physical interpretation of the conformal factor in terms of the electric charge. The rate of change of the conformal factor determines much of the outcome, which ranges from having no solution to wormholes having either one or two throats.

  5. Increasing ablation distance peripheral to the saphenofemoral junction may result in a diminished rate of endothermal heat-induced thrombosis.

    Science.gov (United States)

    Sadek, Mikel; Kabnick, Lowell S; Rockman, Caron B; Berland, Todd L; Zhou, Di; Chasin, Cara; Jacobowitz, Glenn R; Adelman, Mark A

    2013-07-01

    The treatment of venous insufficiency using endovenous laser ablation or radiofrequency ablation may result in endothermal heat-induced thrombosis (EHIT), a form of deep venous thrombosis. This study sought to assess whether increasing the ablation distance peripheral to the deep venous junction would result in a reduction in the incidence of EHIT II. This study was a retrospective review of a prospectively maintained database from April 2007 to December 2011. Consecutive patients undergoing great saphenous vein (GSV) or small saphenous vein (SSV) ablation were evaluated. Previous to February 2011, all venous ablations were performed 2 cm peripheral to the saphenofemoral or saphenopopliteal junction (group I). Subsequent to February 2011, ablations were performed greater than or equal to 2.5 cm peripheral to the respective deep system junction (group II). The primary outcome was the development of EHIT II or greater (ie, thrombus protruding into the deep venous system but comprising less than 50% of the deep vein lumen). Secondary outcomes included procedure-site complications such as thrombophlebitis and hematomas. χ(2) tests were performed for all discrete variables, and unpaired Student's t-tests were performed for all continuous variables. P result was significant (group I: 2.6% ± 0.9% vs group II: 2.8% ± 1.0%; P = .006). The incidence of EHIT II was 76 in group I and 13 in group II. This represented a trend toward diminished frequency in group II as compared with group I (group I: 2.3% vs group II: 1.3%; P = .066). There were no reported cases of EHIT III or IV in this patient cohort. Patients who developed an EHIT II in group I were treated using anticoagulation 54% of the time, and patients who developed an EHIT II in group II were treated using anticoagulation 100% of the time. This study suggests that changing the treatment distance from 2 cm to greater than or equal to 2.5 cm peripheral to the deep venous junction may result in a diminished

  6. [Dosimetric evaluation of conformal radiotherapy: conformity factor].

    Science.gov (United States)

    Oozeer, R; Chauvet, B; Garcia, R; Berger, C; Felix-Faure, C; Reboul, F

    2000-01-01

    The aim of three-dimensional conformal therapy (3DCRT) is to treat the Planning Target Volume (PTV) to the prescribed dose while reducing doses to normal tissues and critical structures, in order to increase local control and reduce toxicity. The evaluation tools used for optimizing treatment techniques are three-dimensional visualization of dose distributions, dose-volume histograms, tumor control probabilities (TCP) and normal tissue complication probabilities (NTCP). These tools, however, do not fully quantify the conformity of dose distributions to the PTV. Specific tools were introduced to measure this conformity for a given dose level. We have extended those definitions to different dose levels, using a conformity index (CI). CI is based on the relative volumes of PTV and outside the PTV receiving more than a given dose. This parameter has been evaluated by a clinical study including 82 patients treated for lung cancer and 82 patients treated for prostate cancer. The CI was low for lung dosimetric studies (0.35 at the prescribed dose 66 Gy) due to build-up around the GTV and to spinal cord sparing. For prostate dosimetric studies, the CI was higher (0.57 at the prescribed dose 70 Gy). The CI has been used to compare treatment plans for lung 3DCRT (2 vs 3 beams) and prostate 3DCRT (4 vs 7 beams). The variation of CI with dose can be used to optimize dose prescription.

  7. On conformally related -waves

    Indian Academy of Sciences (India)

    Varsha Daftardar-Gejji

    2001-05-01

    Brinkmann [1] has shown that conformally related distinct Ricci flat solutions are -waves. Brinkmann's result has been generalized to include the conformally invariant source terms. It has been shown that [4] if $g_{ik}$ and $\\overline{g}_{ik}$ ($=^{-2}g_{ik}$, : a scalar function), are distinct metrics having the same Einstein tensor, $G_{ik}=\\overline{G}_{ik}$, then both represent (generalized) $pp$-waves and $_{i}$ is a null convariantly constant vector of $g_{ik}$. Thus $pp$-waves are the only candidates which yield conformally related nontrivial solutions of $G_{ik}=T_{ik}=\\overline{G}_{ik}$, with $T_{ik}$ being conformally invariant source. In this paper the functional form of the conformal factor for the conformally related $pp$-waves/generalized $pp$-waves has been obtained. It has been shown that the most general $pp$-wave, conformally related to ${\\rm d}s^{2}=-2{\\rm d}u[{\\rm d}v-m{\\rm d}y+H{\\rm d}u]+P^{-2}[{\\rm d}y^{2}+{\\rm d}z^{2}]$, turns out to the $(au+b)^{-2}{\\rm d}s^{2}$, where , are constants. Only in the special case when $m=0$, $H=1$, and $P=P(y,z)$, the conformal factor is $(au+b)^{-2}$ or $(a(u+v)+b)^{-2}$.

  8. Conformational stability of calreticulin

    DEFF Research Database (Denmark)

    Jørgensen, C.S.; Trandum, C.; Larsen, N.

    2005-01-01

    The conformational stability of calreticulin was investigated. Apparent unfolding temperatures (T-m) increased from 31 degrees C at pH 5 to 51 degrees C at pH 9, but electrophoretic analysis revealed that calreticulin oligomerized instead of unfolding. Structural analyses showed that the single C......-terminal a-helix was of major importance to the conformational stability of calreticulin....

  9. Conformal invariance and particle aspects in general relativity

    CERN Document Server

    Salehi, H; Darabi, F

    2000-01-01

    We study the breakdown of conformal symmetry in a conformally invariantgravitational model. The symmetry breaking is introduced by defining apreferred conformal frame in terms of the large scale characteristics of theuniverse. In this context we show that a local change of the preferredconformal frame results in a Hamilton-Jacobi equation describing a particlewith adjustable mass. In this equation the dynamical characteristics of theparticle substantially depends on the applied conformal factor and localgeometry. Relevant interpretations of the results are also discussed.

  10. Interconversion of active and inactive 30 S ribosomal subunits is accompanied by a conformational change in the decoding region of 16 S rRNA

    DEFF Research Database (Denmark)

    Moazed, D; Van Stolk, B J; Douthwaite, S

    1986-01-01

    Zamir, Elson and their co-workers have shown that 30 S ribosomal subunits are reversibly inactivated by depletion of monovalent or divalent cations. We have re-investigated the conformation of 16 S rRNA in the active and inactive forms of the 30 S subunit, using a strategy that is designed...... by end-labeling followed by analine-induced strand scission (in some cases preceded by hybrid selection), or by primer extension using synthetic DNA oligomers. These studies show the following: The transition from the active to the inactive state cannot be described as a simple loosening or unfolding...

  11. Dynamics and structural changes induced by ATP and/or substrate binding in the inward-facing conformation state of P-glycoprotein

    Science.gov (United States)

    Watanabe, Yurika; Hsu, Wei-Lin; Chiba, Shuntaro; Hayashi, Tomohiko; Furuta, Tadaomi; Sakurai, Minoru

    2013-02-01

    P-glycoprotein (P-gp) is a multidrug transporter that catalyzes the transport of a substrate. To elucidate the underlying mechanism of this type of substrate transport, we performed molecular dynamics (MD) simulations using the X-ray crystal structure of P-gp, which has an inward-facing conformation. Our simulations indicated that the dimerization of the nucleotide binding domains (NBDs) is driven by the binding of ATP to the NBDs and/or the binding of the substrate to a cavity in the transmembrane domains (TMDs). Based on these results, we discuss a role of ATP in the allosteric communication that occurs between the NBDs and the TMDs.

  12. Conformational elasticity theory of chain molecules

    Institute of Scientific and Technical Information of China (English)

    YANG; Xiaozhen

    2001-01-01

    This paper develops a conformational elasticity theory of chain molecules, which is based on three key points: (ⅰ) the molecular model is the rotational isomeric state (RIS) model; (ⅱ) the conformational distribution function of a chain molecule is described by a function of two variables, the end-to-end distance of a chain conformation and the energy of the conformation; (ⅲ) the rule of changes in the chain conformational states during deformation is that a number of chain conformations would vanish. The ideal deformation behavior calculated by the theory shows that the change in chain conformations is physically able to make the upward curvature of the stress-strain curve at the large-scale deformation of natural rubber. With the theory, different deformation behaviors between polymers with different chemical structures can be described, the energy term of the stress in the deformations can be predicted, and for natural rubber the fraction of the energy term is around 13%, coinciding with the experimental results.

  13. EC declaration of conformity.

    Science.gov (United States)

    Donawa, M E

    1996-05-01

    The CE-marking procedure requires that manufacturers draw up a written declaration of conformity before placing their products on the market. However, some companies do not realize that this is a requirement for all devices. Also, there is no detailed information concerning the contents and format of the EC declaration of conformity in the medical device Directives or in EC guidance documentation. This article will discuss some important aspects of the EC declaration of conformity and some of the guidance that is available on its contents and format.

  14. The Hemophore HasA from Yersinia pestis (HasAyp) Coordinates Hemin with a Single Residue, Tyr75, and with Minimal Conformational Change

    Science.gov (United States)

    Kumar, Ritesh; Lovell, Scott; Matsumura, Hirotoshi; Battaile, Kevin P.; Moënne-Loccoz, Pierre; Rivera, Mario

    2015-01-01

    Hemophores from Serratia marcescens (HasAsm) and Pseudomonas aeruginosa (HasAp) bind hemin between two loops, which harbor the axial ligands H32 and Y75. Hemin binding to the Y75 loop triggers closing of the H32 loop and enables binding of H32. Because Yersinia pestis HasA (HasAyp) presents a Gln at position 32, we determined the structures of apo-and holo-HasAyp. Surprisingly, the Q32 loop in apo-HasAyp is already in the closed conformation but no residue from the Q32 loop binds hemin in holo-HasAyp. In agreement with the minimal reorganization between the apo-and holo-structures, the hemin on-rate is too fast to detect by conventional stopped-flow measurements. PMID:23578210

  15. The conformal transformation of the night sky

    Science.gov (United States)

    Minguzzi, E.

    2016-12-01

    We give a simple differential geometric proof of the conformal transformation of the night sky under change of observer. The proof does not use the four dimensionality of spacetime or spinor methods. Furthermore, it really shows that the result does not depend on Lorentz transformations. This approach, by giving a transparent covariant expression to the conformal factor, shows that in most situations it is possible to define a thermal sky metric independent of the observer.

  16. Conformal expansions and renormalons

    CERN Document Server

    Gardi, E; Gardi, Einan; Grunberg, Georges

    2001-01-01

    The large-order behaviour of QCD is dominated by renormalons. On the other hand renormalons do not occur in conformal theories, such as the one describing the infrared fixed-point of QCD at small beta_0 (the Banks--Zaks limit). Since the fixed-point has a perturbative realization, all-order perturbative relations exist between the conformal coefficients, which are renormalon-free, and the standard perturbative coefficients, which contain renormalons. Therefore, an explicit cancellation of renormalons should occur in these relations. The absence of renormalons in the conformal limit can thus be seen as a constraint on the structure of the QCD perturbative expansion. We show that the conformal constraint is non-trivial: a generic model for the large-order behaviour violates it. We also analyse a specific example, based on a renormalon-type integral over the two-loop running-coupling, where the required cancellation does occur.

  17. Animal culture: chimpanzee conformity?

    Science.gov (United States)

    van Schaik, Carel P

    2012-05-22

    Culture-like phenomena in wild animals have received much attention, but how good is the evidence and how similar are they to human culture? New data on chimpanzees suggest their culture may even have an element of conformity.

  18. Quantum massive conformal gravity

    Energy Technology Data Exchange (ETDEWEB)

    Faria, F.F. [Universidade Estadual do Piaui, Centro de Ciencias da Natureza, Teresina, PI (Brazil)

    2016-04-15

    We first find the linear approximation of the second plus fourth order derivative massive conformal gravity action. Then we reduce the linearized action to separated second order derivative terms, which allows us to quantize the theory by using the standard first order canonical quantization method. It is shown that quantum massive conformal gravity is renormalizable but has ghost states. A possible decoupling of these ghost states at high energies is discussed. (orig.)

  19. Delineating the conformal window

    DEFF Research Database (Denmark)

    Frandsen, Mads Toudal; Pickup, Thomas; Teper, Michael

    2011-01-01

    We identify and characterise the conformal window in gauge theories relevant for beyond the standard model building, e.g. Technicolour, using the criteria of metric confinement and causal analytic couplings, which are known to be consistent with the phase diagram of supersymmetric QCD from Seiberg...... duality. Using these criteria we find perturbation theory to be consistent throughout the predicted conformal window for several of these gauge theories and we discuss recent lattice results in the light of our findings....

  20. Conformally coupled inflation

    CERN Document Server

    Faraoni, Valerio

    2013-01-01

    A massive scalar field in a curved spacetime can propagate along the light cone, a causal pathology, which can, in principle, be eliminated only if the scalar couples conformally to the Ricci curvature of spacetime. This property mandates conformal coupling for the field driving inflation in the early universe. During slow-roll inflation, this coupling can cause super-acceleration and, as a signature, a blue spectrum of primordial gravitational waves.

  1. Group Size and Conformity

    OpenAIRE

    Bond, Rod

    2005-01-01

    Abstract This paper reviews theory and research on the relationship between group size and conformity and presents a meta-analysis of 125 Asch-type conformity studies. It questions the assumption of a single function made in formal models of social influence and proposes instead that the function will vary depending on which social influence process predominates. It is argued that normative influence is lik...

  2. Conformism and Wealth Distribution

    OpenAIRE

    Mino, Kazuo; Nakamoto, Yasuhiro

    2014-01-01

    This paper explores the role of consumption externalities in a neoclassical growth model in which households have heterogeneous preferences. We fi?nd that the degree of conformism in consumption held by each household signifi?cantly affects the speed of convergence of the aggregate economy as well as the patterns of wealth distribution in the steady state equilibrium. In particular, a higher degree of consumption conformism accelerates the convergence speed of the economy towards the steady s...

  3. Conformally Coupled Inflation

    Directory of Open Access Journals (Sweden)

    Valerio Faraoni

    2013-07-01

    Full Text Available A massive scalar field in a curved spacetime can propagate along the light cone, a causal pathology, which can, in principle, be eliminated only if the scalar couples conformally to the Ricci curvature of spacetime. This property mandates conformal coupling for the field driving inflation in the early universe. During slow-roll inflation, this coupling can cause super-acceleration and, as a signature, a blue spectrum of primordial gravitational waves.

  4. Effect of heat-induced pain stimuli on pulse transit time and pulse wave amplitude in healthy volunteers.

    Science.gov (United States)

    van Velzen, Marit H N; Loeve, Arjo J; Kortekaas, Minke C; Niehof, Sjoerd P; Mik, Egbert G; Stolker, Robert J

    2016-01-01

    Pain is commonly assessed subjectively by interpretations of patient behaviour and/or reports from patients. When this is impossible the availability of a quantitative objective pain assessment tool based on objective physiological parameters would greatly benefit clinical practice and research beside the standard self-report tests. Vasoconstriction is one of the physiological responses to pain. The aim of this study was to investigate whether pulse transit time (PTT) and pulse wave amplitude (PWA) decrease in response to this vasoconstriction when caused by heat-induced pain. The PTT and PWA were measured in healthy volunteers, on both index fingers using photoplethysmography and electrocardiography. Each subject received 3 heat-induced pain stimuli using a Temperature-Sensory Analyzer thermode block to apply a controlled, increasing temperature from 32.0 °C to 50.0 °C to the skin. After reaching 50.0 °C, the thermode was immediately cooled down to 32.0 °C. The study population was divided into 2 groups with a time-interval between the stimuli 20s or 60s. The results showed a significant (p  <  0.05) decrease of both PTT and PWA on the stimulated and contralateral side. Moreover, there was no significant difference between the stimulated and contralateral side. The time-interval of 20s was too short to allow PTT and PWA to return to baseline values and should exceed 40s in future studies. Heat-induced pain causes a decrease of PTT and PWA. Consequently, it is expected that, in the future, PTT and PWA may be applied as objective indicators of pain, either beside the standard self-report test, or when self-report testing is impossible.

  5. Conformational sampling techniques.

    Science.gov (United States)

    Hatfield, Marcus P D; Lovas, Sándor

    2014-01-01

    The potential energy hyper-surface of a protein relates the potential energy of the protein to its conformational space. This surface is useful in determining the native conformation of a protein or in examining a statistical-mechanical ensemble of structures (canonical ensemble). In determining the potential energy hyper-surface of a protein three aspects must be considered; reducing the degrees of freedom, a method to determine the energy of each conformation and a method to sample the conformational space. For reducing the degrees of freedom the choice of solvent, coarse graining, constraining degrees of freedom and periodic boundary conditions are discussed. The use of quantum mechanics versus molecular mechanics and the choice of force fields are also discussed, as well as the sampling of the conformational space through deterministic and heuristic approaches. Deterministic methods include knowledge-based statistical methods, rotamer libraries, homology modeling, the build-up method, self-consistent electrostatic field, deformation methods, tree-based elimination and eigenvector following routines. The heuristic methods include Monte Carlo chain growing, energy minimizations, metropolis monte carlo and molecular dynamics. In addition, various methods to enhance the conformational search including the deformation or smoothing of the surface, scaling of system parameters, and multi copy searching are also discussed.

  6. Electrophysiological precursors of social conformity.

    Science.gov (United States)

    Shestakova, Anna; Rieskamp, Jörg; Tugin, Sergey; Ossadtchi, Alexey; Krutitskaya, Janina; Klucharev, Vasily

    2013-10-01

    Humans often change their beliefs or behavior due to the behavior or opinions of others. This study explored, with the use of human event-related potentials (ERPs), whether social conformity is based on a general performance-monitoring mechanism. We tested the hypothesis that conflicts with a normative group opinion evoke a feedback-related negativity (FRN) often associated with performance monitoring and subsequent adjustment of behavior. The experimental results show that individual judgments of facial attractiveness were adjusted in line with a normative group opinion. A mismatch between individual and group opinions triggered a frontocentral negative deflection with the maximum at 200 ms, similar to FRN. Overall, a conflict with a normative group opinion triggered a cascade of neuronal responses: from an earlier FRN response reflecting a conflict with the normative opinion to a later ERP component (peaking at 380 ms) reflecting a conforming behavioral adjustment. These results add to the growing literature on neuronal mechanisms of social influence by disentangling the conflict-monitoring signal in response to the perceived violation of social norms and the neural signal of a conforming behavioral adjustment.

  7. The Conformal Method and the Conformal Thin-Sandwich Method Are the Same

    CERN Document Server

    Maxwell, David

    2014-01-01

    The conformal method developed in the 1970s and the more recent Lagrangian and Hamiltonian conformal thin-sandwich methods are techniques for finding solutions of the Einstein constraint equations. We show that they are manifestations of a single conformal method: there is a straightforward way to convert back and forth between the parameters for these methods so that the corresponding solutions of the Einstein constraint equations agree. The unifying idea is the need to clearly distinguish tangent and cotangent vectors to the space of conformal classes on a manifold, and we introduce a vocabulary for working with these objects without reference to a particular representative background metric. As a consequence of these conceptual advantages, we demonstrate how to strengthen previous near-CMC existence and non-existence theorems for the original conformal method to include metrics with scalar curvatures that change sign.

  8. Effects of designed sulfhydryl groups and disulfide bonds into soybean proglycinin on its structural stability and heat-induced gelation.

    Science.gov (United States)

    Adachi, Motoyasu; Chunying, Ho; Utsumi, Shigeru

    2004-09-08

    The gel-forming ability of glycinin is one of soybean's most important functional properties. The proglycinin A1aB1b homotrimer was engineered to introduce sulfhydryl groups and disulfide bonds, and their effects on the structural stability and the heat-induced gelation were evaluated. On the basis of the crystal structure, five mutants were designed and prepared: R161C and F163C forming an interprotomer disulfide bond with the inherent free cysteine residue of Cys377, N116C/P248C forming a new intraprotomer disulfide bond, and N116C and P248C introducing a new sulfhydryl group. Mutants of R161C, F163C, and N116C/P248C formed a new disulfide bond as expected. N116C/P248C was significantly more stable than the wild type against chemical and thermal denaturation and more resistant to alpha-chymotrypsin digestion, whereas F163C showed significantly increased thermal stability. All mutants exhibited greater hardness of heat-induced gels than wild type, and in particular, N116C/P248C gave the hardest gel. This result indicates that it is possible to increase hardness of glycinin gel by introduction of cysteine residues using protein engineering.

  9. Structural studies of conformational changes of proteins upon phosphorylation: Structures of activated CheY, CheY-N16-FliM complex, and AAA {sup +} ATPase domain of NtrC1 in both inactive and active states

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seok-Yong

    2003-04-10

    Protein phosphorylation is a general mechanism for signal transduction as well as regulation of cellular function. Unlike phosphorylation in eukaryotic systems that uses Ser/Thr for the sites of modification, two-component signal transduction systems, which are prevalent in bacteria, archea, and lower eukaryotes, use an aspartate as the site of phosphorylation. Two-component systems comprise a histidine kinase and a receiver domain. The conformational change of the receiver domain upon phosphorylation leads to signal transfer to the downstream target, a process that had not been understood well at the molecular level. The transient nature of the phospho-Asp bond had made structural studies difficult. The discovery of an excellent analogue for acylphosphate, BeF{sub 3}{sup -}, enabled structural study of activated receiver domains. The structure of activated Chemotaxis protein Y (CheY) was determined both by NMR spectroscopy and X-ray crystallography. These structures revealed the molecular basis of the conformational change that is coupled to phosphorylation. Phosphorylation of the conserved Asp residue in the active site allows hydrogen bonding of the T87 O{gamma} to phospho-aspartate, which in turn leads to the rotation of Y106 into the ''in'' position (termed Y-T coupling). The structure of activated CheY complexed with the 16 N-terminal residues of FliM (N16-FliM), its target, was also determined by X-ray crystallography and confirmed the proposed mechanism of activation (Y-T coupling). First, N16-FliM binds to the region on CheY that undergoes a significant conformational change. Second, the ''in'' position of Y106 presents a better binding surface for FliM because the sidechain of Y106 in the inactive form of CheY (''out'' position) sterically interferes with binding of N16-FliM. In addition to confirmation of Y-T coupling, the structure of the activated CheY-N16-FliM complex suggested that the N16

  10. Recombinant Expression of the Full-length Ectodomain of LDL Receptor-related Protein 1 (LRP1) Unravels pH-dependent Conformational Changes and the Stoichiometry of Binding with Receptor-associated Protein (RAP).

    Science.gov (United States)

    De Nardis, Camilla; Lössl, Philip; van den Biggelaar, Maartje; Madoori, Pramod K; Leloup, Nadia; Mertens, Koen; Heck, Albert J R; Gros, Piet

    2017-01-20

    LDL receptor-related protein 1 (LRP1) is a highly modular protein and the largest known mammalian endocytic receptor. LRP1 binds and internalizes many plasma components, playing multiple crucial roles as a scavenger and signaling molecule. One major challenge to studying LRP1 has been that it is difficult to express such a large, highly glycosylated, and cysteine-rich protein, limiting structural studies to LRP1 fragments. Here, we report the first recombinant expression of the complete 61 domains of the full-length LRP1 ectodomain. This advance was achieved with a multistep cloning approach and by using DNA dilutions to improve protein yields. We investigated the binding properties of LRP1 using receptor-associated protein (RAP) as a model ligand due to its tight binding interaction. The LRP1 conformation was studied in its bound and unbound state using mass spectrometry, small-angle X-ray scattering, and negative-stain electron microscopy at neutral and acidic pH. Our findings revealed a pH-dependent release of the ligand associated with a conformational change of the receptor. In summary, this investigation of the complete LRP1 ectodomain significantly advances our understanding of this important receptor and provides the basis for further elucidating the mechanism of action of LRP1 in a whole and integrated system. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. The conformational changes induced by ubiquinone binding in the Na+-pumping NADH:ubiquinone oxidoreductase (Na+-NQR) are kinetically controlled by conserved glycines 140 and 141 of the NqrB subunit.

    Science.gov (United States)

    Strickland, Madeleine; Juárez, Oscar; Neehaul, Yashvin; Cook, Darcie A; Barquera, Blanca; Hellwig, Petra

    2014-08-22

    Na(+)-pumping NADH:ubiquinone oxidoreductase (Na(+)-NQR) is responsible for maintaining a sodium gradient across the inner bacterial membrane. This respiratory enzyme, which couples sodium pumping to the electron transfer between NADH and ubiquinone, is not present in eukaryotes and as such could be a target for antibiotics. In this paper it is shown that the site of ubiquinone reduction is conformationally coupled to the NqrB subunit, which also hosts the final cofactor in the electron transport chain, riboflavin. Previous work showed that mutations in conserved NqrB glycine residues 140 and 141 affect ubiquinone reduction and the proper functioning of the sodium pump. Surprisingly, these mutants did not affect the dissociation constant of ubiquinone or its analog HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide) from Na(+)-NQR, which indicates that these residues do not participate directly in the ubiquinone binding site but probably control its accessibility. Indeed, redox-induced difference spectroscopy showed that these mutations prevented the conformational change involved in ubiquinone binding but did not modify the signals corresponding to bound ubiquinone. Moreover, data are presented that demonstrate the NqrA subunit is able to bind ubiquinone but with a low non-catalytically relevant affinity. It is also suggested that Na(+)-NQR contains a single catalytic ubiquinone binding site and a second site that can bind ubiquinone but is not active.

  12. Chemical footprinting reveals conformational changes of 18S and 28S rRNAs at different steps of translation termination on the human ribosome.

    Science.gov (United States)

    Bulygin, Konstantin N; Bartuli, Yulia S; Malygin, Alexey A; Graifer, Dmitri M; Frolova, Ludmila Yu; Karpova, Galina G

    2016-02-01

    Translation termination in eukaryotes is mediated by release factors: eRF1, which is responsible for stop codon recognition and peptidyl-tRNA hydrolysis, and GTPase eRF3, which stimulates peptide release. Here, we have utilized ribose-specific probes to investigate accessibility of rRNA backbone in complexes formed by association of mRNA- and tRNA-bound human ribosomes with eRF1•eRF3•GMPPNP, eRF1•eRF3•GTP, or eRF1 alone as compared with complexes where the A site is vacant or occupied by tRNA. Our data show which rRNA ribose moieties are protected from attack by the probes in the complexes with release factors and reveal the rRNA regions increasing their accessibility to the probes after the factors bind. These regions in 28S rRNA are helices 43 and 44 in the GTPase associated center, the apical loop of helix 71, and helices 89, 92, and 94 as well as 18S rRNA helices 18 and 34. Additionally, the obtained data suggest that eRF3 neither interacts with the rRNA ribose-phosphate backbone nor dissociates from the complex after GTP hydrolysis. Taken together, our findings provide new information on architecture of the eRF1 binding site on mammalian ribosome at various translation termination steps and on conformational rearrangements induced by binding of the release factors. © 2016 Bulygin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  13. Peer influence: neural mechanisms underlying in-group conformity.

    Science.gov (United States)

    Stallen, Mirre; Smidts, Ale; Sanfey, Alan G

    2013-01-01

    People often conform to the behavior of others with whom they identify. However, it is unclear what fundamental mechanisms underlie this type of conformity. Here, we investigate the processes mediating in-group conformity by using functional magnetic resonance imaging (fMRI). Participants completed a perceptual decision-making task while undergoing fMRI, during which they were exposed to the judgments of both in-group and out-group members. Our data suggest that conformity to the in-group is mediated by both positive affect as well as the cognitive capacity of perspective taking. Examining the processes that drive in-group conformity by utilizing a basic decision-making paradigm combined with neuroimaging methods provides important insights into the potential mechanisms of conformity. These results may provide an integral step in developing more effective campaigns using group conformity as a tool for behavioral change.

  14. Peer influence: Neural mechanisms underlying in-group conformity

    Directory of Open Access Journals (Sweden)

    Mirre eStallen

    2013-03-01

    Full Text Available People often conform to the behavior of others with whom they identify. However, it is unclear what fundamental mechanisms underlie this type of conformity. Here, we investigate the processes mediating in-group conformity by using functional magnetic resonance imaging (fMRI. Participants completed a perceptual decision-making task while undergoing fMRI, during which they were exposed to the judgments of both in-group and out-group members. Our data suggest that conformity to the in-group is mediated by both positive affect as well as the cognitive capacity of perspective taking. Examining the processes that drive in-group conformity by utilizing a basic decision-making paradigm combined with neuroimaging methods provides important insights into the potential mechanisms of conformity. These results may provide an integral step in developing more effective campaigns using group conformity as a tool for behavioral change.

  15. Boundary Conformal Field Theory

    CERN Document Server

    Cardy, J L

    2004-01-01

    Boundary conformal field theory (BCFT) is simply the study of conformal field theory (CFT) in domains with a boundary. It gains its significance because, in some ways, it is mathematically simpler: the algebraic and geometric structures of CFT appear in a more straightforward manner; and because it has important applications: in string theory in the physics of open strings and D-branes, and in condensed matter physics in boundary critical behavior and quantum impurity models. In this article, however, I describe the basic ideas from the point of view of quantum field theory, without regard to particular applications nor to any deeper mathematical formulations.

  16. Conformal field theory

    CERN Document Server

    Ketov, Sergei V

    1995-01-01

    Conformal field theory is an elegant and powerful theory in the field of high energy physics and statistics. In fact, it can be said to be one of the greatest achievements in the development of this field. Presented in two dimensions, this book is designed for students who already have a basic knowledge of quantum mechanics, field theory and general relativity. The main idea used throughout the book is that conformal symmetry causes both classical and quantum integrability. Instead of concentrating on the numerous applications of the theory, the author puts forward a discussion of the general

  17. Enhanced conformational sampling technique provides an energy landscape view of large-scale protein conformational transitions.

    Science.gov (United States)

    Shao, Qiang

    2016-10-26

    Large-scale conformational changes in proteins are important for their functions. Tracking the conformational change in real time at the level of a single protein molecule, however, remains a great challenge. In this article, we present a novel in silico approach with the combination of normal mode analysis and integrated-tempering-sampling molecular simulation (NMA-ITS) to give quantitative data for exploring the conformational transition pathway in multi-dimensional energy landscapes starting only from the knowledge of the two endpoint structures of the protein. The open-to-closed transitions of three proteins, including nCaM, AdK, and HIV-1 PR, were investigated using NMA-ITS simulations. The three proteins have varied structural flexibilities and domain communications in their respective conformational changes. The transition state structure in the conformational change of nCaM and the associated free-energy barrier are in agreement with those measured in a standard explicit-solvent REMD simulation. The experimentally measured transition intermediate structures of the intrinsically flexible AdK are captured by the conformational transition pathway measured here. The dominant transition pathways between the closed and fully open states of HIV-1 PR are very similar to those observed in recent REMD simulations. Finally, the evaluated relaxation times of the conformational transitions of three proteins are roughly at the same level as reported experimental data. Therefore, the NMA-ITS method is applicable for a variety of cases, providing both qualitative and quantitative insights into the conformational changes associated with the real functions of proteins.

  18. Conformational properties of oxazoline-amino acids

    Science.gov (United States)

    Staś, Monika; Broda, Małgorzata A.; Siodłak, Dawid

    2016-04-01

    Oxazoline-amino acids (Xaa-Ozn) occur in natural peptides of potentially important bioactivity. The conformations of the model compounds: Ac-(S)-Ala-Ozn(4R-Me), Ac-(S)-Ala-Ozn(4S-Me), and (gauche+, gauche-, anti) Ac-(S)-Val-Ozn(4R-Me) were studied at meta-hybrid M06-2X/6-311++G(d,p) method including solvent effect. Boc-L-Ala-L-Ozn-4-COOMe and Boc-L-Val-L-Ozn-4-COOMe were synthesized and studied by FT-IR and NMR-NOE methods. The conformations in crystal state were gathered from the Cambridge Structural Data Base. The main conformational feature of the oxazoline amino acids is the conformation β2 (ϕ,ψ ∼ -161°, -6°), which predominates in weakly polar environment and still is accessible in polar surrounding. The changes of the conformational preferences towards the conformations αR (ϕ,ψ ∼ -70°, -15°) and then β (ϕ,ψ ∼ -57°, -155°) are observed with increase of the environment polarity.

  19. Conformational stability of calreticulin

    DEFF Research Database (Denmark)

    Jørgensen, Charlotte S; Trandum, Christa; Larsen, Nanna Brink

    2005-01-01

    The conformational stability of calreticulin was investigated. Apparent unfolding temperatures (Tm) increased from 31 degrees C at pH 5 to 51 degrees C at pH 9, but electrophoretic analysis revealed that calreticulin oligomerized instead of unfolding. Structural analyses showed that the single C-...

  20. A CONFORMATIONAL ELASTICITY THEORY

    Institute of Scientific and Technical Information of China (English)

    1998-01-01

    A new statistical theory based on the rotational isomeric state model describing the chain conformational free energy has been proposed. This theory can be used to predict different tensions of rubber elongation for chemically different polymers, and the energy term during the elongation of natural rubber coincides with the experimental one.

  1. Conformal cloak for waves

    CERN Document Server

    Chen, Huanyang; Tyc, Tomas

    2011-01-01

    Conformal invisibility devices are only supposed to work within the validity range of geometrical optics. Here we show by numerical simulations and analytical arguments that for certain quantized frequencies they are nearly perfect even in a regime that clearly violates geometrical optics. The quantization condition follows from the analogy between the Helmholtz equation and the stationary Schrodinger equation.

  2. Conformal supermultiplets without superpartners

    CERN Document Server

    Jarvis, Peter

    2011-01-01

    We consider polynomial deformations of Lie superalgebras and their representations. For the class A(n-1,0) ~ sl(n/1), we identify families of superalgebras of quadratic and cubic type, consistent with Jacobi identities. For such deformed superalgebras we point out the possibility of zero step supermultiplets, carried on a single, irreducible representation of the even (Lie) subalgebra. For the conformal group SU(2,2) in 1+3-dimensional spacetime, such irreducible (unitary) representations correspond to standard conformal fields (j_1,j_2;d), where (j_1,j_2) is the spin and d the conformal dimension; in the massless class j_1 j_2=0, and d=j_1+j_2+1. We show that these repesentations are zero step supermultiplets for the superalgebra SU_(2)(2,2/1), the quadratic deformation of conformal supersymmetry SU(2,2/1). We propose to elevate SU_(2)(2,2/1) to a symmetry of the S-matrix. Under this scenario, low-energy standard model matter fields (leptons, quarks, Higgs scalars and gauge fields) descended from such confor...

  3. Conformal General Relativity

    CERN Document Server

    Pervushin, V

    2001-01-01

    The inflation-free solution of problems of the modern cosmology (horizon, cosmic initial data, Planck era, arrow of time, singularity,homogeneity, and so on) is considered in the conformal-invariant unified theory given in the space with geometry of similarity where we can measure only the conformal-invariant ratio of all quantities. Conformal General Relativity is defined as the $SU_c(3)\\times SU(2)\\times U(1)$-Standard Model where the dimensional parameter in the Higgs potential is replaced by a dilaton scalar field described by the negative Penrose-Chernikov-Tagirov action. Spontaneous SU(2) symmetry breaking is made on the level of the conformal-invariant angle of the dilaton-Higgs mixing, and it allows us to keep the structure of Einstein's theory with the equivalence principle. We show that the lowest order of the linearized equations of motion solves the problems mentioned above and describes the Cold Universe Scenario with the constant temperature T and z-history of all masses with respect to an obser...

  4. Hypothesis for the mechanism for heat-induced antigen retrieval occurring on fresh frozen sections without formalin-fixation in immunohistochemistry.

    Science.gov (United States)

    Kakimoto, Kochi; Takekoshi, Susumu; Miyajima, Katsuhiro; Osamura, R Yoshiyuki

    2008-08-01

    The mechanism involved in heat-induced antigen retrieval (AR) remains unproven but probably utilizes the breaking of formalin-induced cross-linkages. We investigated the effectiveness of heat-induced AR on immunohistochemistry and dot-blot analysis using rat uterus tissue sections and protein extracts without formalin-fixation. The unfixed frozen sections, which did not show immunostaining with nine antibodies, were clearly stained after heating the sections. In the dot-blot analysis, the immunoblot sensitivity of detection was greatly enhanced by heating the protein-blotted membrane. These results indicate that other mechanisms of breaking formalin-induced cross-linkages may be present. We propose that one of the other mechanisms for heat-induced AR is that accessibility to the target epitopes of antigenic proteins is limited by natural steric barriers even in the fresh state caused by the antigenic protein itself.

  5. Using IR spectroscopy and multivariate curve resolution to elucidate mechanism of heat-induced decomposition of an organic complex

    DEFF Research Database (Denmark)

    Karpushkin, Evgeny; Gvozdik, Nataliya; Kucheryavskiy, Sergey V.

    , the evolved compounds structure is relatively similar, and therefore their analysis by means of vibrational spectroscopy is a convenient approach towards elucida- tion of the underlying reactions mechanism as well as optimization of the pro- cess conditions. Modern analytical instruments provide...... the opportunity to carry out simultaneous thermogravimetry/differential scanning calorimetry analysis and IR/Raman/mass spectrometry investigation of the evolving gaseous products. However, elucidation of the mechanism of the reactions occurring upon heat- ing is not completely straightforward, due to a number...... and discuss the results of TGA-DSC-IR study of heat-induced decomposition of tris(acetylacetonato)manganese(III) complex. Using this process as an example, we have indicated the complications of the evolved gas analysis and demonstrated that they can be partially overcome taking advantage of multivariate...

  6. Conforming STOP Violence Against Women Formula Grant Program Regulations to Statutory Change; Definitions and Confidentiality Requirements Applicable to All OVW Grant Programs. Final rule.

    Science.gov (United States)

    2016-11-29

    This rule amends the regulations for the STOP (ServicesTrainingOfficersProsecutors) Violence Against Women Formula Grant Program (STOP Program) and the general provisions governing Office on Violence Against Women (OVW) programs to comply with statutory changes and reduce repetition of statutory language. Also, this rule implements statutory requirements for nondisclosure of confidential or private information relating to all OVW grant programs.

  7. Intramolecular and intermolecular fluorescence resonance energy transfer in fluorescent protein-tagged Na-K-Cl cotransporter (NKCC1): sensitivity to regulatory conformational change and cell volume.

    Science.gov (United States)

    Pedersen, Meike; Carmosino, Monica; Forbush, Biff

    2008-02-01

    To examine the structure and function of the Na-K-Cl cotransporter, NKCC1, we tagged the transporter with cyan (CFP) and yellow (YFP) fluorescent proteins and measured fluorescence resonance energy transfer (FRET) in stably expressing human embryonic kidney cell lines. Fluorescent protein tags were added at the N-terminal residue between the regulatory domain and the membrane domain and within a poorly conserved region of the C terminus. Both singly and doubly tagged NKCC1s were appropriately trafficked to the cell membrane and were fully functional; regulation was normal except when YFP was inserted near the regulatory domain, in which case activation occurred only upon incubation with calyculin A. Quenching of YFP fluorescence by Cl(-) provided a ratiometric indicator of intracellular [Cl(-)]. All of the CFP/YFP NKCC pairs exhibited some level of FRET, demonstrating the presence of dimers or higher multimers in functioning NKCC1. With YFP near the regulatory domain and CFP in the C terminus, we recorded a 6% FRET change signaling the regulatory phosphorylation event. On the other hand, when the probe was placed at the extreme N terminus, such changes were not seen, presumably due to the length and predicted flexibility of the N terminus. Substantial FRET changes were observed contemporaneous with cell volume changes, possibly reflective of an increase in molecular crowding upon cell shrinkage.

  8. Solvent- and Pressure-Induced Phase Changes in Two 3D Copper Glutarate-Based Metal-Organic Frameworks via Glutarate (+gauche ⇄ -gauche) Conformational Isomerism.

    Science.gov (United States)

    Bezuidenhout, Charl X; Smith, Vincent J; Esterhuysen, Catharine; Barbour, Leonard J

    2017-04-26

    Two isoreticular three-dimensional copper(II) glutarate-based pillared-layered metal-organic frameworks (MOFs) with flexible pillars, [Cu2(glu)2(bpa)] and [Cu2(glu)2(bpp)] (bpa = 1,2-bis(4-pyridyl)ethane; bpp = 1,3-bis(4-pyridyl)propane), undergo spontaneous phase changes upon solvent loss at room temperature. Using single-crystal X-ray diffraction analysis (SCXRD), we show that the phase changes result in new narrow-channel forms that experience a large reduction in solvent-accessible volume. Moreover, the [Cu2(glu)2(bpa)] MOF displays a stepped sorption isotherm for the uptake of CO2 at room temperature. This is indicative of reversion of the framework to the wide-channel form under CO2 pressure. Supercritical CO2 was used to isolate the gas-included structures, and by means of SCXRD we were able to determine the positions of the CO2 molecules in the channels of the frameworks. Finally, we report the use of molecular modeling simulations to elucidate the phase-change mechanism, including the energetic changes involved. Structural limitations in both MOFs allow for only direct gauche-gauche enantiomeric interconversion of the glutarate moieties.

  9. Distortion of genetically modified organism quantification in processed foods: influence of particle size compositions and heat-induced DNA degradation.

    Science.gov (United States)

    Moreano, Francisco; Busch, Ulrich; Engel, Karl-Heinz

    2005-12-28

    Milling fractions from conventional and transgenic corn were prepared at laboratory scale and used to study the influence of sample composition and heat-induced DNA degradation on the relative quantification of genetically modified organisms (GMO) in food products. Particle size distributions of the obtained fractions (coarse grits, regular grits, meal, and flour) were characterized using a laser diffraction system. The application of two DNA isolation protocols revealed a strong correlation between the degree of comminution of the milling fractions and the DNA yield in the extracts. Mixtures of milling fractions from conventional and transgenic material (1%) were prepared and analyzed via real-time polymerase chain reaction. Accurate quantification of the adjusted GMO content was only possible in mixtures containing conventional and transgenic material in the form of analogous milling fractions, whereas mixtures of fractions exhibiting different particle size distributions delivered significantly over- and underestimated GMO contents depending on their compositions. The process of heat-induced nucleic acid degradation was followed by applying two established quantitative assays showing differences between the lengths of the recombinant and reference target sequences (A, deltal(A) = -25 bp; B, deltal(B) = +16 bp; values related to the amplicon length of the reference gene). Data obtained by the application of method A resulted in underestimated recoveries of GMO contents in the samples of heat-treated products, reflecting the favored degradation of the longer target sequence used for the detection of the transgene. In contrast, data yielded by the application of method B resulted in increasingly overestimated recoveries of GMO contents. The results show how commonly used food technological processes may lead to distortions in the results of quantitative GMO analyses.

  10. Reactions driving conformational movements (molecular motors) in gels: conformational and structural chemical kinetics.

    Science.gov (United States)

    Otero, Toribio F

    2017-01-18

    In this perspective the empirical kinetics of conducting polymers exchanging anions and solvent during electrochemical reactions to get dense reactive gels is reviewed. The reaction drives conformational movements of the chains (molecular motors), exchange of ions and solvent with the electrolyte and structural (relaxation, swelling, shrinking and compaction) gel changes. Reaction-driven structural changes are identified and quantified from electrochemical responses. The empirical reaction activation energy (Ea), the reaction coefficient (k) and the reaction orders (α and β) change as a function of the conformational energy variation during the reaction. This conformational energy becomes an empirical magnitude. Ea, k, α and β include and provide quantitative conformational and structural information. The chemical kinetics becomes structural chemical kineti