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Sample records for stabilized protein crystals

  1. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins.

    Science.gov (United States)

    Chae, Pil Seok; Rasmussen, Søren G F; Rana, Rohini R; Gotfryd, Kamil; Chandra, Richa; Goren, Michael A; Kruse, Andrew C; Nurva, Shailika; Loland, Claus J; Pierre, Yves; Drew, David; Popot, Jean-Luc; Picot, Daniel; Fox, Brian G; Guan, Lan; Gether, Ulrik; Byrne, Bernadette; Kobilka, Brian; Gellman, Samuel H

    2010-12-01

    The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces of native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose-neopentyl glycol (MNG) amphiphile family show favorable behavior relative to conventional detergents, as manifested in multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied.

  2. Characterization of polyacrylamide-stabilized Pf1 phage liquid crystals for protein NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Trempe, Jean-Francois; Morin, Frederick G.; Xia Zhicheng; Marchessault, Robert H.; Gehring, Kalle [McGill University, Department of Biochemistry and Department of Chemistry (Canada)], E-mail: kalle@bri.nrc.ca

    2002-01-15

    A new polymer-stabilized nematic liquid crystal has been characterized for the measurement of biomolecular residual dipolar couplings. Filamentous Pf1 phage were embedded in a polyacrylamide matrix that fixes the orientation of the particles. The alignment was characterized by the quadrupolar splitting of the {sup 2}H NMR water signal and by the measurement of {sup 1}H-{sup 15}N residual dipolar couplings (RDC) in the archeal translation elongation factor 1{beta}. Protein dissolved in the polymer-stabilized medium orients quantitatively as in media without polyacrylamide. We show that the quadrupolar splitting and RDCs are zero in media in which the Pf1 phage particles are aligned at the magic angle. This allows measurement of J and dipolar couplings in a single sample.

  3. Characterization of polyacrylamide-stabilized Pf1 phage liquid crystals for protein NMR spectroscopy

    International Nuclear Information System (INIS)

    Trempe, Jean-Francois; Morin, Frederick G.; Xia Zhicheng; Marchessault, Robert H.; Gehring, Kalle

    2002-01-01

    A new polymer-stabilized nematic liquid crystal has been characterized for the measurement of biomolecular residual dipolar couplings. Filamentous Pf1 phage were embedded in a polyacrylamide matrix that fixes the orientation of the particles. The alignment was characterized by the quadrupolar splitting of the 2 H NMR water signal and by the measurement of 1 H- 15 N residual dipolar couplings (RDC) in the archeal translation elongation factor 1β. Protein dissolved in the polymer-stabilized medium orients quantitatively as in media without polyacrylamide. We show that the quadrupolar splitting and RDCs are zero in media in which the Pf1 phage particles are aligned at the magic angle. This allows measurement of J and dipolar couplings in a single sample

  4. A Novel Acidic Matrix Protein, PfN44, Stabilizes Magnesium Calcite to Inhibit the Crystallization of Aragonite*

    Science.gov (United States)

    Pan, Cong; Fang, Dong; Xu, Guangrui; Liang, Jian; Zhang, Guiyou; Wang, Hongzhong; Xie, Liping; Zhang, Rongqing

    2014-01-01

    Magnesium is widely used to control calcium carbonate deposition in the shell of pearl oysters. Matrix proteins in the shell are responsible for nucleation and growth of calcium carbonate crystals. However, there is no direct evidence supporting a connection between matrix proteins and magnesium. Here, we identified a novel acidic matrix protein named PfN44 that affected aragonite formation in the shell of the pearl oyster Pinctada fucata. Using immunogold labeling assays, we found PfN44 in both the nacreous and prismatic layers. In shell repair, PfN44 was repressed, whereas other matrix proteins were up-regulated. Disturbing the function of PfN44 by RNAi led to the deposition of porous nacreous tablets with overgrowth of crystals in the nacreous layer. By in vitro circular dichroism spectra and fluorescence quenching, we found that PfN44 bound to both calcium and magnesium with a stronger affinity for magnesium. During in vitro calcium carbonate crystallization and calcification of amorphous calcium carbonate, PfN44 regulated the magnesium content of crystalline carbonate polymorphs and stabilized magnesium calcite to inhibit aragonite deposition. Taken together, our results suggested that by stabilizing magnesium calcite to inhibit aragonite deposition, PfN44 participated in P. fucata shell formation. These observations extend our understanding of the connections between matrix proteins and magnesium. PMID:24302723

  5. Pressure cryocooling protein crystals

    Science.gov (United States)

    Kim, Chae Un [Ithaca, NY; Gruner, Sol M [Ithaca, NY

    2011-10-04

    Preparation of cryocooled protein crystal is provided by use of helium pressurizing and cryocooling to obtain cryocooled protein crystal allowing collection of high resolution data and by heavier noble gas (krypton or xenon) binding followed by helium pressurizing and cryocooling to obtain cryocooled protein crystal for collection of high resolution data and SAD phasing simultaneously. The helium pressurizing is carried out on crystal coated to prevent dehydration or on crystal grown in aqueous solution in a capillary.

  6. Protein stability: a crystallographer’s perspective

    International Nuclear Information System (INIS)

    Deller, Marc C.; Kong, Leopold; Rupp, Bernhard

    2016-01-01

    An understanding of protein stability is essential for optimizing the expression, purification and crystallization of proteins. In this review, discussion will focus on factors affecting protein stability on a somewhat practical level, particularly from the view of a protein crystallographer. Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed

  7. Protein stability: a crystallographer’s perspective

    Energy Technology Data Exchange (ETDEWEB)

    Deller, Marc C., E-mail: mdeller@stanford.edu [Stanford University, Shriram Center, 443 Via Ortega, Room 097, MC5082, Stanford, CA 94305-4125 (United States); Kong, Leopold [National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Building 8, Room 1A03, 8 Center Drive, Bethesda, MD 20814 (United States); Rupp, Bernhard [k.-k. Hofkristallamt, 91 Audrey Place, Vista, CA 92084 (United States); Medical University of Innsbruck, Schöpfstrasse 41, A-6020 Innsbruck (Austria)

    2016-01-26

    An understanding of protein stability is essential for optimizing the expression, purification and crystallization of proteins. In this review, discussion will focus on factors affecting protein stability on a somewhat practical level, particularly from the view of a protein crystallographer. Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed.

  8. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins

    OpenAIRE

    Chae, Pil Seok; Rasmussen, Søren G. F.; Rana, Rohini; Gotfryd, Kamil; Chandra, Richa; Goren, Michael A.; Kruse, Andrew C.; Nurva, Shailika; Loland, Claus J.; Pierre, Yves; Drew, David; Popot, Jean-Luc; Picot, Daniel; Fox, Brian G.; Guan, Lan

    2010-01-01

    The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces displayed by native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each of which is built around a central quaternary carbon atom derived from...

  9. Glucose-Neopentyl Glycol (GNG) Amphiphiles for Membrane Protein Solubilization, Stabilization and Crystallization

    Science.gov (United States)

    Rana, Rohini R.; Gotfryd, Kamil; Rasmussen, Søren G. F.; Kruse, Andrew C.; Cho, Kyung Ho; Capaldi, Stefano; Carlsson, Emil; Kobilka, Brian; Loland, Claus J.; Gether, Ulrik; Banerjee, Surajit

    2012-01-01

    The development of a new class of surfactants for membrane protein manipulation, “GNG amphiphiles”, is reported. These amphiphiles display promising behavior for membrane proteins, as demonstrated recently by the high resolution structure of a sodium-pumping pyrophosphatase reported by Kellosalo et al. PMID:23165475

  10. Glucose-Neopentyl Glycol (GNG) Amphiphiles for Membrane Protein Solubilization, Stabilization and Crystallization

    OpenAIRE

    Chae, Pil Seok; Rana, Rohini R.; Gotfryd, Kamil; Rasmussen, Søren G. F.; Kruse, Andrew C.; Cho, Kyung Ho; Capaldi, Stefano; Carlsson, Emil; Kobilka, Brian; Loland, Claus J.; Gether, Ulrik; Banerjee, Surajit; Byrne, Bernadette; Lee, John K.; Gellman, Samuel H.

    2013-01-01

    The development of a new class of surfactants for membrane protein manipulation, “GNG amphiphiles”, is reported. These amphiphiles display promising behavior for membrane proteins, as demonstrated recently by the high resolution structure of a sodium-pumping pyrophosphatase reported by Kellosalo et al.

  11. Protein Crystal Growth

    Science.gov (United States)

    2003-01-01

    In order to rapidly and efficiently grow crystals, tools were needed to automatically identify and analyze the growing process of protein crystals. To meet this need, Diversified Scientific, Inc. (DSI), with the support of a Small Business Innovation Research (SBIR) contract from NASA s Marshall Space Flight Center, developed CrystalScore(trademark), the first automated image acquisition, analysis, and archiving system designed specifically for the macromolecular crystal growing community. It offers automated hardware control, image and data archiving, image processing, a searchable database, and surface plotting of experimental data. CrystalScore is currently being used by numerous pharmaceutical companies and academic and nonprofit research centers. DSI, located in Birmingham, Alabama, was awarded the patent Method for acquiring, storing, and analyzing crystal images on March 4, 2003. Another DSI product made possible by Marshall SBIR funding is VaporPro(trademark), a unique, comprehensive system that allows for the automated control of vapor diffusion for crystallization experiments.

  12. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins

    DEFF Research Database (Denmark)

    Chae, Pil Seok; Rasmussen, Søren G F; Rana, Rohini R

    2010-01-01

    proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose-neopentyl glycol (MNG) amphiphile...

  13. Disorder in Protein Crystals.

    Science.gov (United States)

    Clarage, James Braun, II

    1990-01-01

    Methods have been developed for analyzing the diffuse x-ray scattering in the halos about a crystal's Bragg reflections as a means of determining correlations in atomic displacements in protein crystals. The diffuse intensity distribution for rhombohedral insulin, tetragonal lysozyme, and triclinic lysozyme crystals was best simulated in terms of exponential displacement correlation functions. About 90% of the disorder can be accounted for by internal movements correlated with a decay distance of about 6A; the remaining 10% corresponds to intermolecular movements that decay in a distance the order of size of the protein molecule. The results demonstrate that protein crystals fit into neither the Einstein nor the Debye paradigms for thermally fluctuating crystalline solids. Unlike the Einstein model, there are correlations in the atomic displacements, but these correlations decay more steeply with distance than predicted by the Debye-Waller model for an elastic solid. The observed displacement correlations are liquid -like in the sense that they decay exponentially with the distance between atoms, just as positional correlations in a liquid. This liquid-like disorder is similar to the disorder observed in 2-D crystals of polystyrene latex spheres, and similar systems where repulsive interactions dominate; hence, these colloidal crystals appear to provide a better analogy for the dynamics of protein crystals than perfectly elastic lattices.

  14. Protein stability: a crystallographer’s perspective

    Science.gov (United States)

    Deller, Marc C.; Kong, Leopold; Rupp, Bernhard

    2016-01-01

    Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed. PMID:26841758

  15. Bacterial Ice Crystal Controlling Proteins

    Science.gov (United States)

    Lorv, Janet S. H.; Rose, David R.; Glick, Bernard R.

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

  16. Stability of Hyperthermophilic Proteins

    DEFF Research Database (Denmark)

    Stiefler-Jensen, Daniel

    stability by randomly generate mutants and lengthy screening processes to identify the best new mutants. However, with the increase in available genomic sequences of thermophilic or hyperthermophilic organisms a world of enzymes with intrinsic high stability are now available. As these organisms are adapted...... to life at high temperatures so are their enzymes, as a result the high stability is accompanied by low activity at moderate temperatures. Thus, much effort had been put into decoding the mechanisms behind the high stability of the thermophilic enzymes. The hope is to enable scientist to design enzymes...... in the high stability of hyperthermophilic enzymes. The thesis starts with an introduction to the field of protein and enzyme stability with special focus on the thermophilic and hyperthermophilic enzymes and proteins. After the introduction three original research manuscripts present the experimental data...

  17. Crystal Structure of Mn2+-bound Escherichia coli L-arabinose Isomerase (ECAI) and Implications in Protein Catalytic Mechanism and Thermo-Stability

    International Nuclear Information System (INIS)

    Zhu, W.; Manjasetty, B.; Chance, M.

    2007-01-01

    The functional properties of proteins depend on their three-dimensional shapes. Protein structures can be determined by X-ray crystallography as a tool. The three-dimensional structure of the apo form of the Escherichia coli L-arabinose isomerase (ECAI) has recently been determined. ECAI is responsible for the initial stage of L-arabinose catabolism, converting arabinose into ribulose in vivo. This enzyme also plays a crucial role in catalyzing the conversion of galactose into tagatose (low calorie natural sugar) in vitro. ECAI utilizes Mn 2+ for its catalytic activity. Crystals of the ECAI + Mn 2+ complex helps to investigate the catalytic properties of the enzyme. Therefore, crystals of ECAI + Mn 2+ complex were grown using hanging drop vapor diffusion method at room temperature. Diffraction data were collected at X4C beamline, National Synchrotron Light Source, Brookhaven National Laboratory. The structure was solved by the molecular replacement technique and has been refined to Rwork of 0.23 at 2.8 (angstrom) resolution using X3A beamline computational facility. The structure was deposited to Protein Data Bank (PDB ID 2HXG). Mn 2+ ion was localized to the previously identified putative active site with octahedral coordination. Comparison of apo and holo form of ECAI structures permits the identification of structural features that are of importance to the intrinsic activity and heat stability of AI

  18. Protein surface shielding agents in protein crystallization

    International Nuclear Information System (INIS)

    Hašek, J.

    2011-01-01

    The crystallization process can be controlled by protein surface shielding agents blocking undesirable competitive adhesion modes during non-equilibrium processes of deposition of protein molecules on the surface of growing crystalline blocks. The hypothesis is based on a number of experimental proofs from diffraction experiments and also retrieved from the Protein Data Bank. The molecules adhering temporarily on the surface of protein molecules change the propensity of protein molecules to deposit on the crystal surface in a definite position and orientation. The concepts of competitive adhesion modes and protein surface shielding agents acting on the surface of molecules in a non-equilibrium process of protein crystallization provide a useful platform for the control of crystallization. The desirable goal, i.e. a transient preference of a single dominating adhesion mode between protein molecules during crystallization, leads to uniform deposition of proteins in a crystal. This condition is the most important factor for diffraction quality and thus also for the accuracy of protein structure determination. The presented hypothesis is a generalization of the experimentally well proven behaviour of hydrophilic polymers on the surface of protein molecules of other compounds

  19. in silico identification of cross affinity towards Cry1Ac pesticidal protein with receptor enzyme in Bos taurus and sequence, structure analysis of crystal proteins for stability.

    Science.gov (United States)

    Ebenezer, King Solomon; Nachimuthu, Ramesh; Thiagarajan, Prabha; Velu, Rajesh Kannan

    2013-01-01

    Any novel protein introduced into the GM crops need to be evaluated for cross affinity on living organisms. Many researchers are currently focusing on the impact of Bacillus thuringiensis cotton on soil and microbial diversity by field experiments. In spite of this, in silico approach might be helpful to elucidate the impact of cry genes. The crystal a protein which was produced by Bt at the time of sporulation has been used as a biological pesticide to target the insectivorous pests like Cry1Ac for Helicoverpa armigera and Cry2Ab for Spodoptera sp. and Heliothis sp. Here, we present the comprehensive in silico analysis of Cry1Ac and Cry2Ab proteins with available in silico tools, databases and docking servers. Molecular docking of Cry1Ac with procarboxypeptidase from Helicoverpa armigera and Cry1Ac with Leucine aminopeptidase from Bos taurus has showed the 125(th) amino acid position to be the preference site of Cry1Ac protein. The structures were compared with each other and it showed 5% of similarity. The cross affinity of this toxin that have confirmed the earlier reports of ill effects of Bt cotton consumed by cattle.

  20. Protein crystal nucleation in pores.

    Science.gov (United States)

    Nanev, Christo N; Saridakis, Emmanuel; Chayen, Naomi E

    2017-01-16

    The most powerful method for protein structure determination is X-ray crystallography which relies on the availability of high quality crystals. Obtaining protein crystals is a major bottleneck, and inducing their nucleation is of crucial importance in this field. An effective method to form crystals is to introduce nucleation-inducing heterologous materials into the crystallization solution. Porous materials are exceptionally effective at inducing nucleation. It is shown here that a combined diffusion-adsorption effect can increase protein concentration inside pores, which enables crystal nucleation even under conditions where heterogeneous nucleation on flat surfaces is absent. Provided the pore is sufficiently narrow, protein molecules approach its walls and adsorb more frequently than they can escape. The decrease in the nucleation energy barrier is calculated, exhibiting its quantitative dependence on the confinement space and the energy of interaction with the pore walls. These results provide a detailed explanation of the effectiveness of porous materials for nucleation of protein crystals, and will be useful for optimal design of such materials.

  1. Protein Crystal Recombinant Human Insulin

    Science.gov (United States)

    1994-01-01

    The comparison of protein crystal, Recombiant Human Insulin; space-grown (left) and earth-grown (right). On STS-60, Spacehab II indicated that space-grown crystals are larger and of greater optical clarity than their earth-grown counterparts. Recombiant Human Insulin facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  2. Proteins and their crystals

    Czech Academy of Sciences Publication Activity Database

    Kutá-Smatanová, Ivana; Hogg, T.; Hilgenfeld, R.; Grandori, R.; Carey, J.; Vácha, František; Štys, Dalibor

    2003-01-01

    Roč. 10, č. 1 (2003), s. 31-32 ISSN 1211-5894 R&D Projects: GA MŠk LN00A141; GA ČR GA206/00/D007 Institutional research plan: CEZ:AV0Z5051902; CEZ:MSM 123100001 Keywords : pokeweed antiviral protein * flavodoxin-like protein * PSII Subject RIV: EB - Genetics ; Molecular Biology

  3. Proteins and their crystals

    Czech Academy of Sciences Publication Activity Database

    Kutá-Smatanová, Ivana; Hogg, T.; Hilgenfeld, R.; Grandori, R.; Carey, J.; Vácha, František; Štys, D.

    2003-01-01

    Roč. 10, - (2003), s. 30-31 ISSN 1211-5894 R&D Projects: GA MŠk LN00A141; GA ČR GA206/00/D007 Institutional research plan: CEZ:MSM 123100001 Keywords : antiviral proteins Subject RIV: CD - Macromolecular Chemistry

  4. Approaches to automated protein crystal harvesting

    Energy Technology Data Exchange (ETDEWEB)

    Deller, Marc C., E-mail: mdeller@scripps.edu; Rupp, Bernhard, E-mail: mdeller@scripps.edu

    2014-01-28

    Approaches to automated and robot-assisted harvesting of protein crystals are critically reviewed. While no true turn-key solutions for automation of protein crystal harvesting are currently available, systems incorporating advanced robotics and micro-electromechanical systems represent exciting developments with the potential to revolutionize the way in which protein crystals are harvested.

  5. Measurements of Protein Crystal Face Growth Rates

    Science.gov (United States)

    Gorti, S.

    2014-01-01

    Protein crystal growth rates will be determined for several hyperthermophile proteins.; The growth rates will be assessed using available theoretical models, including kinetic roughening.; If/when kinetic roughening supersaturations are established, determinations of protein crystal quality over a range of supersaturations will also be assessed.; The results of our ground based effort may well address the existence of a correlation between fundamental growth mechanisms and protein crystal quality.

  6. Taxol crystals can masquerade as stabilized microtubules.

    Directory of Open Access Journals (Sweden)

    Margit Foss

    Full Text Available Taxol is a potent anti-mitotic drug used in chemotherapy, angioplastic stents, and cell biology research. By binding and stabilizing microtubules, Taxol inhibits their dynamics, crucial for cell division, motility, and survival. The drug has also been reported to induce formation of asters and bundles composed of stabilized microtubules. Surprisingly, at commonly used concentrations, Taxol forms crystals that rapidly bind fluorescent tubulin subunits, generating structures with an uncanny resemblance to microtubule asters and bundles. Kinetic and topological considerations suggest that tubulin subunits, rather than microtubules, bind the crystals. This sequestration of tubulin from the subunit pool would be expected to shift the equilibrium of free to polymerized tubulin to disfavor assembly. Our results imply that some previously reported Taxol-induced asters or bundles could include or be composed of tubulin-decorated Taxol crystals. Thus, reevaluation of certain morphological, chemical, and physical properties of Taxol-treated microtubules may be necessary. Moreover, our findings suggest a novel mechanism for chemotherapy-induced cytotoxicity in non-dividing cells, with far-reaching medical implications.

  7. Protein-crystal growth experiment (planned)

    Science.gov (United States)

    Fujita, S.; Asano, K.; Hashitani, T.; Kitakohji, T.; Nemoto, H.; Kitamura, S.

    1988-01-01

    To evaluate the effectiveness of a microgravity environment on protein crystal growth, a system was developed using 5 cubic feet Get Away Special payload canister. In the experiment, protein (myoglobin) will be simultaneously crystallized from an aqueous solution in 16 crystallization units using three types of crystallization methods, i.e., batch, vapor diffusion, and free interface diffusion. Each unit has two compartments: one for the protein solution and the other for the ammonium sulfate solution. Compartments are separated by thick acrylic or thin stainless steel plates. Crystallization will be started by sliding out the plates, then will be periodically recorded up to 120 hours by a still camera. The temperature will be passively controlled by a phase transition thermal storage component and recorded in IC memory throughout the experiment. Microgravity environment can then be evaluated for protein crystal growth by comparing crystallization in space with that on Earth.

  8. The MORPHEUS II protein crystallization screen

    International Nuclear Information System (INIS)

    Gorrec, Fabrice

    2015-01-01

    MORPHEUS II is a 96-condition initial crystallization screen formulated de novo. The screen incorporates reagents selected from the Protein Data Bank to yield crystals that are not observed in traditional conditions. In addition, the formulation facilitates the optimization and cryoprotection of crystals. High-quality macromolecular crystals are a prerequisite for the process of protein structure determination by X-ray diffraction. Unfortunately, the relative yield of diffraction-quality crystals from crystallization experiments is often very low. In this context, innovative crystallization screen formulations are continuously being developed. In the past, MORPHEUS, a screen in which each condition integrates a mix of additives selected from the Protein Data Bank, a cryoprotectant and a buffer system, was developed. Here, MORPHEUS II, a follow-up to the original 96-condition initial screen, is described. Reagents were selected to yield crystals when none might be observed in traditional initial screens. Besides, the screen includes heavy atoms for experimental phasing and small polyols to ensure the cryoprotection of crystals. The suitability of the resulting novel conditions is shown by the crystallization of a broad variety of protein samples and their efficiency is compared with commercially available conditions

  9. The MORPHEUS II protein crystallization screen

    Energy Technology Data Exchange (ETDEWEB)

    Gorrec, Fabrice, E-mail: fgorrec@mrc-lmb.cam.ac.uk [MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH (United Kingdom)

    2015-06-27

    MORPHEUS II is a 96-condition initial crystallization screen formulated de novo. The screen incorporates reagents selected from the Protein Data Bank to yield crystals that are not observed in traditional conditions. In addition, the formulation facilitates the optimization and cryoprotection of crystals. High-quality macromolecular crystals are a prerequisite for the process of protein structure determination by X-ray diffraction. Unfortunately, the relative yield of diffraction-quality crystals from crystallization experiments is often very low. In this context, innovative crystallization screen formulations are continuously being developed. In the past, MORPHEUS, a screen in which each condition integrates a mix of additives selected from the Protein Data Bank, a cryoprotectant and a buffer system, was developed. Here, MORPHEUS II, a follow-up to the original 96-condition initial screen, is described. Reagents were selected to yield crystals when none might be observed in traditional initial screens. Besides, the screen includes heavy atoms for experimental phasing and small polyols to ensure the cryoprotection of crystals. The suitability of the resulting novel conditions is shown by the crystallization of a broad variety of protein samples and their efficiency is compared with commercially available conditions.

  10. The role of shape vs. patches in protein crystallization

    Science.gov (United States)

    Glaser, Jens; Glotzer, Sharon C.

    Proteins fold into a multitude of three-dimensional native structures. The structures of over 100,000 known proteins are deposited in the protein data bank, and most of them have been determined through X-ray crystallography. We ask the question if the role of shape in protein crystallization can be isolated using simulation. Current computational studies show that patchy complementary contacts stabilize experimentally observed P212121 crystal structures for relatively globular protein using spherical protein models. Here we study an anisotropic rigid shape model of green fluorescent protein based on a coarse-grained representation of the atomic coordinates. Using GPU-accelerated molecular dynamics simulations, we find that the experimentally found crystal structure can be stabilized in self-assembly by using complementary attractive patches, confirming the earlier findings. However, we discuss the additional roles of solvent mediated and electrostatic interactions, depletion effects and the self-assembly properties of a purely hard shape model in stabilizing different assemblies. Our findings shed light on fundamental assembly mechanisms in colloidal systems with many competing interactions.

  11. Saturation and stability of nonlinear photonic crystals

    International Nuclear Information System (INIS)

    Franco-Ortiz, M; Corella-Madueño, A; Rosas-Burgos, R A; Adrian Reyes, J; Avendaño, Carlos G

    2017-01-01

    We consider a one-dimensional photonic crystal made by an infinite set of nonlinear nematic films immersed in a linear dielectric medium. The thickness of each equidistant film is negligible and its refraction index depends continuously on the electric field intensity, giving rise to all the involved nonlinear terms, which joints from a starting linear index for negligible amplitudes to a final saturation index for extremely large field intensities. We show that the nonlinear exact solutions of this system form an intensity-dependent band structure which we calculate and analyze. Next, we ponder a finite version of this system; that is, we take a finite array of linear dielectric stacks of the same size separated by the same nonlinear extremely thin nematic slabs and find the reflection coefficients for this arrangement and obtain the dependence on the wave number and intensity of the incident wave. As a final step we analyze the stability of the analytical solutions of the nonlinear crystal by following the evolution of an additive amplitude to the analytical nonlinear solution we have found here. We discuss our results and state our conclusions. (paper)

  12. Electron crystallography of three dimensional protein crystals

    NARCIS (Netherlands)

    Georgieva, Dilyana

    2008-01-01

    This thesis describes an investigation of the potential of electron diffraction for studying three dimensional sub-micro-crystals of proteins and pharmaceuticals. A prerequisite for using electron diffraction for structural studies is the predictable availability of tiny crystals. A method for

  13. Protein crystal growth in low gravity

    Science.gov (United States)

    Feigelson, Robert S.

    1993-01-01

    This Final Technical Report for NASA Grant NAG8-774 covers the period from April 27, 1989 through December 31, 1992. It covers five main topics: fluid flow studies, the influence of growth conditions on the morphology of isocitrate lyase crystals, control of nucleation, the growth of lysozyme by the temperature gradient method and graphoepitaxy of protein crystals. The section on fluid flow discusses the limits of detectability in the Schlieren imaging of fluid flows around protein crystals. The isocitrate lyase study compares crystals grown terrestrially under a variety of conditions with those grown in space. The controlling factor governing the morphology of the crystals is the supersaturation. The lack of flow in the interface between the drop and the atmosphere in microgravity causes protein precipitation in the boundary layer and a lowering of the supersaturation in the drop. This lowered supersaturation leads to improved crystal morphology. Preliminary experiments with lysozyme indicated that localized temperature gradients could be used to nucleate crystals in a controlled manner. An apparatus (thermonucleator) was designed to study the controlled nucleation of protein crystals. This apparatus has been used to nucleate crystals of materials with both normal (ice-water, Rochelle salt and lysozyme) and retrograde (horse serum albumin and alpha chymotrypsinogen A) solubility. These studies have lead to the design of an new apparatus that small and more compatible with use in microgravity. Lysozyme crystals were grown by transporting nutrient from a source (lysozyme powder) to the crystal in a temperature gradient. The influence of path length and cross section on the growth rate was demonstrated. This technique can be combined with the thermonucleator to control both nucleation and growth. Graphoepitaxy utilizes a patterned substrate to orient growing crystals. In this study, silicon substrates with 10 micron grooves were used to grow crystals of catalase

  14. Stability of Disclinations in Nematic Liquid Crystals

    International Nuclear Information System (INIS)

    Wang Yusheng; Yang Guohong; Tian Lijun; Duan Yishi

    2006-01-01

    In the light of φ-mapping method and topological current theory, the stability of disclinations around a spherical particle in nematic liquid crystals is studied. We consider two different defect structures around a spherical particle: disclination ring and point defect at the north or south pole of the particle. We calculate the free energy of these different defects in the elastic theory. It is pointed out that the total Frank free energy density can be divided into two parts. One is the distorted energy density of director field around the disclinations. The other is the free energy density of disclinations themselves, which is shown to be concentrated at the defect and to be topologically quantized in the unit of (k-k 24 )π/2. It is shown that in the presence of saddle-splay elasticity a dipole (radial and hyperbolic hedgehog) configuration that accompanies a particle with strong homeotropic anchoring takes the structure of a small disclination ring, not a point defect.

  15. Protein Crystals as Novel Catalytic Materials.

    Science.gov (United States)

    Margolin, Alexey L.; Navia, Manuel A.

    2001-06-18

    In this era of molecular biology, protein crystallization is often considered to be a necessary first step in obtaining structural information through X-ray diffraction analysis. In a different light, protein crystals can also be thought of as materials, whose chemical and physical properties make them broadly attractive and useful across a larger spectrum of disciplines. The full potential of these protein crystalline materials has been severely restricted in practice, however, both by their inherent fragility, and by strongly held skepticism concerning their routine and predictable growth, formulation, and practical application. Fortunately, these problems have turned out to be solvable. A systematic exploration of the biophysics and biochemistry of protein crystallization has shown that one can dependably create new protein crystalline materials more or less at will. In turn, these crystals can be readily strengthened, both chemically and mechanically, to make them suitable for practical commercialization. Today, these novel materials are used as industrial catalysts on a commercial scale, in bioremediation and "green chemistry" applications, and in enantioselective chromatography of pharmaceuticals and fine chemicals. In the near future, their utility will expand, to include the purification of protein drugs, formulation of direct protein therapeutics, and development of adjuvant-less vaccines.

  16. Liquid nitrogen dewar for protein crystal growth

    Science.gov (United States)

    2001-01-01

    Gaseous Nitrogen Dewar apparatus developed by Dr. Alex McPherson of the University of California, Irvine for use aboard Mir and the International Space Station allows large quantities of protein samples to be crystallized in orbit. The specimens are contained either in plastic tubing (heat-sealed at each end). Biological samples are prepared with a precipitating agent in either a batch or liquid-liquid diffusion configuration. The samples are then flash-frozen in liquid nitrogen before crystallization can start. On orbit, the Dewar is placed in a quiet area of the station and the nitrogen slowly boils off (it is taken up by the environmental control system), allowing the proteins to thaw to begin crystallization. The Dewar is returned to Earth after one to four months on orbit, depending on Shuttle flight opportunities. The tubes then are analyzed for crystal presence and quality

  17. Controlled in meso phase crystallization--a method for the structural investigation of membrane proteins.

    Directory of Open Access Journals (Sweden)

    Jan Kubicek

    Full Text Available We investigated in meso crystallization of membrane proteins to develop a fast screening technology which combines features of the well established classical vapor diffusion experiment with the batch meso phase crystallization, but without premixing of protein and monoolein. It inherits the advantages of both methods, namely (i the stabilization of membrane proteins in the meso phase, (ii the control of hydration level and additive concentration by vapor diffusion. The new technology (iii significantly simplifies in meso crystallization experiments and allows the use of standard liquid handling robots suitable for 96 well formats. CIMP crystallization furthermore allows (iv direct monitoring of phase transformation and crystallization events. Bacteriorhodopsin (BR crystals of high quality and diffraction up to 1.3 Å resolution have been obtained in this approach. CIMP and the developed consumables and protocols have been successfully applied to obtain crystals of sensory rhodopsin II (SRII from Halobacterium salinarum for the first time.

  18. The plug-based nanovolume Microcapillary Protein Crystallization System (MPCS)

    International Nuclear Information System (INIS)

    Gerdts, Cory J.; Elliott, Mark; Lovell, Scott; Mixon, Mark B.; Napuli, Alberto J.; Staker, Bart L.; Nollert, Peter; Stewart, Lance

    2008-01-01

    The Microcapillary Protein Crystallization System (MPCS) is a new protein-crystallization technology used to generate nanolitre-sized crystallization experiments for crystal screening and optimization. Using the MPCS, diffraction-ready crystals were grown in the plastic MPCS CrystalCard and were used to solve the structure of methionine-R-sulfoxide reductase. The Microcapillary Protein Crystallization System (MPCS) embodies a new semi-automated plug-based crystallization technology which enables nanolitre-volume screening of crystallization conditions in a plasticware format that allows crystals to be easily removed for traditional cryoprotection and X-ray diffraction data collection. Protein crystals grown in these plastic devices can be directly subjected to in situ X-ray diffraction studies. The MPCS integrates the formulation of crystallization cocktails with the preparation of the crystallization experiments. Within microfluidic Teflon tubing or the microfluidic circuitry of a plastic CrystalCard, ∼10–20 nl volume droplets are generated, each representing a microbatch-style crystallization experiment with a different chemical composition. The entire protein sample is utilized in crystallization experiments. Sparse-matrix screening and chemical gradient screening can be combined in one comprehensive ‘hybrid’ crystallization trial. The technology lends itself well to optimization by high-granularity gradient screening using optimization reagents such as precipitation agents, ligands or cryoprotectants

  19. Passive Temperature Stabilization of Silicon Photonic Devices Using Liquid Crystals

    Directory of Open Access Journals (Sweden)

    Joanna Ptasinski

    2014-03-01

    Full Text Available In this work we explore the negative thermo-optic properties of liquid crystal claddings for passive temperature stabilization of silicon photonic integrated circuits. Photonic circuits are playing an increasing role in communications and computing, but they suffer from temperature dependent performance variation. Most existing techniques aimed at compensation of thermal effects rely on power hungry Joule heating. We show that integrating a liquid crystal cladding helps to minimize the effects of a temperature dependent drift. The advantage of liquid crystals lies in their high negative thermo-optic coefficients in addition to low absorption at the infrared wavelengths.

  20. Polymer Stabilization of Liquid-Crystal Blue Phase II toward Photonic Crystals.

    Science.gov (United States)

    Jo, Seong-Yong; Jeon, Sung-Wook; Kim, Byeong-Cheon; Bae, Jae-Hyun; Araoka, Fumito; Choi, Suk-Won

    2017-03-15

    The temperature ranges where a pure simple-cubic blue phase (BPII) emerges are quite narrow compared to the body-centered-cubic BP (BPI) such that the polymer stabilization of BPII is much more difficult. Hence, a polymer-stabilized BPII possessing a wide temperature range has been scarcely reported. Here, we fabricate a polymer-stabilized BPII over a temperature range of 50 °C including room temperature. The fabricated polymer-stabilized BPII is confirmed via polarized optical microscopy, Bragg reflection, and Kossel diagram observations. Furthermore, we demonstrate reflective BP liquid-crystal devices utilizing the reflectance-voltage performance as a potential application of the polymer-stabilized BPII. Our work demonstrates the possibility of practical application of the polymer-stabilized BPII to photonic crystals.

  1. Nonclassical nucleation pathways in protein crystallization.

    Science.gov (United States)

    Zhang, Fajun

    2017-11-08

    Classical nucleation theory (CNT), which was established about 90 years ago, has been very successful in many research fields, and continues to be the most commonly used theory in describing the nucleation process. For a fluid-to-solid phase transition, CNT states that the solute molecules in a supersaturated solution reversibly form small clusters. Once the cluster size reaches a critical value, it becomes thermodynamically stable and favored for further growth. One of the most important assumptions of CNT is that the nucleation process is described by one reaction coordinate and all order parameters proceed simultaneously. Recent studies in experiments, computer simulations and theory have revealed nonclassical features in the early stage of nucleation. In particular, the decoupling of order parameters involved during a fluid-to-solid transition leads to the so-called two-step nucleation mechanism, in which a metastable intermediate phase (MIP) exists between the initial supersaturated solution and the final crystals. Depending on the exact free energy landscapes, the MIPs can be a high density liquid phase, mesoscopic clusters, or a pre-ordered state. In this review, we focus on the studies of nonclassical pathways in protein crystallization and discuss the applications of the various scenarios of two-step nucleation theory. In particular, we focus on protein solutions in the presence of multivalent salts, which serve as a model protein system to study the nucleation pathways. We wish to point out the unique features of proteins as model systems for further studies.

  2. Nonclassical nucleation pathways in protein crystallization

    Science.gov (United States)

    Zhang, Fajun

    2017-11-01

    Classical nucleation theory (CNT), which was established about 90 years ago, has been very successful in many research fields, and continues to be the most commonly used theory in describing the nucleation process. For a fluid-to-solid phase transition, CNT states that the solute molecules in a supersaturated solution reversibly form small clusters. Once the cluster size reaches a critical value, it becomes thermodynamically stable and favored for further growth. One of the most important assumptions of CNT is that the nucleation process is described by one reaction coordinate and all order parameters proceed simultaneously. Recent studies in experiments, computer simulations and theory have revealed nonclassical features in the early stage of nucleation. In particular, the decoupling of order parameters involved during a fluid-to-solid transition leads to the so-called two-step nucleation mechanism, in which a metastable intermediate phase (MIP) exists between the initial supersaturated solution and the final crystals. Depending on the exact free energy landscapes, the MIPs can be a high density liquid phase, mesoscopic clusters, or a pre-ordered state. In this review, we focus on the studies of nonclassical pathways in protein crystallization and discuss the applications of the various scenarios of two-step nucleation theory. In particular, we focus on protein solutions in the presence of multivalent salts, which serve as a model protein system to study the nucleation pathways. We wish to point out the unique features of proteins as model systems for further studies.

  3. Do protein crystals nucleate within dense liquid clusters?

    International Nuclear Information System (INIS)

    Maes, Dominique; Vorontsova, Maria A.; Potenza, Marco A. C.; Sanvito, Tiziano; Sleutel, Mike; Giglio, Marzio; Vekilov, Peter G.

    2015-01-01

    The evolution of protein-rich clusters and nucleating crystals were characterized by dynamic light scattering (DLS), confocal depolarized dynamic light scattering (cDDLS) and depolarized oblique illumination dark-field microscopy. Newly nucleated crystals within protein-rich clusters were detected directly. These observations indicate that the protein-rich clusters are locations for crystal nucleation. Protein-dense liquid clusters are regions of high protein concentration that have been observed in solutions of several proteins. The typical cluster size varies from several tens to several hundreds of nanometres and their volume fraction remains below 10 −3 of the solution. According to the two-step mechanism of nucleation, the protein-rich clusters serve as locations for and precursors to the nucleation of protein crystals. While the two-step mechanism explained several unusual features of protein crystal nucleation kinetics, a direct observation of its validity for protein crystals has been lacking. Here, two independent observations of crystal nucleation with the proteins lysozyme and glucose isomerase are discussed. Firstly, the evolutions of the protein-rich clusters and nucleating crystals were characterized simultaneously by dynamic light scattering (DLS) and confocal depolarized dynamic light scattering (cDDLS), respectively. It is demonstrated that protein crystals appear following a significant delay after cluster formation. The cDDLS correlation functions follow a Gaussian decay, indicative of nondiffusive motion. A possible explanation is that the crystals are contained inside large clusters and are driven by the elasticity of the cluster surface. Secondly, depolarized oblique illumination dark-field microscopy reveals the evolution from liquid clusters without crystals to newly nucleated crystals contained in the clusters to grown crystals freely diffusing in the solution. Collectively, the observations indicate that the protein-rich clusters in

  4. Stability and servo-control of the crystal pulling process

    International Nuclear Information System (INIS)

    Johansen, T.H.

    1990-11-01

    The paper analyzes why the crystal pulling process needs servo-control, and how it can be implemented. Special emphasis is put on the fundamental question of inherent stability, and how to interpret the signal from a balance when the weighing method is used for cystal diameter detection. 15 refs., 13 figs

  5. Ultratight crystal packing of a 10 kDa protein

    Energy Technology Data Exchange (ETDEWEB)

    Trillo-Muyo, Sergio [Molecular Biology Institute of Barcelona, Spanish Research Council CSIC, Barcelona Science Park, c/Baldiri Reixac 15-21, 08028 Barcelona (Spain); Jasilionis, Andrius [Vilnius University, M. K. Čiurlionio 21/27, 03101 Vilnius (Lithuania); Domagalski, Marcin J. [University of Virginia, 1340 Jefferson Park Avenue, Charlottesville, VA 22908-0736 (United States); Chruszcz, Maksymilian [University of South Carolina, 631 Sumter Street, Columbia, SC 29208 (United States); Minor, Wladek [University of Virginia, 1340 Jefferson Park Avenue, Charlottesville, VA 22908-0736 (United States); Kuisiene, Nomeda [Vilnius University, M. K. Čiurlionio 21/27, 03101 Vilnius (Lithuania); Arolas, Joan L.; Solà, Maria; Gomis-Rüth, F. Xavier, E-mail: xgrcri@ibmb.csic.es [Molecular Biology Institute of Barcelona, Spanish Research Council CSIC, Barcelona Science Park, c/Baldiri Reixac 15-21, 08028 Barcelona (Spain)

    2013-03-01

    The crystal structure of the C-terminal domain of a putative U32 peptidase from G. thermoleovorans is reported; it is one of the most tightly packed protein structures reported to date. While small organic molecules generally crystallize forming tightly packed lattices with little solvent content, proteins form air-sensitive high-solvent-content crystals. Here, the crystallization and full structure analysis of a novel recombinant 10 kDa protein corresponding to the C-terminal domain of a putative U32 peptidase are reported. The orthorhombic crystal contained only 24.5% solvent and is therefore among the most tightly packed protein lattices ever reported.

  6. Analysis of crystallization data in the Protein Data Bank.

    Science.gov (United States)

    Kirkwood, Jobie; Hargreaves, David; O'Keefe, Simon; Wilson, Julie

    2015-10-01

    The Protein Data Bank (PDB) is the largest available repository of solved protein structures and contains a wealth of information on successful crystallization. Many centres have used their own experimental data to draw conclusions about proteins and the conditions in which they crystallize. Here, data from the PDB were used to reanalyse some of these results. The most successful crystallization reagents were identified, the link between solution pH and the isoelectric point of the protein was investigated and the possibility of predicting whether a protein will crystallize was explored.

  7. The contribution of polystyrene nanospheres towards the crystallization of proteins.

    Directory of Open Access Journals (Sweden)

    Johanna M Kallio

    Full Text Available BACKGROUND: Protein crystallization is a slow process of trial and error and limits the amount of solved protein structures. Search of a universal heterogeneous nucleant is an effort to facilitate crystallizability of proteins. METHODOLOGY: The effect of polystyrene nanospheres on protein crystallization were tested with three commercial proteins: lysozyme, xylanase, xylose isomerase, and with five research target proteins: hydrophobins HFBI and HFBII, laccase, sarcosine dimethylglycine N-methyltransferase (SDMT, and anti-testosterone Fab fragment 5F2. The use of nanospheres both in screening and as an additive for known crystallization conditions was studied. In screening, the addition of an aqueous solution of nanosphere to the crystallization drop had a significant positive effect on crystallization success in comparison to the control screen. As an additive in hydrophobin crystallization, the nanospheres altered the crystal packing, most likely due to the amphiphilic nature of hydrophobins. In the case of laccase, nanospheres could be used as an alternative for streak-seeding, which insofar had remained the only technique to produce high-diffracting crystals. With methyltransferase SDMT the nanospheres, used also as an additive, produced fewer, larger crystals in less time. Nanospheres, combined with the streak-seeding method, produced single 5F2 Fab crystals in shorter equilibration times. CONCLUSIONS: All in all, the use of nanospheres in protein crystallization proved to be beneficial, both when screening new crystallization conditions to promote nucleation and when used as an additive to produce better quality crystals, faster. The polystyrene nanospheres are easy to use, commercially available and close to being inert, as even with amphiphilic proteins only the crystal packing is altered and the nanospheres do not interfere with the structure and function of the protein.

  8. Structuring detergents for extracting and stabilizing functional membrane proteins.

    Directory of Open Access Journals (Sweden)

    Rima Matar-Merheb

    Full Text Available BACKGROUND: Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. METHODOLOGY/PRINCIPAL FINDINGS: Anionic calix[4]arene based detergents (C4Cn, n=1-12 were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein, a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM. They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux much more efficiently than SDS (sodium dodecyl sulphate, FC12 (Foscholine 12 or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. CONCLUSION/SIGNIFICANCE: These compounds seem promising to extract in a functional state

  9. Analysis of crystallization data in the Protein Data Bank

    International Nuclear Information System (INIS)

    Kirkwood, Jobie; Hargreaves, David; O’Keefe, Simon; Wilson, Julie

    2015-01-01

    In a large-scale study using data from the Protein Data Bank, some of the many reported findings regarding the crystallization of proteins were investigated. The Protein Data Bank (PDB) is the largest available repository of solved protein structures and contains a wealth of information on successful crystallization. Many centres have used their own experimental data to draw conclusions about proteins and the conditions in which they crystallize. Here, data from the PDB were used to reanalyse some of these results. The most successful crystallization reagents were identified, the link between solution pH and the isoelectric point of the protein was investigated and the possibility of predicting whether a protein will crystallize was explored

  10. Analysis of crystallization data in the Protein Data Bank

    Energy Technology Data Exchange (ETDEWEB)

    Kirkwood, Jobie [University of York, York YO10 5DD (United Kingdom); Hargreaves, David [AstraZeneca, Darwin Building, Cambridge Science Park, Cambridge CB4 0WG (United Kingdom); O’Keefe, Simon [University of York, York YO10 5DD (United Kingdom); Wilson, Julie, E-mail: julie.wilson@york.ac.uk [University of York, York YO10 5DD (United Kingdom); University of York, York YO10 5DD (United Kingdom)

    2015-09-23

    In a large-scale study using data from the Protein Data Bank, some of the many reported findings regarding the crystallization of proteins were investigated. The Protein Data Bank (PDB) is the largest available repository of solved protein structures and contains a wealth of information on successful crystallization. Many centres have used their own experimental data to draw conclusions about proteins and the conditions in which they crystallize. Here, data from the PDB were used to reanalyse some of these results. The most successful crystallization reagents were identified, the link between solution pH and the isoelectric point of the protein was investigated and the possibility of predicting whether a protein will crystallize was explored.

  11. Protein nanocrystallography: growth mechanism and atomic structure of crystals induced by nanotemplates.

    Science.gov (United States)

    Pechkova, E; Vasile, F; Spera, R; Fiordoro, S; Nicolini, C

    2005-11-01

    Protein nanocrystallography, a new technology for crystal growth based on protein nanotemplates, has recently been shown to produce diffracting, stable and radiation-resistant lysozyme crystals. This article, by computing these lysozyme crystals' atomic structures, obtained by the diffraction patterns of microfocused synchrotron radiation, provides a possible mechanism for this increased stability, namely a significant decrease in water content accompanied by a minor but significant alpha-helix increase. These data are shown to be compatible with the circular dichroism and two-dimensional Fourier transform spectra of high-resolution H NMR of proteins dissolved from the same nanotemplate-based crystal versus those from a classical crystal. Finally, evidence for protein direct transfer from the nanotemplate to the drop and the participation of the template proteins in crystal nucleation and growth is provided by high-resolution NMR spectrometry and mass spectrometry. Furthermore, the lysozyme nanotemplate appears stable up to 523 K, as confirmed by a thermal denaturation study using spectropolarimetry. The overall data suggest that heat-proof lysozyme presence in the crystal provides a possible explanation of the crystal's resistance to synchrotron radiation.

  12. Isomeric Detergent Comparison for Membrane Protein Stability

    DEFF Research Database (Denmark)

    Cho, Kyung Ho; Hariharan, Parameswaran; Mortensen, Jonas S.

    2016-01-01

    and utility, particularly for eukaryotic membrane proteins and membrane protein complexes. Thus, a number of new agents have been devised; some have made significant contributions to membrane protein structural studies. However, few detergent design principles are available. In this study, we prepared meta...... and ortho isomers of the previously reported para-substituted xylene-linked maltoside amphiphiles (XMAs), along with alkyl chain-length variation. The isomeric XMAs were assessed with three membrane proteins, and the meta isomer with a C12 alkyl chain was most effective at maintaining solubility....../stability of the membrane proteins. We propose that interplay between the hydrophile–lipophile balance (HLB) and alkyl chain length is of central importance for high detergent efficacy. In addition, differences in inter-alkyl-chain distance between the isomers influence the ability of the detergents to stabilise membrane...

  13. Automating the application of smart materials for protein crystallization

    International Nuclear Information System (INIS)

    Khurshid, Sahir; Govada, Lata; EL-Sharif, Hazim F.; Reddy, Subrayal M.; Chayen, Naomi E.

    2015-01-01

    The first semi-liquid, non-protein nucleating agent for automated protein crystallization trials is described. This ‘smart material’ is demonstrated to induce crystal growth and will provide a simple, cost-effective tool for scientists in academia and industry. The fabrication and validation of the first semi-liquid nonprotein nucleating agent to be administered automatically to crystallization trials is reported. This research builds upon prior demonstration of the suitability of molecularly imprinted polymers (MIPs; known as ‘smart materials’) for inducing protein crystal growth. Modified MIPs of altered texture suitable for high-throughput trials are demonstrated to improve crystal quality and to increase the probability of success when screening for suitable crystallization conditions. The application of these materials is simple, time-efficient and will provide a potent tool for structural biologists embarking on crystallization trials

  14. Stabilization of liquid crystal dispersions with acrylamide copolymers

    Energy Technology Data Exchange (ETDEWEB)

    Park, S.J.; Kim, M.H.; Lee, J.R. [Korea Research Institute of Chemical Technology, Taejon (Korea, Republic of)

    1999-03-01

    The effects of hydrophobic moieties(styrene and methyl methacrylate) on the stability of a liquid crystal(LC, E-7)-in-water dispersion stabilized by copolymers of hydrophilic acrylamide with hydrophobic monomers have been studied in terms of nematic curvilinear aligned phase(NCAP) system. It was observed that the preferential adsorption hydrophobic moieties onto LC droplet surface resulted in steric stabilization of the dispersion, due to increasing the interfacial tension of LC and reducing the LC droplet size. According to the interfacial tension, coalescence time, and sedimented layer thickness measurements, it was proposed that the presence of hydrophobic moieties allows to form the apolar microenvironment in the round of LC droplet and finally reduces the anchoring effect between LC and the polymeric wall. 16 refs., 10 figs.

  15. Protein crystals as scanned probes for recognition atomic force microscopy.

    Science.gov (United States)

    Wickremasinghe, Nissanka S; Hafner, Jason H

    2005-12-01

    Lysozyme crystal growth has been localized at the tip of a conventional silicon nitride cantilever through seeded nucleation. After cross-linking with glutaraldehyde, lysozyme protein crystal tips image gold nanoparticles and grating standards with a resolution comparable to that of conventional tips. Force spectra between the lysozyme crystal tips and surfaces covered with antilysozyme reveal an adhesion force that drops significantly upon blocking with free lysozyme, thus confirming that lysozyme crystal tips can detect molecular recognition interactions.

  16. Crystal structure of the Japanese encephalitis virus envelope protein.

    Science.gov (United States)

    Luca, Vincent C; AbiMansour, Jad; Nelson, Christopher A; Fremont, Daved H

    2012-02-01

    Japanese encephalitis virus (JEV) is the leading global cause of viral encephalitis. The JEV envelope protein (E) facilitates cellular attachment and membrane fusion and is the primary target of neutralizing antibodies. We have determined the 2.1-Å resolution crystal structure of the JEV E ectodomain refolded from bacterial inclusion bodies. The E protein possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies onto the structure reveals determinants that correspond to the domain I lateral ridge, fusion loop, domain III lateral ridge, and domain I-II hinge. While monomeric in solution, JEV E assembles as an antiparallel dimer in the crystal lattice organized in a highly similar fashion as seen in cryo-electron microscopy models of mature flavivirus virions. The dimer interface, however, is remarkably small and lacks many of the domain II contacts observed in other flavivirus E homodimers. In addition, uniquely conserved histidines within the JEV serocomplex suggest that pH-mediated structural transitions may be aided by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.

  17. Simulation of diffusion time of small molecules in protein crystals.

    Science.gov (United States)

    Geremia, Silvano; Campagnolo, Mara; Demitri, Nicola; Johnson, Louise N

    2006-03-01

    A simple model for evaluation of diffusion times of small molecule into protein crystals has been developed, which takes into account the physical and chemical properties both of protein crystal and the diffusing molecules. The model also includes consideration of binding and the binding affinity of a ligand to the protein. The model has been validated by simulation of experimental set-ups of several examples found in the literature. These experiments cover a wide range of situations: from small to relatively large diffusing molecules, crystals having low, medium, or high protein density, and different size. The reproduced experiments include ligand exchange in protein crystals by soaking techniques. Despite the simplifying assumptions of the model, theoretical and experimental data are in agreement with available data, with experimental diffusion times ranging from a few seconds to several hours. The method has been used successfully for planning intermediate cryotrapping experiments in maltodextrin phosphorylase crystals.

  18. Lab-on-a-Chip Based Protein Crystallization

    Science.gov (United States)

    vanderWoerd, Mark J.; Brasseur, Michael M.; Spearing, Scott F.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    We are developing a novel technique with which we will grow protein crystals in very small volumes, utilizing chip-based, microfluidic ("LabChip") technology. This development, which is a collaborative effort between NASA's Marshall Space Flight Center and Caliper Technologies Corporation, promises a breakthrough in the field of protein crystal growth. Our initial results obtained from two model proteins, Lysozyme and Thaumatin, show that it is feasible to dispense and adequately mix protein and precipitant solutions on a nano-liter scale. The mixtures have shown crystal growth in volumes in the range of 10 nanoliters to 5 microliters. In addition, large diffraction quality crystals were obtained by this method. X-ray data from these crystals were shown to be of excellent quality. Our future efforts will include the further development of protein crystal growth with LabChip(trademark) technology for more complex systems. We will initially address the batch growth method, followed by the vapor diffusion method and the liquid-liquid diffusion method. The culmination of these chip developments is to lead to an on orbit protein crystallization facility on the International Space Station. Structural biologists will be invited to utilize the on orbit Iterative Biological Crystallization facility to grow high quality macromolecular crystals in microgravity.

  19. Protein crystal structure analysis using synchrotron radiation at atomic resolution

    International Nuclear Information System (INIS)

    Nonaka, Takamasa

    1999-01-01

    We can now obtain a detailed picture of protein, allowing the identification of individual atoms, by interpreting the diffraction of X-rays from a protein crystal at atomic resolution, 1.2 A or better. As of this writing, about 45 unique protein structures beyond 1.2 A resolution have been deposited in the Protein Data Bank. This review provides a simplified overview of how protein crystallographers use such diffraction data to solve, refine, and validate protein structures. (author)

  20. A Proposed Model for Protein Crystal Nucleation and Growth

    Science.gov (United States)

    Pusey, Marc; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    How does one take a molecule, strongly asymmetric in both shape and charge distribution, and assemble it into a crystal? We propose a model for the nucleation and crystal growth process for tetragonal lysozyme, based upon fluorescence, light, neutron, and X-ray scattering data, size exclusion chromatography experiments, dialysis kinetics, AFM, and modeling of growth rate data, from this and other laboratories. The first species formed is postulated to be a 'head to side' dimer. Through repeating associations involving the same intermolecular interactions this grows to a 4(sub 3) helix structure, that in turn serves as the basic unit for nucleation and subsequent crystal growth. High salt attenuates surface charges while promoting hydrophobic interactions. Symmetry facilitates subsequent helix-helix self-association. Assembly stability is enhanced when a four helix structure is obtained, with each bound to two neighbors. Only two unique interactions are required. The first are those for helix formation, where the dominant interaction is the intermolecular bridging anion. The second is the anti-parallel side-by-side helix-helix interaction, guided by alternating pairs of symmetry related salt bridges along each side. At this stage all eight unique positions of the P4(sub3)2(sub 1),2(sub 1) unit cell are filled. The process is one of a) attenuating the most strongly interacting groups, such that b) the molecules begin to self-associate in defined patterns, so that c) symmetry is obtained, which d) propagates as a growing crystal. Simple and conceptually obvious in hindsight, this tells much about what we are empirically doing when we crystallize macromolecules. By adjusting the growth parameters we are empirically balancing the intermolecular interactions, preferentially attenuating the dominant strong (for lysozyme the charged groups) while strengthening the lesser strong (hydrophobic) interactions. In the general case for proteins the lack of a singularly defined

  1. Automating the application of smart materials for protein crystallization.

    Science.gov (United States)

    Khurshid, Sahir; Govada, Lata; El-Sharif, Hazim F; Reddy, Subrayal M; Chayen, Naomi E

    2015-03-01

    The fabrication and validation of the first semi-liquid nonprotein nucleating agent to be administered automatically to crystallization trials is reported. This research builds upon prior demonstration of the suitability of molecularly imprinted polymers (MIPs; known as `smart materials') for inducing protein crystal growth. Modified MIPs of altered texture suitable for high-throughput trials are demonstrated to improve crystal quality and to increase the probability of success when screening for suitable crystallization conditions. The application of these materials is simple, time-efficient and will provide a potent tool for structural biologists embarking on crystallization trials.

  2. Crystallization and initial X-ray analysis of polyhydroxyalkanoate granule-associated protein from Aeromonas hydrophila

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Minglian; Li, Zhenguo; Zheng, Wei; Lou, Zhiyong [MOE Key Laboratory of Protein Science, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084 (China); Chen, Guo-Qiang, E-mail: chengq@stu.edu.cn [MOE Key Laboratory of Protein Science, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084 (China); Multidisciplinary Research Center, Shantou University, Shantou 515063, Guangdong (China)

    2006-08-01

    The phasin PhaP{sub Ah} from A. hydrophila strain 4AK4 was crystallized using the hanging-drop vapour-diffusion method. Polyhydroxyalkanoate (PHA) granule-associated proteins (phasins) were discovered in PHA-accumulating bacteria. They play a crucial role as a structural protein during initial PHA-granule formation and granule growth and also serve as interfaces for granule stabilization in vivo. The phasin PhaP{sub Ah} from Aeromonas hydrophila strain 4AK4 was crystallized using the hanging-drop vapour-diffusion method. Single crystals were cryocooled for X-ray diffraction analysis. The phasin crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 80.8, b = 108.9, c = 134.4 Å.

  3. The crystal structure and stability of molybdenum at ultrahigh pressures

    International Nuclear Information System (INIS)

    Jona, F; Marcus, P M

    2005-01-01

    Crystal structures and their stabilities for molybdenum under increasing hydrostatic pressures are investigated by first-principles calculations of the Gibbs free energy. Three structures are considered: body-centred cubic (bcc, the ground state at zero pressure), hexagonal close-packed (hcp) and face-centred cubic (fcc). For each structure and each pressure (up to 8 Mbar) the equilibrium states are found from minima of the Gibbs free energy at zero temperature. The stability is tested by calculating the elastic constants and checking whether they satisfy the appropriate stability conditions. The bcc structure is confirmed to be stable at zero pressure and at 6 Mbar. At and above 6.2 M-bar the ground-state structure changes to hcp, which is found to be stable at 7 M-bar. At 7.7 Mbar another transition occurs, and the ground-state structure changes from hcp to fcc. The fcc structure, which is unstable at zero pressure, becomes metastable over the range from 3 to 7.7 M-bar and becomes the ground state at higher pressures (at least up to 8 Mbar). Direct confirmation of these calculated transition pressures with experiment is not now possible, as the maximum static pressure currently reached experimentally is 5.6 Mbar, where Mo is found to be still in the bcc phase

  4. Cloning and characterization of an insecticidal crystal protein gene ...

    Indian Academy of Sciences (India)

    Unknown

    The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene from Bacillus .... diet or by topical application on food substrates as .... has very high similarity (99.74%) at DNA level with.

  5. The biochemistry of the protein crystal toxin of Bacillus thuringiensis

    Science.gov (United States)

    Paul G. Fast

    1985-01-01

    The crystal consists of dimeric protein subunits. The monomer peptide chains are held together in the subunit and the subunit in the crystal by disulfide and non-covalent bonds. The monomer peptide has a molecular weight of about 130 kdaltons which, in the presence of proteases, is hydrolyzed to a protease-resistant-protein of 65 kda that is toxic both to larvae by...

  6. Investigating the nucleation of protein crystals with hydrostatic pressure

    Energy Technology Data Exchange (ETDEWEB)

    Kadri, A [Departement ' Mecanismes et Macromolecules de la Synthese Proteique et Cristallogenese' UPR 9002, Institut de Biologie Moleculaire et Cellulaire du CNRS, 15 rue Rene Descartes, F-67084 Strasbourg Cedex (France); Damak, M [Laboratoire de Chimie des Substances Naturelles, Faculte des Sciences de Sfax, BP 802, 3018 Sfax (Tunisia); Jenner, G [Laboratoire de Piezochimie Organique, UMR 7123, Faculte de Chimie, Universite Louis Pasteur, 1 rue Blaise Pascal, F-67008 Strasbourg Cedex (France); Lorber, B [Departement ' Mecanismes et Macromolecules de la Synthese Proteique et Cristallogenese' UPR 9002, Institut de Biologie Moleculaire et Cellulaire du CNRS, 15 rue Rene Descartes, F-67084 Strasbourg Cedex (France); Giege, R [Departement ' Mecanismes et Macromolecules de la Synthese Proteique et Cristallogenese' UPR 9002, Institut de Biologie Moleculaire et Cellulaire du CNRS, 15 rue Rene Descartes, F-67084 Strasbourg Cedex (France)

    2003-12-17

    Hydrostatic pressure in the 0.1-75 MPa range has been used as a non-invasive tool to study the crystallization process of the tetragonal crystal form of the protein thaumatin (M{sub r} 22 200). Crystals were prepared within agarose gel and at temperatures in the range from 283 to 303 K. The solubility, i.e. the concentration of soluble macromolecules remaining in equilibrium with the crystals, decreases when the pressure increases and when the temperature decreases. High pressure was used to probe the nucleation behaviour of thaumatin. The pressure dependence of the nucleation rate leads to an activation volume of -46.5cm{sup 3} mol{sup -1}. It is shown that an increase in pressure decreases the enthalpy, the entropy and the free energy of crystallization of thaumatin. The data are discussed in the light of the results of crystallographic analyses and of the structure of the protein.

  7. Investigating the nucleation of protein crystals with hydrostatic pressure

    International Nuclear Information System (INIS)

    Kadri, A; Damak, M; Jenner, G; Lorber, B; Giege, R

    2003-01-01

    Hydrostatic pressure in the 0.1-75 MPa range has been used as a non-invasive tool to study the crystallization process of the tetragonal crystal form of the protein thaumatin (M r 22 200). Crystals were prepared within agarose gel and at temperatures in the range from 283 to 303 K. The solubility, i.e. the concentration of soluble macromolecules remaining in equilibrium with the crystals, decreases when the pressure increases and when the temperature decreases. High pressure was used to probe the nucleation behaviour of thaumatin. The pressure dependence of the nucleation rate leads to an activation volume of -46.5cm 3 mol -1 . It is shown that an increase in pressure decreases the enthalpy, the entropy and the free energy of crystallization of thaumatin. The data are discussed in the light of the results of crystallographic analyses and of the structure of the protein

  8. Two-photon excited UV fluorescence for protein crystal detection

    International Nuclear Information System (INIS)

    Madden, Jeremy T.; DeWalt, Emma L.; Simpson, Garth J.

    2011-01-01

    Complementary measurements using SONICC and TPE-UVF allow the sensitive and selective detection of protein crystals. Two-photon excited ultraviolet fluorescence (TPE-UVF) microscopy is explored for sensitive protein-crystal detection as a complement to second-order nonlinear optical imaging of chiral crystals (SONICC). Like conventional ultraviolet fluorescence (UVF), TPE-UVF generates image contrast based on the intrinsic fluorescence of aromatic residues, generally producing higher fluorescence emission within crystals than the mother liquor by nature of the higher local protein concentration. However, TPE-UVF has several advantages over conventional UVF, including (i) insensitivity to optical scattering, allowing imaging in turbid matrices, (ii) direct compatibility with conventional optical plates and windows by using visible light for excitation, (iii) elimination of potentially damaging out-of-plane UV excitation, (iv) improved signal to noise through background reduction from out-of-plane excitation and (v) relatively simple integration into instrumentation developed for SONICC

  9. Conservation of Oxidative Protein Stabilization in an Insect Homologue of Parkinsonism-Associated Protein DJ-1

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Jiusheng; Prahlad, Janani; Wilson, Mark A. (UNL)

    2012-08-21

    DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well-conserved in other DJ-1 homologues, including two (DJ-1{alpha} and DJ-1{beta}) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1{beta}. The structure of D. melanogaster DJ-1{beta} is similar to that of human DJ-1, although two important residues in the human protein, Met26 and His126, are not conserved in DJ-1{beta}. His126 in human DJ-1 is substituted with a tyrosine in DJ-1{beta}, and this residue is not able to compose a putative catalytic dyad with Cys106 that was proposed to be important in the human protein. The reactive cysteine in DJ-1 is oxidized readily to the cysteine-sulfinic acid in both flies and humans, and this may regulate the cytoprotective function of the protein. We show that the oxidation of this conserved cysteine residue to its sulfinate form (Cys-SO{sub 2{sup -}}) results in considerable thermal stabilization of both Drosophila DJ-1{beta} and human DJ-1. Therefore, protein stabilization is one potential mechanism by which cysteine oxidation may regulate DJ-1 function in vivo. More generally, most close DJ-1 homologues are likely stabilized by cysteine-sulfinic acid formation but destabilized by further oxidation, suggesting that they are biphasically regulated by oxidative modification.

  10. Protein crystal growth studies at the Center for Macromolecular Crystallography

    International Nuclear Information System (INIS)

    DeLucas, Lawrence J.; Long, Marianna M.; Moore, Karen M.; Harrington, Michael; McDonald, William T.; Smith, Craig D.; Bray, Terry; Lewis, Johanna; Crysel, William B.; Weise, Lance D.

    2000-01-01

    The Center for Macromolecular Crystallography (CMC) has been involved in fundamental studies of protein crystal growth (PCG) in microgravity and in our earth-based laboratories. A large group of co-investigators from academia and industry participated in these experiments by providing protein samples and by performing the x-ray crystallographic analysis. These studies have clearly demonstrated the usefulness of a microgravity environment for enhancing the quality and size of protein crystals. Review of the vapor diffusion (VDA) PCG results from nineteen space shuttle missions is given in this paper

  11. Blue Shifting Tuning of the Selective Reflection of Polymer Stabilized Cholesteric Liquid Crystals (Postprint)

    Science.gov (United States)

    2017-08-08

    crystal (MLC-2079, Merck). The polymer stabi- lizing network was formed within the samples by photoinitiated polymerization with 50–700 mW cm2 of 365...AFRL-RX-WP-JA-2017-0347 BLUE-SHIFTING TUNING OF THE SELECTIVE REFLECTION OF POLYMER STABILIZED CHOLESTERIC LIQUID CRYSTALS (POSTPRINT...BLUE-SHIFTING TUNING OF THE SELECTIVE REFLECTION OF POLYMER STABILIZED CHOLESTERIC LIQUID CRYSTALS (POSTPRINT) 5a. CONTRACT NUMBER FA8650-16-F

  12. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    Energy Technology Data Exchange (ETDEWEB)

    Assenberg, René [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Delmas, Olivier [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J. [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Bourhy, Hervé [UPRE Lyssavirus Dynamics and Host Adaptation, WHO Collaborating Centre for Reference and Research on Rabies, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris CEDEX 15 (France); Grimes, Jonathan M., E-mail: jonathan@strubi.ox.ac.uk [Division of Structural Biology and Oxford Protein Production Facility, The Henry Wellcome Building for Genomic Medicine, Oxford University, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

    2008-04-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

  13. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    International Nuclear Information System (INIS)

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-01-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6 1 22 or P6 5 22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress

  14. Liquid Crystal Formation from Sunflower Oil: Long Term Stability Studies.

    Science.gov (United States)

    da Rocha-Filho, Pedro Alves; Maruno, Mônica; Ferrari, Márcio; Topan, José Fernando

    2016-06-09

    The Brazilian biodiversity offers a multiplicity of raw materials with great potential in cosmetics industry applications. Some vegetable oils and fatty esters increase skin hydration by occlusivity, keeping the skin hydrated and with a shiny appearance. Sunflower (Helianthus annus L.) oil is widely employed in cosmetic emulsions in the form of soaps, creams, moisturizers and skin cleansers due to the presence of polyphenols and its high vitamin E content. Liquid crystals are systems with many applications in both pharmaceutical and cosmetic formulations and are easily detected by microscopy under polarized light due to their birefringence properties. The aim of this research was to develop emulsions from natural sunflower oil for topical uses. Sunflower oil (75.0% w/w) was combined with liquid vaseline (25.0% w/w) employing a natural self-emulsifying base (SEB) derivative. The high temperature of the emulsification process did not influence the antioxidant properties of sunflower oil. Fatty esters were added to cosmetic formulations and extended stability tests were performed to characterize the emulsions. Fatty esters like cetyl palmitate and cetyl ester increase the formation of anisotropic structures. O/W emulsions showed acidic pH values and pseudoplastic behavior. The presence of a lamellar phase was observed after a period of 90 days under different storage conditions.

  15. Split green fluorescent protein as a modular binding partner for protein crystallization

    International Nuclear Information System (INIS)

    Nguyen, Hau B.; Hung, Li-Wei; Yeates, Todd O.; Terwilliger, Thomas C.; Waldo, Geoffrey S.

    2013-01-01

    A strategy using a new split green fluorescent protein (GFP) as a modular binding partner to form stable protein complexes with a target protein is presented. The modular split GFP may open the way to rapidly creating crystallization variants. A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP β-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10–11) hairpin in complex with GFP(1–9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10–11) hairpin with a variety of GFP(1–9) mutants engineered for favorable crystallization

  16. Methodology for fast evaluation of Bacillus thuringiensis crystal protein content

    Directory of Open Access Journals (Sweden)

    Alves Lúcia M. Carareto

    2000-01-01

    Full Text Available The development of the production and use of Bacillus thuringiensis in Brazil at a commercial scale faces certain difficulties, among them the establishment of efficient methodologies for the quantitation of toxic products to be commercialized. Presently, the amount of toxin is given in percentage by analyzing the samples total protein content. Such methodology however, does not measure the actual amount of active protein present in the product, since most strains express different endotoxin genes and might even produce b-toxin. Since the various types of toxins exhibit different antigenic characteristics, this work has as objective the utilization of fast immunological techniques to quantify the level of crystal protein. Crystal protein produced by a subspecies of Bacillus thuringiensis var. israelensis was purified by ultracentrifugation and utilized to immunize rabbits and to produce hiperimmune sera. Such sera were latter used to evaluate the level of proteins on commercial bioinsecticide and on laboratory cultures of B. thuringiensis through the immunodot technique. The results were obtained by comparison of data obtained from reactions with known concentrations of crystal protein permitting to evaluate the level of such protein on various materials.

  17. Crystallization of bi-functional ligand protein complexes.

    Science.gov (United States)

    Antoni, Claudia; Vera, Laura; Devel, Laurent; Catalani, Maria Pia; Czarny, Bertrand; Cassar-Lajeunesse, Evelyn; Nuti, Elisa; Rossello, Armando; Dive, Vincent; Stura, Enrico Adriano

    2013-06-01

    Homodimerization is important in signal transduction and can play a crucial role in many other biological systems. To obtaining structural information for the design of molecules able to control the signalization pathways, the proteins involved will have to be crystallized in complex with ligands that induce dimerization. Bi-functional drugs have been generated by linking two ligands together chemically and the relative crystallizability of complexes with mono-functional and bi-functional ligands has been evaluated. There are problems associated with crystallization with such ligands, but overall, the advantages appear to be greater than the drawbacks. The study involves two matrix metalloproteinases, MMP-12 and MMP-9. Using flexible and rigid linkers we show that it is possible to control the crystal packing and that by changing the ligand-enzyme stoichiometric ratio, one can toggle between having one bi-functional ligand binding to two enzymes and having the same ligand bound to each enzyme. The nature of linker and its point of attachment on the ligand can be varied to aid crystallization, and such variations can also provide valuable structural information about the interactions made by the linker with the protein. We report here the crystallization and structure determination of seven ligand-dimerized complexes. These results suggest that the use of bi-functional drugs can be extended beyond the realm of protein dimerization to include all drug design projects. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. The 2DX robot: a membrane protein 2D crystallization Swiss Army knife.

    Science.gov (United States)

    Iacovache, Ioan; Biasini, Marco; Kowal, Julia; Kukulski, Wanda; Chami, Mohamed; van der Goot, F Gisou; Engel, Andreas; Rémigy, Hervé-W

    2010-03-01

    Among the state-of-the-art techniques that provide experimental information at atomic scale for membrane proteins, electron crystallography, atomic force microscopy and solid state NMR make use of two-dimensional crystals. We present a cyclodextrin-driven method for detergent removal implemented in a fully automated robot. The kinetics of the reconstitution processes is precisely controlled, because the detergent complexation by cyclodextrin is of stoichiometric nature. The method requires smaller volumes and lower protein concentrations than established 2D crystallization methods, making it possible to explore more conditions with the same amount of protein. The method yielded highly ordered 2D crystals diffracting to high resolution from the pore-forming toxin Aeromonas hydrophila aerolysin (2.9A), the plant aquaporin SoPIP2;1 (3.1A) and the human aquaporin-8 (hAQP8; 3.3A). This new method outperforms traditional 2D crystallization approaches in terms of accuracy, flexibility, throughput, and allows the usage of detergents having low critical micelle concentration (CMC), which stabilize the structure of membrane proteins in solution. (c) 2009 Elsevier Inc. All rights reserved.

  19. A chemical library to screen protein and protein-ligand crystallization using a versatile microfluidic platform

    OpenAIRE

    Gerard , Charline ,; Ferry , Gilles; Vuillard , Laurent ,; Boutin , Jean ,; Ferte , Nathalie ,; Grossier , Romain ,; Candoni , Nadine ,; Veesler , Stéphane ,

    2018-01-01

    Here, we describe a plug-and-play microfluidic platform, suitable for protein crystallization. The droplet factory is designed to generate hundreds of droplets as small as a few nanoliters (2 to 10nL) for screening and optimization of crystallization conditions. Commercially-available microfluidic junctions and tubing are combined to create the appropriate geometry. In addition, a " chemical library " is produced in tubing. The microfluidic geometry for a " crystallization agent-based chemica...

  20. Two types of amorphous protein particles facilitate crystal nucleation.

    Science.gov (United States)

    Yamazaki, Tomoya; Kimura, Yuki; Vekilov, Peter G; Furukawa, Erika; Shirai, Manabu; Matsumoto, Hiroaki; Van Driessche, Alexander E S; Tsukamoto, Katsuo

    2017-02-28

    Nucleation, the primary step in crystallization, dictates the number of crystals, the distribution of their sizes, the polymorph selection, and other crucial properties of the crystal population. We used time-resolved liquid-cell transmission electron microscopy (TEM) to perform an in situ examination of the nucleation of lysozyme crystals. Our TEM images revealed that mesoscopic clusters, which are similar to those previously assumed to consist of a dense liquid and serve as nucleation precursors, are actually amorphous solid particles (ASPs) and act only as heterogeneous nucleation sites. Crystalline phases never form inside them. We demonstrate that a crystal appears within a noncrystalline particle assembling lysozyme on an ASP or a container wall, highlighting the role of heterogeneous nucleation. These findings represent a significant departure from the existing formulation of the two-step nucleation mechanism while reaffirming the role of noncrystalline particles. The insights gained may have significant implications in areas that rely on the production of protein crystals, such as structural biology, pharmacy, and biophysics, and for the fundamental understanding of crystallization mechanisms.

  1. X-ray transparent Microfluidics for Protein Crystallization and Biomineralization

    Science.gov (United States)

    Opathalage, Achini

    Protein crystallization demands the fundamental understanding of nucleation and applying techniques to find the optimal conditions to achieve the kinetic pathway for a large and defect free crystal. Classical nucleation theory predicts that the nucleation occurs at high supersaturation conditions. In this dissertation we sought out to develop techniques to attain optimal supersaturation profile to a large defect free crystal and subject it to in-situ X-ray diffraction using microfluidics. We have developed an emulsion-based serial crystallographic technology in nanolitre-sized droplets of protein solution encapsulated in to nucleate one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different un-oriented crystals. As proof of concept, the structure of Glucose Isomerase was solved to 2.1 A. We have developed a suite of X-ray semi-transparent micrfluidic devices which enables; controlled evaporation as a method of increasing supersaturation and manipulating the phase space of proteins and small molecules. We exploited the inherently high water permeability of the thin X-ray semi-transparent devices as a mean of increasing the supersaturation by controlling the evaporation. We fabricated the X-ray semi-transparent version of the PhaseChip with a thin PDMS membrane by which the storage and the reservoir layers are separated, and studies the phase transition of amorphous CaCO3.

  2. Biaxial potential of surface-stabilized ferroelectric liquid crystals

    Science.gov (United States)

    Kaznacheev, Anatoly; Pozhidaev, Evgeny; Rudyak, Vladimir; Emelyanenko, Alexander V.; Khokhlov, Alexei

    2018-04-01

    A biaxial surface potential Φs of smectic-C* surface-stabilized ferroelectric liquid crystals (SSFLCs) is introduced in this paper to explain the experimentally observed electric-field dependence of polarization P˜cell(E ) , in particular the shape of the static hysteresis loops. Our potential consists of three independent parts. The first nonpolar part Φn describes the deviation of the prime director n (which is the most probable orientation of the long molecular axes) from the easy alignment axis R , which is located in the boundary surface plane. It is introduced in the same manner as the uniaxial Rapini potential. The second part Φp of the potential is a polar term associated with the presence of the polar axis in a FLC. The third part Φm relates to the inherent FLC biaxiality, which has not been taken into consideration previously. The Φm part takes into account the deviations of the secondary director m (which is the most probable orientation of the short molecular axes) from the normal to the boundary surface. The overall surface potential Φs, which is a sum of Φn,Φp , and Φm, allows one to model the conditions when either one, two, or three minima of the SSFLC cell free energy are realized depending on the biaxiality extent. A monodomain or polydomain structure, as well as the bistability or monostability of SSFLC cells, depends on the number of free-energy minima, as confirmed experimentally. In this paper, we analyze the biaxiality impact on the FLC alignment. We also answer the question of whether the bistable or monostable structure can be formed in an SSFLC cell. Our approach is essentially based on a consideration of the biaxial surface potential, while the uniaxial surface potential cannot adequately describe the experimental observations in the FLC.

  3. Protein sensing by nanofluidic crystal and its signal enhancement

    Science.gov (United States)

    Sang, Jianming; Du, Hongtan; Wang, Wei; Chu, Ming; Wang, Yuedan; Li, Haichao; Alice Zhang, Haixia; Wu, Wengang; Li, Zhihong

    2013-01-01

    Nanofluidics has a unique property that ionic conductance across a nanometer-sized confined space is strongly affected by the space surface charge density, which can be utilized to construct electrical read-out biosensor. Based on this principle, this work demonstrated a novel protein sensor along with a sandwich signal enhancement approach. Nanoparticles with designed aptamer onside are assembled in a suspended micropore to form a 3-dimensional network of nanometer-sized interstices, named as nanofluidic crystal hereafter, as the basic sensing unit. Proteins captured by aptamers will change the surface charge density of nanoparticles and thereby can be detected by monitoring the ionic conductance across this nanofluidic crystal. Another aptamer can further enlarge the variations of the surface charge density by forming a sandwich structure (capturing aptamer/protein/signal enhancement aptamer) and the read-out conductance as well. The preliminary experimental results indicated that human α-thrombin was successfully detected by the corresponding aptamer modified nanofluidic crystal with the limit of detection of 5 nM (0.18 μg/ml) and the read-out signal was enhanced up to 3 folds by using another thrombin aptamer. Being easy to graft probe, facile and low-cost to prepare the nano-device, and having an electrical read-out, the present nanofluidic crystal scheme is a promising and universal strategy for protein sensing. PMID:24404017

  4. Heterogeneous nucleation of protein crystals on fluorinated layered silicate.

    Directory of Open Access Journals (Sweden)

    Keita Ino

    Full Text Available Here, we describe an improved system for protein crystallization based on heterogeneous nucleation using fluorinated layered silicate. In addition, we also investigated the mechanism of nucleation on the silicate surface. Crystallization of lysozyme using silicates with different chemical compositions indicated that fluorosilicates promoted nucleation whereas the silicates without fluorine did not. The use of synthesized saponites for lysozyme crystallization confirmed that the substitution of hydroxyl groups contained in the lamellae structure for fluorine atoms is responsible for the nucleation-inducing property of the nucleant. Crystallization of twelve proteins with a wide range of pI values revealed that the nucleation promoting effect of the saponites tended to increase with increased substitution rate. Furthermore, the saponite with the highest fluorine content promoted nucleation in all the test proteins regardless of their overall net charge. Adsorption experiments of proteins on the saponites confirmed that the density of adsorbed molecules increased according to the substitution rate, thereby explaining the heterogeneous nucleation on the silicate surface.

  5. Computer simulations of radiation damage in protein crystals

    International Nuclear Information System (INIS)

    Zehnder, M.

    2007-03-01

    The achievable resolution and the quality of the dataset of an intensity data collection for structure analysis of protein crystals with X-rays is limited among other factors by radiation damage. The aim of this work is to obtain a better quantitative understanding of the radiation damage process in proteins. Since radiation damage is unavoidable it was intended to look for the optimum ratio between elastically scattered intensity and radiation damage. Using a Monte Carlo algorithm physical processes after an inelastic photon interaction are studied. The main radiation damage consists of ionizations of the atoms through the electron cascade following any inelastic photon interaction. Results of the method introduced in this investigation and results of an earlier theoretical studies of the influence of Auger-electron transport in diamond are in a good agreement. The dependence of the radiation damage as a function of the energy of the incident photon was studied by computer-aided simulations. The optimum energy range for diffraction experiments on the protein myoglobin is 10-40 keV. Studies of radiation damage as a function of crystal volume and shape revealed that very small plate or rod shaped crystals suffer less damage than crystals formed like a cube with the same volume. Furthermore the influence of a few heavy atoms in the protein molecule on radiation damage was examined. Already two iron atoms in the unit cell of myoglobin increase radiation damage significantly. (orig.)

  6. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    Science.gov (United States)

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-01-01

    The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6122 or P6522, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress. PMID:18391421

  7. Protein kinesis: The dynamics of protein trafficking and stability

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  8. Symmetry, stability, and diffraction properties of icosahedral crystals

    International Nuclear Information System (INIS)

    Bak, P.

    1985-01-01

    In a remarkable experiment on an Mn-Al alloy, Shechtman et al. observed a diffraction spectrum with icosahedral symmetry. This is inconsistent with discrete translational invariance since the symmetry includes a five-fold axis. In this paper, it is shown that the crystallography and diffraction pattern can be described by a six-dimensional space group. The crystal structure in 3d is obtained as a cut along a 3d hyperplane in a regular 6d crystal. Displacements of the 6d crystal along 6 orthogonal directions define 6 continuous symmetries for the icosahedral crystal, three of which are phase symmetries describing internal rearrangements of the atoms

  9. Design of a high-resolution high-stability positioning mechanism for crystal optics

    International Nuclear Information System (INIS)

    Shu, D.; Toellner, T. S.; Alp, E. E.

    1999-01-01

    The authors present a novel miniature multi-axis driving structure that will allow positioning of two crystals with better than 50-nrad angular resolution and nanometer linear driving sensitivity.The precision and stability of this structure allow the user to align or adjust an assembly of crystals to achieve the same performance as does a single channel-cut crystal, so they call it an artificial channel-cut crystal. In this paper, the particular designs and specifications, as well as the test results,for a two-axis driving structure for a high-energy-resolution artificial channel-cut crystal monochromator are presented

  10. The Growth of Protein Crystals Using McDUCK

    Science.gov (United States)

    Ewing, Felicia; Wilson, Lori; Nadarajah, Arunan; Pusey, Marc

    1998-01-01

    Most of the current microgravity crystal growth hardware is optimized to produce crystals within the limited time available on orbit. This often results in the actual nucleation and growth process being rushed or the system not coming to equilibrium within the limited time available. Longer duration hardware exists, but one cannot readily pick out crystals grown early versus those which nucleated and grew more slowly. We have devised a long duration apparatus, the Multi-chamber Dialysis Unit for Crystallization Kinetics, or McDUCK. This apparatus-is a series of protein chambers, stacked upon a precipitant reservoir chamber. All chambers are separated by a dialysis membrane, which serves to pass small molecules while retaining the protein. The volume of the Precipitant chamber is equal to the sum of the volumes of the protein chamber. In operation, the appropriate chambers are filled with precipitant solution or protein solution, and the McDUCK is placed standing upright, with the precipitant chamber on the bottom. The precipitant diffuses upwards over time, with the time to reach equilibration a function of the diffusivity of the precipitant and the overall length of the diffusion pathway. Typical equilibration times are approximately 2-4 months, and one can readily separate rapid from slow nucleation and growth crystals. An advantage on Earth is that the vertical precipitant concentration gradient dominates that of the solute, thus dampening out solute density gradient driven convective flows. However, large Earth-grown crystals have so far tended to be more two dimensional. Preliminary X-ray diffraction analysis of lysozyme crystals grown in McDUCK have indicated that the best, and largest, come from the middle chambers, suggesting that there is an optimal growth rate. Further, the improvements in diffraction resolution have been better signal to noise ratios in the low resolution data, not an increase in resolution overall. Due to the persistently large crystals

  11. The crystallization of a solid solution in a solvent and the stability of a growth interface

    International Nuclear Information System (INIS)

    Malmejac, Yves

    1971-03-01

    The potential uses of germanium-silicon alloys as thermoelectric generators in hitherto unexploited temperature ranges initiated the present study. Many delicate problems are encountered in the classical methods of preparation. An original technique was sought for crystallization in a metallic solvent. The thermodynamic equilibria between the various phases of the ternary System used were studied in order to justify the method used. The conditions (temperature and composition) were determined in which the cooling of a ternary liquid mixture induces the precipitation of a binary solid solution with the desired composition. If large crystals are to be obtained from the solid solution, metallic solvent precipitation must be replaced by a mono-directional solvent crystallization. The combined effect of a certain number of simple physical phenomena on the stability of a crystal liquid interface was studied: the morphological stability of the crystal growth interface is the first step towards obtaining perfect crystals. (author) [fr

  12. Four crystal forms of a Bence-Jones protein

    Energy Technology Data Exchange (ETDEWEB)

    Makino, Debora L.; Henschen-Edman, Agnes H.; McPherson, Alexander, E-mail: amcphers@uci.edu [Molecular Biology and Biochemistry, University of California, Irvine, 560 Steinhaus Hall, Irvine, CA 92697-3900 (United States)

    2005-01-01

    Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 y ago. The trigonal crystal form may shed some light on the formation of fibrils common to certain storage diseases. Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 years ago. Closely related tetragonal and orthorhombic forms belonging to space groups P4{sub 3}2{sub 1}2 and P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = b = 68.7, c = 182.1 and a = 67.7, b = 69.4, c = 87.3 Å, diffract to 1.5 and 1.9 Å, respectively. Two closely related trigonal forms, both belonging to space group P3{sub 1}21 with unit-cell parameters a = b = 154.3 Å but differing by a doubling of the c axis, one 46.9 Å and the other 94.0 Å, diffract to 2.9 and 2.6 Å resolution, respectively. The trigonal crystal of short c-axis length shows a positive indication of twinning. The trigonal crystal of longer c axis, which appeared only after eight months of incubation at room temperature, is likely to be composed of proteolytically degraded molecules and unlike the other crystal forms contains two entire Bence-Jones dimers in the asymmetric unit. This latter crystal form may shed some light on the formation of fibrils common to certain storage diseases.

  13. Four crystal forms of a Bence-Jones protein

    International Nuclear Information System (INIS)

    Makino, Debora L.; Henschen-Edman, Agnes H.; McPherson, Alexander

    2004-01-01

    Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 y ago. The trigonal crystal form may shed some light on the formation of fibrils common to certain storage diseases. Four crystal forms have been grown and characterized by X-ray diffraction of a Bence-Jones protein collected from the urine of a multiple myeloma patient more than 40 years ago. Closely related tetragonal and orthorhombic forms belonging to space groups P4 3 2 1 2 and P2 1 2 1 2 1 , with unit-cell parameters a = b = 68.7, c = 182.1 and a = 67.7, b = 69.4, c = 87.3 Å, diffract to 1.5 and 1.9 Å, respectively. Two closely related trigonal forms, both belonging to space group P3 1 21 with unit-cell parameters a = b = 154.3 Å but differing by a doubling of the c axis, one 46.9 Å and the other 94.0 Å, diffract to 2.9 and 2.6 Å resolution, respectively. The trigonal crystal of short c-axis length shows a positive indication of twinning. The trigonal crystal of longer c axis, which appeared only after eight months of incubation at room temperature, is likely to be composed of proteolytically degraded molecules and unlike the other crystal forms contains two entire Bence-Jones dimers in the asymmetric unit. This latter crystal form may shed some light on the formation of fibrils common to certain storage diseases

  14. Models of protein-ligand crystal structures: trust, but verify.

    Science.gov (United States)

    Deller, Marc C; Rupp, Bernhard

    2015-09-01

    X-ray crystallography provides the most accurate models of protein-ligand structures. These models serve as the foundation of many computational methods including structure prediction, molecular modelling, and structure-based drug design. The success of these computational methods ultimately depends on the quality of the underlying protein-ligand models. X-ray crystallography offers the unparalleled advantage of a clear mathematical formalism relating the experimental data to the protein-ligand model. In the case of X-ray crystallography, the primary experimental evidence is the electron density of the molecules forming the crystal. The first step in the generation of an accurate and precise crystallographic model is the interpretation of the electron density of the crystal, typically carried out by construction of an atomic model. The atomic model must then be validated for fit to the experimental electron density and also for agreement with prior expectations of stereochemistry. Stringent validation of protein-ligand models has become possible as a result of the mandatory deposition of primary diffraction data, and many computational tools are now available to aid in the validation process. Validation of protein-ligand complexes has revealed some instances of overenthusiastic interpretation of ligand density. Fundamental concepts and metrics of protein-ligand quality validation are discussed and we highlight software tools to assist in this process. It is essential that end users select high quality protein-ligand models for their computational and biological studies, and we provide an overview of how this can be achieved.

  15. Surface (glyco-)proteins: primary structure and crystallization under microgravity conditions

    Science.gov (United States)

    Claus, H.; Akca, E.; Schultz, N.; Karbach, G.; Schlott, B.; Debaerdemaeker, T.; De Clercq, J.-P.; König, H.

    2001-08-01

    The Archaea comprise microorganisms that live under environmental extremes, like high temperature, low pH value or high salt concentration. Their cells are often covered by a single layer of (glyco)protein subunits (S-layer) in hexagonal arrangement. In order to get further hints about the molecular mechanisms of protein stabilization we compared the primary and secondary structures of archaeal S-layer (glyco)proteins. We found an increase of charged amino acids in the S-layer proteins of the extreme thermophilic species compared to their mesophilic counterparts. Our data and those of other authors suggest that ionic interactions, e.g., salt bridges seem to be played a major role in protein stabilization at high temperatures. Despite the differences in the growth optima and the predominance of some amino acids the primary structures of S-layers revealed also a significant degree of identity between phylogenetically related archaea. These obervations indicate that protein sequences of S-layers have been conserved during the evolution from extremely thermophilic to mesophilic life. To support these findings the three-dimensional structure of the S-layer proteins has to be elucidated. Recently, we described the first successful crystallization of an extreme thermophilic surface(glyco)protein under microgravity conditions.

  16. Increasing protein stability by improving beta-turns.

    Science.gov (United States)

    Fu, Hailong; Grimsley, Gerald R; Razvi, Abbas; Scholtz, J Martin; Pace, C Nick

    2009-11-15

    Our goal was to gain a better understanding of how protein stability can be increased by improving beta-turns. We studied 22 beta-turns in nine proteins with 66-370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some beta-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein, Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase alpha-subunit, and Maltose binding protein. Of the 15 single proline mutations, 11 increased stability (Average = 0.8 +/- 0.3; Range = 0.3-1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. On the basis of this and our previous work, we conclude that proteins can generally be stabilized by replacing nonproline residues with proline residues at the i + 1 position of Type I and II beta-turns and at the i position in Type II beta-turns. Other turn positions can sometimes be used if the phi angle is near -60 degrees for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in beta-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in beta-turns that could be replaced by Gly to increase protein stability. Improving beta-turns by substituting Pro residues is a generally useful way of increasing protein stability. 2009 Wiley-Liss, Inc.

  17. Screening and Crystallization Plates for Manual and High-throughput Protein Crystal Growth

    Science.gov (United States)

    Thorne, Robert E. (Inventor); Berejnov, Viatcheslav (Inventor); Kalinin, Yevgeniy (Inventor)

    2010-01-01

    In one embodiment, a crystallization and screening plate comprises a plurality of cells open at a top and a bottom, a frame that defines the cells in the plate, and at least two films. The first film seals a top of the plate and the second film seals a bottom of the plate. At least one of the films is patterned to strongly pin the contact lines of drops dispensed onto it, fixing their position and shape. The present invention also includes methods and other devices for manual and high-throughput protein crystal growth.

  18. Crystal structure of human protein kinase CK2

    DEFF Research Database (Denmark)

    Niefind, K; Guerra, B; Ermakowa, I

    2001-01-01

    The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 A resolution. In the CK2 complex the regulatory subunits form a stable dimer linking the two catalyt...... as a docking partner for various protein kinases. Furthermore it shows an inter-domain mobility in the catalytic subunit known to be functionally important in protein kinases and detected here for the first time directly within one crystal structure.......The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 A resolution. In the CK2 complex the regulatory subunits form a stable dimer linking the two catalytic...... subunits, which make no direct contact with one another. Each catalytic subunit interacts with both regulatory chains, predominantly via an extended C-terminal tail of the regulatory subunit. The CK2 structure is consistent with its constitutive activity and with a flexible role of the regulatory subunit...

  19. Effects of protein phosphorylation on color stability of ground meat.

    Science.gov (United States)

    Li, Meng; Li, Xin; Xin, Jianzeng; Li, Zheng; Li, Guixia; Zhang, Yan; Du, Manting; Shen, Qingwu W; Zhang, Dequan

    2017-03-15

    The influence of protein phosphorylation on meat color stability was investigated in this study. Phosphatase and protein kinase inhibitors were added to minced ovine Longissimus thoracis et lumborum (LTL) muscle to manipulate the global phosphorylation of sarcoplasmic proteins. The data obtained show that the rate and extent of pH decline, along with lactate accumulation in postmortem muscle, were related to protein phosphorylation. Analysis of meat color and the relative content of myoglobin redox forms revealed that meat color stability was inversely related to the phosphorylation of sarcoplasmic proteins. Thus, this study suggests that protein phosphorylation may be involved in meat color development by regulating glycolysis and the redox stability of myoglobin. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Applications of Protein Thermodynamic Database for Understanding Protein Mutant Stability and Designing Stable Mutants.

    Science.gov (United States)

    Gromiha, M Michael; Anoosha, P; Huang, Liang-Tsung

    2016-01-01

    Protein stability is the free energy difference between unfolded and folded states of a protein, which lies in the range of 5-25 kcal/mol. Experimentally, protein stability is measured with circular dichroism, differential scanning calorimetry, and fluorescence spectroscopy using thermal and denaturant denaturation methods. These experimental data have been accumulated in the form of a database, ProTherm, thermodynamic database for proteins and mutants. It also contains sequence and structure information of a protein, experimental methods and conditions, and literature information. Different features such as search, display, and sorting options and visualization tools have been incorporated in the database. ProTherm is a valuable resource for understanding/predicting the stability of proteins and it can be accessed at http://www.abren.net/protherm/ . ProTherm has been effectively used to examine the relationship among thermodynamics, structure, and function of proteins. We describe the recent progress on the development of methods for understanding/predicting protein stability, such as (1) general trends on mutational effects on stability, (2) relationship between the stability of protein mutants and amino acid properties, (3) applications of protein three-dimensional structures for predicting their stability upon point mutations, (4) prediction of protein stability upon single mutations from amino acid sequence, and (5) prediction methods for addressing double mutants. A list of online resources for predicting has also been provided.

  1. Nanostructured Mineral Coatings Stabilize Proteins for Therapeutic Delivery.

    Science.gov (United States)

    Yu, Xiaohua; Biedrzycki, Adam H; Khalil, Andrew S; Hess, Dalton; Umhoefer, Jennifer M; Markel, Mark D; Murphy, William L

    2017-09-01

    Proteins tend to lose their biological activity due to their fragile structural conformation during formulation, storage, and delivery. Thus, the inability to stabilize proteins in controlled-release systems represents a major obstacle in drug delivery. Here, a bone mineral inspired protein stabilization strategy is presented, which uses nanostructured mineral coatings on medical devices. Proteins bound within the nanostructured coatings demonstrate enhanced stability against extreme external stressors, including organic solvents, proteases, and ethylene oxide gas sterilization. The protein stabilization effect is attributed to the maintenance of protein conformational structure, which is closely related to the nanoscale feature sizes of the mineral coatings. Basic fibroblast growth factor (bFGF) released from a nanostructured mineral coating maintains its biological activity for weeks during release, while it maintains activity for less than 7 d during release from commonly used polymeric microspheres. Delivery of the growth factors bFGF and vascular endothelial growth factor using a mineral coated surgical suture significantly improves functional Achilles tendon healing in a rabbit model, resulting in increased vascularization, more mature collagen fiber organization, and a two fold improvement in mechanical properties. The findings of this study demonstrate that biomimetic interactions between proteins and nanostructured minerals provide a new, broadly applicable mechanism to stabilize proteins in the context of drug delivery and regenerative medicine. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Thermal precipitation fluorescence assay for protein stability screening.

    Science.gov (United States)

    Fan, Junping; Huang, Bo; Wang, Xianping; Zhang, Xuejun C

    2011-09-01

    A simple and reliable method of protein stability assessment is desirable for high throughput expression screening of recombinant proteins. Here we described an assay termed thermal precipitation fluorescence (TPF) which can be used to compare thermal stabilities of recombinant protein samples directly from cell lysate supernatants. In this assay, target membrane proteins are expressed as recombinant fusions with a green fluorescence protein tag and solubilized with detergent, and the fluorescence signals are used to report the quantity of the fusion proteins in the soluble fraction of the cell lysate. After applying a heat shock, insoluble protein aggregates are removed by centrifugation. Subsequently, the amount of remaining protein in the supernatant is quantified by in-gel fluorescence analysis and compared to samples without a heat shock treatment. Over 60 recombinant membrane proteins from Escherichia coli were subject to this screening in the presence and absence of a few commonly used detergents, and the results were analyzed. Because no sophisticated protein purification is required, this TPF technique is suitable to high throughput expression screening of recombinant membrane proteins as well as soluble ones and can be used to prioritize target proteins based on their thermal stabilities for subsequent large scale expression and structural studies. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Tandem Facial Amphiphiles for Membrane Protein Stabilization

    DEFF Research Database (Denmark)

    Chae, Pil Seok; Gotfryd, Kamil; Pacyna, Jennifer

    2010-01-01

    We describe a new type of synthetic amphiphile that is intended to support biochemical characterization of intrinsic membrane proteins. Members of this new family displayed favorable behavior with four of five membrane proteins tested, and these amphiphiles formed relatively small micelles....

  4. Crystal structure of Homo sapiens protein LOC79017

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Euiyoung; Bingman, Craig A.; Aceti, David J.; Phillips, Jr., George N. (UW)

    2010-02-08

    LOC79017 (MW 21.0 kDa, residues 1-188) was annotated as a hypothetical protein encoded by Homo sapiens chromosome 7 open reading frame 24. It was selected as a target by the Center for Eukaryotic Structural Genomics (CESG) because it did not share more than 30% sequence identity with any protein for which the three-dimensional structure is known. The biological function of the protein has not been established yet. Parts of LOC79017 were identified as members of uncharacterized Pfam families (residues 1-95 as PB006073 and residues 104-180 as PB031696). BLAST searches revealed homologues of LOC79017 in many eukaryotes, but none of them have been functionally characterized. Here, we report the crystal structure of H. sapiens protein LOC79017 (UniGene code Hs.530024, UniProt code O75223, CESG target number go.35223).

  5. Stabilization of Proteins and Noncovalent Protein Complexes during Electrospray Ionization by Amino Acid Additives.

    Science.gov (United States)

    Zhang, Hua; Lu, Haiyan; Chingin, Konstantin; Chen, Huanwen

    2015-07-21

    Ionization of proteins and noncovalent protein complexes with minimal disturbance to their native structure presents a great challenge for biological mass spectrometry (MS). In living organisms, the native structure of intracellular proteins is commonly stabilized by solute amino acids (AAs) accumulated in cells at very high concentrations. Inspired by nature, we hypothesized that AAs could also pose a stabilizing effect on the native structure of proteins and noncovalent protein complexes during ionization. To test this hypothesis, here we explored MS response for various protein complexes upon the addition of free AAs at mM concentrations into the electrospray ionization (ESI) solution. Thermal activation of ESI droplets in the MS inlet capillary was employed as a model destabilizing factor during ionization. Our results indicate that certain AAs, in particular proline (Pro), pose considerable positive effect on the stability of noncovalent protein complexes in ESI-MS without affecting the signal intensity of protein ions and original protein-ligand equilibrium, even when added at the 20 mM concentration. The data suggest that the degree of protein stabilization is primarily determined by the osmolytic and ampholytic characteristics of AA solutes. The highest stability and visibility of noncovalent protein complexes in ESI-MS are achieved using AA additives with neutral isoelectric point, moderate proton affinity, and unfavorable interaction with the native protein state. Overall, our results indicate that the simple addition of free amino acids into the working solution can notably improve the stability and accuracy of protein analysis by native ESI-MS.

  6. H-Bond stabilized columnar discotic liquid crystals

    NARCIS (Netherlands)

    Paraschiv, I.

    2007-01-01

    Since 1977, more than 2300 publications on discotic (disk-like) liquid crystalline materials have appeared. Discotic liquid crystals, which usually consist of polyaromatic molecules surrounded by long peripheral alkyl tails, can form liquid crystalline mesophases in a wide temperature range. Within

  7. Improvement of the Crystal Stability and Dissolution Profile of ...

    African Journals Online (AJOL)

    This study was undertaken to improve the solubility of metronidazole by modifying its crystal characteristics using pharmaceutical excipients. Metronidazole granules were formulated with cashew gum (2 – 8% w/w) and microcrystalline cellulose (10% w/w) via kneading, solid dispersion, or physical mixing. Resulting ...

  8. Physicochemical stability of lycopene-loaded emulsions stabilized by plant or dairy proteins

    NARCIS (Netherlands)

    Ho, Kacie; Schroen, C.G.P.H.; San Martín-González, M.F.; Berton-Carabin, C.C.

    2017-01-01

    Lycopene is a lipophilic bioactive compound that can be challenging to deliver in vivo. To mediate this, delivery strategies, such as protein-stabilized oil-in-water (O/W) emulsions, have been suggested to improve the physicochemical stability and bioavailability of lycopene. In this research, the

  9. A Comparison of Protein Stability in Prefillable Syringes Made of Glass and Plastic.

    Science.gov (United States)

    Waxman, Lloyd; Vilivalam, Vinod D

    2017-01-01

    The development of protein therapeutics requires stabilization of these labile molecules during shipment and storage. Biologics, particularly monoclonal antibodies, are frequently packaged at high concentration in prefillable syringes traditionally made of glass. However, some biologics are unstable in glass due to sensitivity to silicone oil, tungsten, glue, or metal ions. Syringes made from the plastic cyclic olefin polymer, Daikyo Crystal Zenith® (CZ), with a Flurotec-laminated piston, have none of these issues. This study compared the stability of several proteins including biotherapeutics when stored up to 14 months at 5 °C and 25 °C in prefillable siliconized syringes made of glass or silicone oil-free CZ syringes, and when subjected to mild agitation by end-over-end rotation at room temperature. At each time point, proteins were analyzed by several techniques including turbidity, size exclusion high-performance liquid chromatography, reversed phase high-performance liquid chromatography, ion-exchange chromatography, electrophoresis, and light scattering to monitor changes in aggregation and degradation. The results show that proteins have comparable stability when stored in glass syringes or in syringes made of CZ sterilized by E-beam or autoclave. In addition, proteins stressed by agitation were generally more stable and aggregated less in syringes made of CZ than in ones made of glass. LAY ABSTRACT: Biotherapeutic protein drugs such as monoclonal antibodies are frequently packaged at high concentration in prefillable syringes, which allows the drug to be directly administered by the patient or caregiver. Protein drugs, or biologics, can be unstable, and may aggregate, particularly when shaken. These aggregates can be immunogenic, stimulating the body's immune system to produce antibodies that can reduce the drug's efficacy. Although prefillable syringes are traditionally made of glass, some biologics are unstable in glass syringes due to the presence of

  10. Liquid crystal blue phases: stability, field effects and alignment

    OpenAIRE

    Gleeson, HF; Miller, RJ; Tian, L; Görtz, V; Goodby, JW

    2015-01-01

    The blue phases are fascinating structures in liquid crystals, fluids that exhibit cubic structures that have true crystalline order. The blue phases were discovered in the 1970s and were the subject of extensive research in the 1980s, when a deep understanding of many of their properties was established. The discovery that the blue phases could be stabilised to exist over wide temperature ranges meant that they became more than scientific curiosities and led to a recent resurgence in researc...

  11. Expression, crystallization and preliminary crystallographic study of mouse hepatitis virus (MHV) nucleocapsid protein C-terminal domain

    International Nuclear Information System (INIS)

    Tong, Xiaohang; Ma, Yanlin; Li, Xuemei

    2010-01-01

    The C-terminal domain of mouse hepatitis virus nucleocapsid protein has been overexpressed in E. coli, purified and crystallized. The crystal belonged to space group P422, with unit-cell parameters a = 66.6, c = 50.8 Å, and diffracted to 2.20 Å resolution. Mouse hepatitis virus (MHV) belongs to the group II coronaviruses. The virus produces nine genes encoding 11 proteins that could be recognized as structural proteins and nonstructural proteins and are crucial for viral RNA synthesis. The nucleocapsid (N) protein, one of the structural proteins, interacts with the 30.4 kb virus genomic RNA to form the helical nucleocapsid and associates with the membrane glycoprotein via its C-terminus to stabilize virion assembly. Here, the expression and crystallization of the MHV nucleocapsid protein C-terminal domain are reported. The crystals diffracted to 2.20 Å resolution and belonged to space group P422, with unit-cell parameters a = 66.6, c = 50.8 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content is 43.0% (V M = 2.16 Å 3 Da −1 )

  12. Solid dispersions of Myricetin with enhanced solubility: Formulation, characterization and crystal structure of stability-impeding Myricetin monohydrate crystals

    Science.gov (United States)

    Mureşan-Pop, M.; Pop, M. M.; Borodi, G.; Todea, M.; Nagy-Simon, T.; Simon, S.

    2017-08-01

    Three solid dispersion forms of Myricetin combined with the Polyvinylpyrrolidone were successfully prepared by spray drying method, and characterized by X-ray powder diffraction, thermal analysis, infrared spectroscopy and optical microscopy. Zeta potential measurements provided indications on solid dispersions stability in aqueous suspension related to their storage at elevated temperature and relative humidity, which depends on the Myricetin load. By increase of Myricetin load, the stability of the solid dispersion is impeded due to growth of Myricetin monohydrate crystals. The amorphous dispersions with 10% and 50% Myricetin load are stable and, compared to pure Myricetin, their aqueous solubility is enhanced by a factor of 47 and 13, respectively. The dispersion with 80% Myricetin load is unstable on storage, and this behavior acts in conjunction with the development of Myricetin monohydrate crystals. Single-crystal X-ray diffraction results obtained for Myricetin monohydrate reveal a structure of an infinite 2D network of hydrogen-bonded molecules involving all six hydroxyl groups of Myricetin. The water molecules are positioned in between the infinite chains, and contribute via H-bonds to robust crystal packing. The calculated needle-like morphology of monohydrate form is in agreement with the optical microscopy results. The study shows that the solid amorphous dispersions with up to 50% Myricetin load are a viable option for achieving substantial solubility improvement of Myricetin, and supports their potential use in pharmaceutical applications.

  13. Systematic comparison of crystal and NMR protein structures deposited in the protein data bank.

    Science.gov (United States)

    Sikic, Kresimir; Tomic, Sanja; Carugo, Oliviero

    2010-09-03

    Nearly all the macromolecular three-dimensional structures deposited in Protein Data Bank were determined by either crystallographic (X-ray) or Nuclear Magnetic Resonance (NMR) spectroscopic methods. This paper reports a systematic comparison of the crystallographic and NMR results deposited in the files of the Protein Data Bank, in order to find out to which extent these information can be aggregated in bioinformatics. A non-redundant data set containing 109 NMR - X-ray structure pairs of nearly identical proteins was derived from the Protein Data Bank. A series of comparisons were performed by focusing the attention towards both global features and local details. It was observed that: (1) the RMDS values between NMR and crystal structures range from about 1.5 Å to about 2.5 Å; (2) the correlation between conformational deviations and residue type reveals that hydrophobic amino acids are more similar in crystal and NMR structures than hydrophilic amino acids; (3) the correlation between solvent accessibility of the residues and their conformational variability in solid state and in solution is relatively modest (correlation coefficient = 0.462); (4) beta strands on average match better between NMR and crystal structures than helices and loops; (5) conformational differences between loops are independent of crystal packing interactions in the solid state; (6) very seldom, side chains buried in the protein interior are observed to adopt different orientations in the solid state and in solution.

  14. Stability of Coulomb crystals in a linear Paul trap with storage-ring-like confinement

    DEFF Research Database (Denmark)

    Kjærgaard, Niels; Mølhave, Kristian; Drewsen, Michael

    2002-01-01

    We report experiments on the stability of ion Coulomb crystals in a linear Paul trap with storage-ring-like confinement. The transverse dynamics of charged particles in a trap of this type is analogous to that of a fast beam traveling through a channel with periodic, magnetic alternating gradient...... confinement. The experimentally observed stability conditions for stationary crystals comply remarkably well with current theory of crystalline plasmas and beams.......We report experiments on the stability of ion Coulomb crystals in a linear Paul trap with storage-ring-like confinement. The transverse dynamics of charged particles in a trap of this type is analogous to that of a fast beam traveling through a channel with periodic, magnetic alternating gradient...

  15. Modeling of monolayer charge-stabilized colloidal crystals with static hexagonal crystal lattice

    Science.gov (United States)

    Nagatkin, A. N.; Dyshlovenko, P. E.

    2018-01-01

    The mathematical model of monolayer colloidal crystals of charged hard spheres in liquid electrolyte is proposed. The particles in the monolayer are arranged into the two-dimensional hexagonal crystal lattice. The model enables finding elastic constants of the crystals from the stress-strain dependencies. The model is based on the nonlinear Poisson-Boltzmann differential equation. The Poisson-Boltzmann equation is solved numerically by the finite element method for any spatial configuration. The model has five geometrical and electrical parameters. The model is used to study the crystal with particles comparable in size with the Debye length of the electrolyte. The first- and second-order elastic constants are found for a broad range of densities. The model crystal turns out to be stable relative to small uniform stretching and shearing. It is also demonstrated that the Cauchy relation is not fulfilled in the crystal. This means that the pair effective interaction of any kind is not sufficient to proper model the elasticity of colloids within the one-component approach.

  16. Structure of a second crystal form of Bence-Jones protein Loc: Strikingly different domain associations in two crystal forms of a single protein

    International Nuclear Information System (INIS)

    Schiffer, M.; Ainsworth, C.; Xu, Z.B.; Carperos, W.; Olsen, K.; Solomon, A.; Stevens, F.J.; Chang, C.H.

    1989-01-01

    The authors have determined the structure of the immunoglobulin light-chain dimer Loc in a second crystal form that was grown from distilled water. The crystal structure was determined to 2.8-angstrom resolution; the R factor is 0.22. The two variable domains are related by local 2-fold axes and form an antigen binding pocket. The variable domain-variable domain interaction observed in this crystal form differs from the one exhibited by the protein when crystallized from ammonium sulfate in which the two variable domains formed a protrusion. The structure attained in the distilled water crystals is similar to, but not identical with, the one observed for the Mcg light-chain dimer in crystals grown from ammonium sulfate. Thus, two strikingly different structures were attained by this multisubunit protein in crystals grown under two different, commonly used, crystallization techniques. The quaternary interactions exhibited by the protein in the two crystal forms are sufficiently different to suggest fundamentally different interpretations of the structural basis for the function of this protein. This observation may have general implications regarding the use of single crystallographic determinations for detailed identification of structural and functional relationships. On the other hand, proteins whose structures can be altered by manipulation of crystallization conditions may provide useful systems for study of fundamental structural chemistry

  17. Powder diffraction from a continuous microjet of submicrometer protein crystals.

    Science.gov (United States)

    Shapiro, D A; Chapman, H N; Deponte, D; Doak, R B; Fromme, P; Hembree, G; Hunter, M; Marchesini, S; Schmidt, K; Spence, J; Starodub, D; Weierstall, U

    2008-11-01

    Atomic-resolution structures from small proteins have recently been determined from high-quality powder diffraction patterns using a combination of stereochemical restraints and Rietveld refinement [Von Dreele (2007), J. Appl. Cryst. 40, 133-143; Margiolaki et al. (2007), J. Am. Chem. Soc. 129, 11865-11871]. While powder diffraction data have been obtained from batch samples of small crystal-suspensions, which are exposed to X-rays for long periods of time and undergo significant radiation damage, the proof-of-concept that protein powder diffraction data from nanocrystals of a membrane protein can be obtained using a continuous microjet is shown. This flow-focusing aerojet has been developed to deliver a solution of hydrated protein nanocrystals to an X-ray beam for diffraction analysis. This method requires neither the crushing of larger polycrystalline samples nor any techniques to avoid radiation damage such as cryocooling. Apparatus to record protein powder diffraction in this manner has been commissioned, and in this paper the first powder diffraction patterns from a membrane protein, photosystem I, with crystallite sizes of less than 500 nm are presented. These preliminary patterns show the lowest-order reflections, which agree quantitatively with theoretical calculations of the powder profile. The results also serve to test our aerojet injector system, with future application to femtosecond diffraction in free-electron X-ray laser schemes, and for serial crystallography using a single-file beam of aligned hydrated molecules.

  18. Protein-detergent interactions in single crystals of membrane proteins studied by neutron crystallography

    International Nuclear Information System (INIS)

    Timmins, P.A.; Pebay-Peyroula, E.

    1994-01-01

    The detergent micelles surrounding membrane protein molecules in single crystals can be investigated using neutron crystallography combined with H 2 O/D 2 O contrast variation. If the protein structure is known then the contrast variation method allows phases to be determined at a contrast where the detergent dominates the scattering. The application of various constraints allows the resulting scattering length density map to be realistically modeled. The method has been applied to two different forms of the membrane protein porin. In one case both hydrogenated and partially deuterated protein were used, allowing the head group and tail to be distinguished

  19. Protein-detergent interactions in single crystals of membrane proteins studied by neutron crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Timmins, P.A. [ILL, Grenoble (France); Pebay-Peyroula, E. [IBS-UJF Grenoble (France)

    1994-12-31

    The detergent micelles surrounding membrane protein molecules in single crystals can be investigated using neutron crystallography combined with H{sub 2}O/D{sub 2}O contrast variation. If the protein structure is known then the contrast variation method allows phases to be determined at a contrast where the detergent dominates the scattering. The application of various constraints allows the resulting scattering length density map to be realistically modeled. The method has been applied to two different forms of the membrane protein porin. In one case both hydrogenated and partially deuterated protein were used, allowing the head group and tail to be distinguished.

  20. Protein stability and enzyme activity at extreme biological temperatures

    International Nuclear Information System (INIS)

    Feller, Georges

    2010-01-01

    Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 0 C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins. (topical review)

  1. Escherichia coli PII protein: purification, crystallization and oligomeric structure.

    Science.gov (United States)

    Vasudevan, S G; Gedye, C; Dixon, N E; Cheah, E; Carr, P D; Suffolk, P M; Jeffrey, P D; Ollis, D L

    1994-01-17

    The Escherichia coli signal transduction protein PII, product of the glnB gene, was overproduced and purified. The predicted molecular weight of the protein based on the correct nucleotide sequence is 12,427 and is very close to the value 12,435 obtained by matrix-assisted laser desorption mass spectrometry. Hexagonal crystals of the unuridylylated form of PII with dimensions 0.2 x 0.2 x 0.3 mm were grown and analysed by X-ray diffraction. The crystals belong to space group P6(3) with a = b = 61.6 A, c = 56.3 A and Vm of 2.5 for one subunit in the asymmetric unit. A low-resolution electron density map showed electron density concentrated around a three-fold axis, suggesting the molecule to be a trimer. A sedimentation equilibrium experiment of the meniscus depletion type was used to estimate a molecular weight of 35,000 +/- 1,000 for PII in solution. This result is consistent with the native protein being a homotrimer.

  2. Stability of equilibrium states in finite samples of smectic C* liquid crystals

    International Nuclear Information System (INIS)

    Stewart, I W

    2005-01-01

    Equilibrium solutions for a sample of ferroelectric smectic C (SmC*) liquid crystal in the 'bookshelf' geometry under the influence of a tilted electric field will be presented. A linear stability criterion is identified and used to confirm stability for typical materials possessing either positive or negative dielectric anisotropy. The theoretical response times for perturbations to the equilibrium solutions are calculated numerically and found to be consistent with estimates for response times in ferroelectric smectic C liquid crystals reported elsewhere in the literature for non-tilted fields

  3. Stability of equilibrium states in finite samples of smectic C* liquid crystals

    Energy Technology Data Exchange (ETDEWEB)

    Stewart, I W [Department of Mathematics, University of Strathclyde, Livingstone Tower, 26 Richmond Street, Glasgow G1 1XH (United Kingdom)

    2005-03-04

    Equilibrium solutions for a sample of ferroelectric smectic C (SmC*) liquid crystal in the 'bookshelf' geometry under the influence of a tilted electric field will be presented. A linear stability criterion is identified and used to confirm stability for typical materials possessing either positive or negative dielectric anisotropy. The theoretical response times for perturbations to the equilibrium solutions are calculated numerically and found to be consistent with estimates for response times in ferroelectric smectic C liquid crystals reported elsewhere in the literature for non-tilted fields.

  4. Citrate effects on amorphous calcium carbonate (ACC) structure, stability, and crystallization

    DEFF Research Database (Denmark)

    Tobler, Dominique Jeanette; Rodriguez Blanco, Juan Diego; Dideriksen, Knud

    2015-01-01

    Understanding the role of citrate in the crystallization kinetics of amorphous calcium carbonate (ACC) is essential to explain the formation mechanisms, stabilities, surface properties, and morphologies of CaCO3 biominerals. It also contributes to deeper insight into fluid-mineral inte......Understanding the role of citrate in the crystallization kinetics of amorphous calcium carbonate (ACC) is essential to explain the formation mechanisms, stabilities, surface properties, and morphologies of CaCO3 biominerals. It also contributes to deeper insight into fluid...

  5. Protein engineering of subtilisins to improve stability in detergent formulations.

    Science.gov (United States)

    von der Osten, C; Branner, S; Hastrup, S; Hedegaard, L; Rasmussen, M D; Bisgård-Frantzen, H; Carlsen, S; Mikkelsen, J M

    1993-03-01

    Microbial proteases are used extensively in a large number of industrial processes and most importantly in detergent formulations facilitating the removal of proteinaceous stains. Site-directed mutagenesis has been employed in the construction of subtilisin variants with improved storage and oxidation stabilities. It is shown that in spite of significant structural homology between subtilisins subjected to protein engineering the effects of specific mutations can be quite different. Mutations that stabilize one subtilisin may destabilize another.

  6. Determination of protein and solvent volumes in protein crystals from contrast variation data

    Energy Technology Data Exchange (ETDEWEB)

    Badger, J. [Brandeis Univ., Waltham, MA (United States)

    1994-12-31

    By varying the relative values of protein and solvent scattering densities in a crystal, it is possible to obtain information on the shape and dimensions of protein molecular envelopes. Neutron diffraction methods are ideally suited to these contrast variation experiments because H/D exchange leads to large differential changes in the protein and solvent scattering densities and is structurally non-perturbing. Low resolution structure factors have been measured from cubic insulin crystals with differing H/D contents. Structure factors calculated from a simple binary density model, in which uniform scattering densities represent the protein and solvent volumes in the crystals, were compared with these data. The contrast variation differences in the sets of measured structure factors were found to be accurately fitted by this simple model. Trial applications to two problems in crystal structure determination illustrate how this fact may be exploited. (1) A translation function that employs contrast variation data gave a sharp minimum within 1-9{Angstrom} of the correctly positioned insulin molecule and is relatively insensitive to errors in the atomic model. (2) An ab initio phasing method for the contrast variation data, based on analyzing histograms of the density distributions in trial maps, was found to recover the correct molecular envelope.

  7. Crystallization and evaluation of hen egg-white lysozyme crystals for protein pH titration in the crystalline state.

    Science.gov (United States)

    Iwai, Wakari; Yagi, Daichi; Ishikawa, Takuya; Ohnishi, Yuki; Tanaka, Ichiro; Niimura, Nobuo

    2008-05-01

    To observe the ionized status of the amino acid residues in proteins at different pH (protein pH titration in the crystalline state) by neutron diffraction, hen egg-white lysozyme was crystallized over a wide pH range (2.5-8.0). Crystallization phase diagrams at pH 2.5, 6.0 and 7.5 were determined. At pH diagram, and at pH > 4.5 the border shifted to the right (higher precipitant concentration). The qualities of these crystals were characterized using the Wilson plot method. The qualities of all crystals at different pH were more or less equivalent (B-factor values within 25-40). It is expected that neutron diffraction analysis of these crystals of different pH provides equivalent data in quality for discussions of protein pH titration in the crystalline state of hen egg-white lysozyme.

  8. Positively selected sites in cetacean myoglobins contribute to protein stability

    DEFF Research Database (Denmark)

    Dasmeh, Pouria; Serohijos, Adrian W R; Kepp, Kasper P

    2013-01-01

    Since divergence ∼50 Ma ago from their terrestrial ancestors, cetaceans underwent a series of adaptations such as a ∼10-20 fold increase in myoglobin (Mb) concentration in skeletal muscle, critical for increasing oxygen storage capacity and prolonging dive time. Whereas the O2-binding affinity...... between Mb folding stability and protein abundance, suggesting that a selection pressure for stability acts proportionally to higher expression. We also identify a major divergence event leading to the common ancestor of whales, during which major stabilization occurred. Most of the positively selected...

  9. Equilibrium Kinetics Studies and Crystallization Aboard the International Space Station (ISS) Using the Protein Crystallization Apparatus for Microgravity (PCAM)

    Science.gov (United States)

    Achari, Aniruddha; Roeber, Dana F.; Barnes, Cindy L.; Kundrot, Craig E.; Stinson, Thomas N. (Technical Monitor)

    2002-01-01

    Protein Crystallization Apparatus in Microgravity (PCAM) trays have been used in Shuttle missions to crystallize proteins in a microgravity environment. The crystallization experiments are 'sitting drops' similar to that in Cryschem trays, but the reservoir solution is soaked in a wick. From early 2001, crystallization experiments are conducted on the International Space Station using mission durations of months rather than two weeks on previous shuttle missions. Experiments were set up in April 2001 on Flight 6A to characterize the time crystallization experiments will take to reach equilibrium in a microgravity environment using salts, polyethylene glycols and an organic solvent as precipitants. The experiments were set up to gather data for a series of days of activation with different droplet volumes and precipitants. The experimental set up on ISS and results of this study will be presented. These results will help future users of PCAM to choose precipitants to optimize crystallization conditions for their target macromolecules for a particular mission with known mission duration. Changes in crystal morphology and size between the ground and space grown crystals of a protein and a protein -DNA complex flown on the same mission will also be presented.

  10. Protein crystal growth on board Shenzhou 3: a concerted effort improves crystal diffraction quality and facilitates structure determination

    International Nuclear Information System (INIS)

    Han, Y.; Cang, H.-X.; Zhou, J.-X.; Wang, Y.-P.; Bi, R.-C.; Colelesage, J.; Delbaere, L.T.J.; Nahoum, V.; Shi, R.; Zhou, M.; Zhu, D.-W.; Lin, S.-X.

    2004-01-01

    The crystallization of 16 proteins was carried out using 60 wells on board Shenzhou 3 in 2002. Although the mission was only 7 days, careful and concerted planning at all stages made it possible to obtain crystals of improved quality compared to their ground controls for some of the proteins. Significantly improved resolutions were obtained from diffracted crystals of 4 proteins. A complete data set from a space crystal of the PEP carboxykinase yielded significantly higher resolution (1.46 A vs. 1.87 A), I/sigma (22.4 vs. 15.5), and a lower average temperature factor (29.2 A 2 vs. 42.9 A 2 ) than the best ground-based control crystal. The 3-D structure of the enzyme is well improved with significant ligand density. It has been postulated that the reduced convection and absence of macromolecule sedimentation under microgravity have advantages/benefits for protein crystal growth. Improvements in experimental design for protein crystal growth in microgravity are ongoing

  11. Stabilizing additives added during cell lysis aid in the solubilization of recombinant proteins.

    Directory of Open Access Journals (Sweden)

    David J Leibly

    Full Text Available Insoluble recombinant proteins are a major issue for both structural genomics and enzymology research. Greater than 30% of recombinant proteins expressed in Escherichia coli (E. coli appear to be insoluble. The prevailing view is that insolubly expressed proteins cannot be easily solubilized, and are usually sequestered into inclusion bodies. However, we hypothesize that small molecules added during the cell lysis stage can yield soluble protein from insoluble protein previously screened without additives or ligands. We present a novel screening method that utilized 144 additive conditions to increase the solubility of recombinant proteins expressed in E. coli. These selected additives are natural ligands, detergents, salts, buffers, and chemicals that have been shown to increase the stability of proteins in vivo. We present the methods used for this additive solubility screen and detailed results for 41 potential drug target recombinant proteins from infectious organisms. Increased solubility was observed for 80% of the recombinant proteins during the primary and secondary screening of lysis with the additives; that is 33 of 41 target proteins had increased solubility compared with no additive controls. Eleven additives (trehalose, glycine betaine, mannitol, L-Arginine, potassium citrate, CuCl(2, proline, xylitol, NDSB 201, CTAB and K(2PO(4 solubilized more than one of the 41 proteins; these additives can be easily screened to increase protein solubility. Large-scale purifications were attempted for 15 of the proteins using the additives identified and eight (40% were prepared for crystallization trials during the first purification attempt. Thus, this protocol allowed us to recover about a third of seemingly insoluble proteins for crystallography and structure determination. If recombinant proteins are required in smaller quantities or less purity, the final success rate may be even higher.

  12. Microphase Separation Controlled beta-Sheet Crystallization Kinetics in Fibrous Proteins

    International Nuclear Information System (INIS)

    Hu, X.; Lu, Q.; Kaplan, D.; Cebe, P.

    2009-01-01

    Silk is a naturally occurring fibrous protein with a multiblock chain architecture. As such, it has many similarities with synthetic block copolymers, including the possibility for e-sheet crystallization restricted within the crystallizable blocks. The mechanism of isothermal crystallization kinetics of e-sheet crystals in silk multiblock fibrous proteins is reported in this study. Kinetics theories, such as Avrami analysis which was established for studies of synthetic polymer crystal growth, are for the first time extended to investigate protein self-assembly in e-sheet rich Bombyx mori silk fibroin samples, using time-resolved Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and synchrotron real-time wide-angle X-ray scattering (WAXS). The Avrami exponent, n, was close to 2 for all methods and crystallization temperatures, indicating formation of e-sheet crystals in silk proteins is different from the 3-D spherulitic crystal growth found in synthetic polymers. Observations by scanning electron microscopy support the view that the protein structures vary during the different stages of crystal growth, and show a microphase separation pattern after chymotrypsin enzyme biodegradation. We present a model to explain the crystallization of the multiblock silk fibroin protein, by analogy to block copolymers: crystallization of e-sheets occurs under conditions of geometrical restriction caused by phase separation of the crystallizable and uncrystallizable blocks. This crystallization model could be widely applicable in other proteins with multiblock (i.e., crystallizable and noncrystallizable) domains.

  13. The Feasibility of Bulk Crystallization as an Industrial Purification and Production Technique for Proteins

    Science.gov (United States)

    Judge, Russell A.; Forsythe, Elizabeth L.; Johns, Michael R.; Pusey, Marc L.; White, Edward T.

    1998-01-01

    Bulk crystallization in stirred vessels is used industrially for the recovery and purification of many inorganic and organic materials. Although much has been written on the crystallization of proteins for X-ray diffraction analysis, very little has been reported on the application of bulk crystallization in stirred vessels. In this study, a 1-liter, seeded, stirred, batch crystallizer was used with ovalbumin as a model protein to test the feasibility of this crystallization method as a recovery and purification process for proteins. Results were obtained for ovalbumin solubility, nucleation thresholds, crystal breakage and crystal growth kinetics in bulk solution under a range of operating conditions of pH and ammonium sulphate concentration (Judge et al., 1996). Experiments were also performed to determine the degree of purification that can be achieved by the crystallization of ovalbumin from a mixture of proteins. The effect of the presence of these proteins upon the ovalbumin crystal growth kinetics was also investigated (Judge et al., 1995). All of these aspects are essential for the design of bulk crystallization processes which have not previously been reported for proteins. Results from a second study that investigated the effect of structurally different proteins on the solubility, crystal growth rates and crystal purity of chicken egg white lysozyme are also presented (Judge et al., 1997). In this case face growth rates were measured using lysozyme purified by liquid chromatography and the effect of the addition of specific protein impurities were observed on the (110) and (101) crystal faces. In these two studies the results are presented to show the feasibility and purifying ability of crystallization as a production process for proteins.

  14. Enhanced Stability of a Protein with Increasing Temperature

    DEFF Research Database (Denmark)

    Vinther, Joachim Møllesøe; Kristensen, Søren M; Led, Jens J

    2010-01-01

    The unusual stability of a structured but locally flexible protein, human growth hormone (hGH) at pH 2.7, was investigated using the temperature dependence of the nanosecond-picosecond dynamics of the backbone amide groups obtained from (15)N NMR relaxation data. It is found that the flexibility ...

  15. Salinity induced changes in cell membrane stability, protein and ...

    African Journals Online (AJOL)

    control), 4.7, 9.4 and 14.1 dS m-1 to determine the effect of salt on vegetative growth, relative water content, cell membrane stability, protein and RNA contents in sand culture experiment. Fresh and dry weights of plants, shoots and roots decreased ...

  16. High-throughput method for optimum solubility screening for homogeneity and crystallization of proteins

    Science.gov (United States)

    Kim, Sung-Hou [Moraga, CA; Kim, Rosalind [Moraga, CA; Jancarik, Jamila [Walnut Creek, CA

    2012-01-31

    An optimum solubility screen in which a panel of buffers and many additives are provided in order to obtain the most homogeneous and monodisperse protein condition for protein crystallization. The present methods are useful for proteins that aggregate and cannot be concentrated prior to setting up crystallization screens. A high-throughput method using the hanging-drop method and vapor diffusion equilibrium and a panel of twenty-four buffers is further provided. Using the present methods, 14 poorly behaving proteins have been screened, resulting in 11 of the proteins having highly improved dynamic light scattering results allowing concentration of the proteins, and 9 were crystallized.

  17. Crystallization and evaluation of hen egg-white lysozyme crystals for protein pH titration in the crystalline state

    International Nuclear Information System (INIS)

    Iwai, Wakari; Yagi, Daichi; Ishikawa, Takuya; Ohnishi, Yuki; Tanaka, Ichiro; Niimura, Nobuo

    2008-01-01

    Hen egg-white lysozyme was crystallized over a wide pH range (2.5–8.0) and the quality of the crystals was characterized. Crystallization phase diagrams at pH 2.5, 6.0 and 7.5 were determined To observe the ionized status of the amino acid residues in proteins at different pH (protein pH titration in the crystalline state) by neutron diffraction, hen egg-white lysozyme was crystallized over a wide pH range (2.5–8.0). Crystallization phase diagrams at pH 2.5, 6.0 and 7.5 were determined. At pH < 4.5 the border between the metastable region and the nucleation region shifted to the left (lower precipitant concentration) in the phase diagram, and at pH > 4.5 the border shifted to the right (higher precipitant concentration). The qualities of these crystals were characterized using the Wilson plot method. The qualities of all crystals at different pH were more or less equivalent (B-factor values within 25–40). It is expected that neutron diffraction analysis of these crystals of different pH provides equivalent data in quality for discussions of protein pH titration in the crystalline state of hen egg-white lysozyme

  18. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

    International Nuclear Information System (INIS)

    Cuttitta, Christina M.; Ericson, Daniel L.; Scalia, Alexander; Roessler, Christian G.; Teplitsky, Ella; Joshi, Karan; Campos, Olven; Agarwal, Rakhi; Allaire, Marc; Orville, Allen M.; Sweet, Robert M.; Soares, Alexei S.

    2015-01-01

    An acoustic high-throughput screening method is described for harvesting protein crystals and combining the protein crystals with chemicals such as a fragment library. Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s −1 ) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening

  19. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

    Energy Technology Data Exchange (ETDEWEB)

    Cuttitta, Christina M. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); The City University of New York, 2800 Victory Boulevard, Staten Island, NY 10314 (United States); Ericson, Daniel L. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); University at Buffalo, SUNY, 12 Capen Hall, Buffalo, NY 14260 (United States); Scalia, Alexander [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Binghamton University, 4400 Vestal Parkway East, Binghamton, NY 11973-5000 (United States); Roessler, Christian G. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Teplitsky, Ella [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Stony Brook University, Stony Brook, NY 11794-5215 (United States); Joshi, Karan [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); PEC University of Technology, Chandigarh (India); Campos, Olven [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33414 (United States); Agarwal, Rakhi; Allaire, Marc [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Orville, Allen M. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Sweet, Robert M.; Soares, Alexei S., E-mail: soares@bnl.gov [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States)

    2015-01-01

    An acoustic high-throughput screening method is described for harvesting protein crystals and combining the protein crystals with chemicals such as a fragment library. Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s{sup −1}) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.

  20. Self-assembling peptides form nanodiscs that stabilize membrane proteins

    DEFF Research Database (Denmark)

    Midtgaard, Søren Roi; Pedersen, Martin Cramer; Kirkensgaard, Jacob Judas Kain

    2014-01-01

    -ray scattering (SAXS) and small-angle neutron scattering (SANS) supported by coarse-grained molecular dynamics simulations. The detailed structure of the discs was determined in unprecedented detail and it was found that they adopt a discoidal structure very similar to the ApoA1 based nanodiscs. We furthermore...... show that, like the ApoA1 and derived nanodiscs, these peptide discs can accommodate and stabilize a membrane protein. Finally, we exploit their dynamic properties and show that the 18A discs may be used for transferring membrane proteins and associated phospholipids directly and gently......New methods to handle membrane bound proteins, e.g. G-protein coupled receptors (GPCRs), are highly desirable. Recently, apoliprotein A1 (ApoA1) based lipoprotein particles have emerged as a new platform for studying membrane proteins, and it has been shown that they can self...

  1. Water polygons in high-resolution protein crystal structures.

    Science.gov (United States)

    Lee, Jonas; Kim, Sung-Hou

    2009-07-01

    We have analyzed the interstitial water (ISW) structures in 1500 protein crystal structures deposited in the Protein Data Bank that have greater than 1.5 A resolution with less than 90% sequence similarity with each other. We observed varieties of polygonal water structures composed of three to eight water molecules. These polygons may represent the time- and space-averaged structures of "stable" water oligomers present in liquid water, and their presence as well as relative population may be relevant in understanding physical properties of liquid water at a given temperature. On an average, 13% of ISWs are localized enough to be visible by X-ray diffraction. Of those, averages of 78% are water molecules in the first water layer on the protein surface. Of the localized ISWs beyond the first layer, almost half of them form water polygons such as trigons, tetragons, as well as expected pentagons, hexagons, higher polygons, partial dodecahedrons, and disordered networks. Most of the octagons and nanogons are formed by fusion of smaller polygons. The trigons are most commonly observed. We suggest that our observation provides an experimental basis for including these water polygon structures in correlating and predicting various water properties in liquid state.

  2. Crystal Structure of a Lipid G Protein-Coupled Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Hanson, Michael A; Roth, Christopher B; Jo, Euijung; Griffith, Mark T; Scott, Fiona L; Reinhart, Greg; Desale, Hans; Clemons, Bryan; Cahalan, Stuart M; Schuerer, Stephan C; Sanna, M Germana; Han, Gye Won; Kuhn, Peter; Rosen, Hugh; Stevens, Raymond C [Scripps; (Receptos)

    2012-03-01

    The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein-coupled sphingosine 1-phosphate receptors. Here, we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P1-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data, and modeling, provides a detailed view of the molecular recognition and requirement for hydrophobic volume that activates S1P1, resulting in the modulation of immune and stromal cell responses.

  3. Towards protein-crystal centering using second-harmonic generation (SHG) microscopy

    International Nuclear Information System (INIS)

    Kissick, David J.; Dettmar, Christopher M.; Becker, Michael; Mulichak, Anne M.; Cherezov, Vadim; Ginell, Stephan L.; Battaile, Kevin P.; Keefe, Lisa J.; Fischetti, Robert F.; Simpson, Garth J.

    2013-01-01

    The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-ray diffraction of protein crystals has been explored. The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-ray diffraction of protein crystals was explored. These studies included (i) comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and (ii) X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging. In studies using β 2 adrenergic receptor membrane-protein crystals prepared in lipidic mesophase, the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X-ray minibeam. SHG imaging was found to provide about 2 µm spatial resolution and shorter image-acquisition times. The general insensitivity of SHG images to optical scatter enabled the reliable identification of microcrystals within opaque cryocooled lipidic mesophases that were not identified by conventional bright-field imaging. The potential impact of extended exposure of protein crystals to five times a typical imaging dose from an ultrafast laser source was also assessed. Measurements of myoglobin and thaumatin crystals resulted in no statistically significant differences between structures obtained from diffraction data acquired from exposed and unexposed regions of single crystals. Practical constraints for integrating SHG imaging into an active beamline for routine automated crystal centering are discussed

  4. Thermodynamic effects of proline introduction on protein stability.

    Science.gov (United States)

    Prajapati, Ravindra Singh; Das, Mili; Sreeramulu, Sridhar; Sirajuddin, Minhajuddin; Srinivasan, Sankaranarayanan; Krishnamurthy, Vaishnavi; Ranjani, Ranganathan; Ramakrishnan, C; Varadarajan, Raghavan

    2007-02-01

    The amino acid Pro is more rigid than other naturally occurring amino acids and, in proteins, lacks an amide hydrogen. To understand the structural and thermodynamic effects of Pro substitutions, it was introduced at 13 different positions in four different proteins, leucine-isoleucine-valine binding protein, maltose binding protein, ribose binding protein, and thioredoxin. Three of the maltose binding protein mutants were characterized by X-ray crystallography to confirm that no structural changes had occurred upon mutation. In the remaining cases, fluorescence and CD spectroscopy were used to show the absence of structural change. Stabilities of wild type and mutant proteins were characterized by chemical denaturation at neutral pH and by differential scanning calorimetry as a function of pH. The mutants did not show enhanced stability with respect to chemical denaturation at room temperature. However, 6 of the 13 single mutants showed a small but significant increase in the free energy of thermal unfolding in the range of 0.3-2.4 kcal/mol, 2 mutants showed no change, and 5 were destabilized. In five of the six cases, the stabilization was because of reduced entropy of unfolding. However, the magnitude of the reduction in entropy of unfolding was typically several fold larger than the theoretical estimate of -4 cal K(-1) mol(-1) derived from the relative areas in the Ramachandran map accessible to Pro and Ala residues, respectively. Two double mutants were constructed. In both cases, the effects of the single mutations on the free energy of thermal unfolding were nonadditive. Copyright 2006 Wiley-Liss, Inc.

  5. Approach for growth of high-quality and large protein crystals

    Energy Technology Data Exchange (ETDEWEB)

    Matsumura, Hiroyoshi, E-mail: matsumura@chem.eng.osaka-u.ac.jp [Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871 (Japan); JST (Japan); SOSHO Inc., Osaka 541-0053 (Japan); Sugiyama, Shigeru; Hirose, Mika; Kakinouchi, Keisuke; Maruyama, Mihoko; Murai, Ryota [Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871 (Japan); JST (Japan); Adachi, Hiroaki; Takano, Kazufumi [Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871 (Japan); JST (Japan); SOSHO Inc., Osaka 541-0053 (Japan); Murakami, Satoshi [JST (Japan); SOSHO Inc., Osaka 541-0053 (Japan); Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan); Mori, Yusuke; Inoue, Tsuyoshi [Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871 (Japan); JST (Japan); SOSHO Inc., Osaka 541-0053 (Japan)

    2011-01-01

    Three crystallization methods, including crystallization in the presence of a semi-solid agarose gel, top-seeded solution growth (TSSG) and a large-scale hanging-drop method, have previously been presented. In this study, crystallization has been further evaluated in the presence of a semi-solid agarose gel by crystallizing additional proteins. A novel crystallization method combining TSSG and the large-scale hanging-drop method has also been developed. Three crystallization methods for growing large high-quality protein crystals, i.e. crystallization in the presence of a semi-solid agarose gel, top-seeded solution growth (TSSG) and a large-scale hanging-drop method, have previously been presented. In this study the effectiveness of crystallization in the presence of a semi-solid agarose gel has been further evaluated by crystallizing additional proteins in the presence of 2.0% (w/v) agarose gel, resulting in complete gelification with high mechanical strength. In TSSG the seed crystals are hung by a seed holder protruding from the top of the growth vessel to prevent polycrystallization. In the large-scale hanging-drop method, a cut pipette tip was used to maintain large-scale droplets consisting of protein–precipitant solution. Here a novel crystallization method that combines TSSG and the large-scale hanging-drop method is reported. A large and single crystal of lysozyme was obtained by this method.

  6. Three-Dimentional Structures of Autophosphorylation Complexes in Crystals of Protein Kinases

    KAUST Repository

    Dumbrack, Roland

    2016-01-01

    Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Several autophosphorylation complexes have been identified in crystals of protein kinases, with a known serine, threonine, or tyrosine

  7. The effect of exchange-correlation on change and stability of crystal structure

    International Nuclear Information System (INIS)

    Yazdani, A.; Niazi, M.; Alimardan, V.

    2007-01-01

    Since exchange interaction energy has effect on band structure via polarization of spin of free electron, then can directly effects formation crystal structure. Therefore exchange-correlation is able to have an effect on determination of crystal structure or its change and stability. This energy is subject to fluctuation range of electrons between conduction band and valance band or density of electrons which due to increase the entropy of system, via Gibss Energy .We investigated these factors: 1) Size of ions 2) Density of States 3) Range of inter atomic and pair-potential.

  8. Graphene as a protein crystal mounting material to reduce background scatter.

    Science.gov (United States)

    Wierman, Jennifer L; Alden, Jonathan S; Kim, Chae Un; McEuen, Paul L; Gruner, Sol M

    2013-10-01

    The overall signal-to-noise ratio per unit dose for X-ray diffraction data from protein crystals can be improved by reducing the mass and density of all material surrounding the crystals. This article demonstrates a path towards the practical ultimate in background reduction by use of atomically thin graphene sheets as a crystal mounting platform for protein crystals. The results show the potential for graphene in protein crystallography and other cases where X-ray scatter from the mounting material must be reduced and specimen dehydration prevented, such as in coherent X-ray diffraction imaging of microscopic objects.

  9. Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.

    Science.gov (United States)

    Johnson, Jennifer L; Entzminger, Kevin C; Hyun, Jeongmin; Kalyoncu, Sibel; Heaner, David P; Morales, Ivan A; Sheppard, Aly; Gumbart, James C; Maynard, Jennifer A; Lieberman, Raquel L

    2015-04-01

    Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.

  10. Expression, purification and crystallization of the catalytic subunit of protein kinase CK2 from Zea mays

    DEFF Research Database (Denmark)

    Guerra, B; Niefind, K; Pinna, L A

    1998-01-01

    The catalytic (alpha) subunit of protein kinase CK2 (CK2alpha) was originally cloned and overexpressed in the Escherichia coli strain pT7-7/BL21(DE3). The protein has been purified to homogeneity and crystallized. The crystals belong to the monoclinic space group C2, they have unit-cell parameter...

  11. USING BIOPOLYMERS TO STABILIZE THE PROTEIN OXYGEN FOAM

    Directory of Open Access Journals (Sweden)

    N. V. Nepovinnyh

    2013-01-01

    Full Text Available The cottage cheese whey as an oxygen cocktail foaming base and natural juices as a flavoring ingredient are analyzed. The lifetime of foam generated by the serum proteins is not long: foam falls off rapidly; because from the foam liquid is released (syneresis. The effects of plant polysaccharides on the stabilization of the protein foam oxygen cocktail is studied. It was shown that the use of plant polysaccharides (guar gum, high methoxyl citrus pectin, locust been gum prolong the life of the foam up to 20 times, compared with conventional blowing agents. It was found that oxygen foam properties depend on the molecular weight of guar gum.

  12. Direct measurements of protein-stabilized gold nanoparticle interactions.

    Science.gov (United States)

    Eichmann, Shannon L; Bevan, Michael A

    2010-09-21

    We report integrated video and total internal reflection microscopy measurements of protein stabilized 110 nm Au nanoparticles confined in 280 nm gaps in physiological media. Measured potential energy profiles display quantitative agreement with Brownian dynamic simulations that include hydrodynamic interactions and camera exposure time and noise effects. Our results demonstrate agreement between measured nonspecific van der Waals and adsorbed protein interactions with theoretical potentials. Confined, lateral nanoparticle diffusivity measurements also display excellent agreement with predictions. These findings provide a basis to interrogate specific biomacromolecular interactions in similar experimental configurations and to design future improved measurement methods.

  13. Mechanism of Stabilization of Labile Compounds by Silk Fibroin Proteins

    Science.gov (United States)

    2017-04-05

    saliva, or urine , and their collection and storage is critical to obtain reliable results. Without proper temperature regulation protein biomarkers in... samples for long-term ambient storage and subsequent on-demand recovery and laboratory analysis. Air dried silks provide a protective barrier that...silk in the stabilization of a range of different analytes, including entrapment, storage and recovery. Here, we successfully used silk fibroin as a

  14. Crystallization and preliminary crystallographic analysis of merohedrally twinned crystals of MJ0729, a CBS-domain protein from Methanococcus jannaschii

    International Nuclear Information System (INIS)

    Fernández-Millán, Pablo; Kortazar, Danel; Lucas, María; Martínez-Chantar, María Luz; Astigarraga, Egoitz; Fernández, José Andrés; Sabas, Olatz; Albert, Armando; Mato, Jose M.; Martínez-Cruz, Luis Alfonso

    2008-01-01

    Trigonal crystals of MJ0729 showing different degrees of merohedral twinning that may vary from perfect hemihedral twinning to perfect tetartohedral twinning were obtained upon slight variation of the pH. CBS domains are small protein motifs, usually associated in tandem, that are implicated in binding to adenosyl groups. Several genetic diseases in humans have been associated with mutations in CBS sequences, which has made them very promising targets for rational drug design. Trigonal crystals of the CBS-domain protein MJ0729 from Methanococcus jannaschii were grown by the vapour-diffusion method at acidic pH. Preliminary analysis of nine X-ray diffraction data sets using Yeates statistics and Britton plots showed that slight variation in the pH as well as in the buffer used in the crystallization experiments led to crystals with different degrees of merohedral twinning that may vary from perfect hemihedral twinning to perfect tetartohedral twinning

  15. Non-isothermal Crystallization, Thermal Stability, and Mechanical Performance of Poly(L-lactic acid/Barium Phenylphosphonate Systems

    Directory of Open Access Journals (Sweden)

    Cai Yan-Hua

    2017-11-01

    Full Text Available The introduction of a nucleating agent in semi-crystalline polymers is a frequently utilized way to improve the crystallization performance, and the use of a nucleating agent has a very great effect on the performance of the polymer in other areas including thermal stability and mechanical properties. In this investigation, barium phenylphosphonate (BaP was prepared as a crystallization accelerator for Poly(L-lactic acid (PLLA, and the non-isothermal crystallization behavior, thermal stability, and mechanical properties of PLLA modified by BaP were investigated using differential scanning calorimetry (DSC, X-ray diffraction (XRD, thermogravimetric analysis (TGA, and electronic tensile testing. Non-isothermal crystallization analysis showed that the BaP could significantly accelerate the crystallization of PLLA, and the non-isothermal crystallization peak shifted to a higher temperature with increasing concentration of BaP, however, the corresponding crystallization peak became wider. XRD results after non-isothermal crystallization confirmed the non-isothermal crystallization DSC results. Additionally, the addition of BaP did not change the crystal form of PLLA. A comparative study on thermal stability indicated that BaP decreased the onset decomposition temperature of PLLA, resulting from the formation of more tiny and imperfect crystals. Whereas the influence of BaP on the thermal decomposition profile of PLLA was negligible. In terms of mechanical properties, the tensile strength and elastic modulus of PLLA/BaP increased compared to the virgin PLLA, unfortunately, the elongation at break decreased.

  16. An Overview of Hardware for Protein Crystallization in a Magnetic Field

    Directory of Open Access Journals (Sweden)

    Er-Kai Yan

    2016-11-01

    Full Text Available Protein crystallization under a magnetic field is an interesting research topic because a magnetic field may provide a special environment to acquire improved quality protein crystals. Because high-quality protein crystals are very useful in high-resolution structure determination using diffraction techniques (X-ray, neutron, and electron diffraction, research using magnetic fields in protein crystallization has attracted substantial interest; some studies have been performed in the past two decades. In this research field, the hardware is especially essential for successful studies because the environment is special and the design and utilization of the research apparatus in such an environment requires special considerations related to the magnetic field. This paper reviews the hardware for protein crystallization (including the magnet systems and the apparatus designed for use in a magnetic field and progress in this area. Future prospects in this field will also be discussed.

  17. Numerical computation of the linear stability of the diffusion model for crystal growth simulation

    Energy Technology Data Exchange (ETDEWEB)

    Yang, C.; Sorensen, D.C. [Rice Univ., Houston, TX (United States); Meiron, D.I.; Wedeman, B. [California Institute of Technology, Pasadena, CA (United States)

    1996-12-31

    We consider a computational scheme for determining the linear stability of a diffusion model arising from the simulation of crystal growth. The process of a needle crystal solidifying into some undercooled liquid can be described by the dual diffusion equations with appropriate initial and boundary conditions. Here U{sub t} and U{sub a} denote the temperature of the liquid and solid respectively, and {alpha} represents the thermal diffusivity. At the solid-liquid interface, the motion of the interface denoted by r and the temperature field are related by the conservation relation where n is the unit outward pointing normal to the interface. A basic stationary solution to this free boundary problem can be obtained by writing the equations of motion in a moving frame and transforming the problem to parabolic coordinates. This is known as the Ivantsov parabola solution. Linear stability theory applied to this stationary solution gives rise to an eigenvalue problem of the form.

  18. Crystallization behaviour and thermal stability of two aluminium-based metallic glass powder materials

    Energy Technology Data Exchange (ETDEWEB)

    Li, X.P.; Yan, M. [University of Queensland, School of Mechanical and Mining Engineering, ARC Centre of Excellence for Design in Light Metals, Brisbane, QLD 4072 (Australia); Yang, B.J. [Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016 (China); Wang, J.Q., E-mail: jqwang@imr.ac.cn [Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016 (China); Schaffer, G.B. [University of Queensland, School of Mechanical and Mining Engineering, ARC Centre of Excellence for Design in Light Metals, Brisbane, QLD 4072 (Australia); Qian, M., E-mail: ma.qian@uq.edu.au [University of Queensland, School of Mechanical and Mining Engineering, ARC Centre of Excellence for Design in Light Metals, Brisbane, QLD 4072 (Australia)

    2011-12-15

    Highlights: Black-Right-Pointing-Pointer The crystallization paths and products of Al{sub 86}Ni{sub 7}Y{sub 4.5}Co{sub 1}La{sub 1.5} powder have been identified. Black-Right-Pointing-Pointer The thermal stability of Al{sub 86}Ni{sub 7}Y{sub 4.5}Co{sub 1}La{sub 1.5} powder has been assessed. Black-Right-Pointing-Pointer The Al{sub 86}Ni{sub 7}Y{sub 4.5}Co{sub 1}La{sub 1.5} powder shows a wide processing window of 75 K. Black-Right-Pointing-Pointer The powder has the potential to be consolidated into thick BMG components based on the findings. Black-Right-Pointing-Pointer The Al{sub 85}Ni{sub 5}Y{sub 6}Co{sub 2}Fe{sub 2} powder shows similar characteristics but inferior thermal stability. - Abstract: The crystallization behaviour and thermal stability of two Al-based metallic glass powder materials, Al{sub 85}Ni{sub 5}Y{sub 6}Co{sub 2}Fe{sub 2} and Al{sub 86}Ni{sub 6}Y{sub 4.5}Co{sub 2}La{sub 1.5}, have been investigated using differential scanning calorimetry (DSC), X-ray diffraction (XRD) and electron microscopy. Both alloy powders show a distinct three-stage crystallization process with a similar gap of {approx}75 K between the onset crystallization temperature (T{sub x}) and the second crystallization temperature. Crystallization occurs by the precipitation and growth of fcc-Al, without intermetallic formation. The apparent activation energy for each stage of crystallization was determined from DSC analyses and the phases resulting from each crystallization stage were identified by XRD and electron microscopy. The critical cooling rate for each alloy powder was calculated from the DSC data. These results are necessary to inform the consolidation of amorphous powder particles of Al{sub 85}Ni{sub 5}Y{sub 6}Co{sub 2}Fe{sub 2} or Al{sub 86}Ni{sub 6}Y{sub 4.5}Co{sub 2}La{sub 1.5} into thick (>1 mm) metallic glass components.

  19. Salvage and storage of infectious disease protein targets in the SSGCID high-throughput crystallization pathway using microfluidics

    International Nuclear Information System (INIS)

    Christensen, Jeff; Gerdts, Cory J.; Clifton, Mathew C.; Stewart, Lance

    2011-01-01

    SSGCID protein crystals were salvaged and stored using the MPCS Plug Maker and CrystalCards when high-throughput traditional sitting-drop vapor diffusion initially failed. The MPCS Plug Maker is a microcapillary-based protein-crystallization system for generating diffraction-ready crystals from nanovolumes of protein. Crystallization screening using the Plug Maker was used as a salvage pathway for proteins that failed to crystallize during the initial observation period using the traditional sitting-drop vapor-diffusion method. Furthermore, the CrystalCards used to store the crystallization experiments set up by the Plug Maker are shown be a viable container for long-term storage of protein crystals without a discernable loss of diffraction quality with time. Use of the Plug Maker with SSGCID proteins is demonstrated to be an effective crystal-salvage and storage method

  20. Electron paramagnetic resonance study of Ce doped partially stabilized ZrO2 crystals

    Directory of Open Access Journals (Sweden)

    Mikhail А. Borik

    2017-09-01

    Full Text Available ZrO2 (PSZ solid solutions crystals stabilized with yttrium and cerium oxides have been studied using electron paramagnetic resonance (EPR in the X and Q ranges. Zr3+ have been observed centers in the as-annealed ZrO2 crystals stabilized only by yttrium oxide (2.8 mol% Y2O3. Another type of paramagnetic-O-centers appear as a result of CeO2 addition to ZrO2 crystals along with yttrium oxide. To estimate the concentration of Ce3+ ions in PZS crystals, we recorded the EPR spectra in the presence of a reference at 7 K. Paramagnetic Ce3+ ions have been identified and their relative amount in the PSZ crystals before and after high-temperature heat treatment has been assessed. Annealing in air leads decreases the concentration of Ce3+ ions for all the test compositions and changes the color of the crystals from red to white. After annealing of the sample 2.0Y0.8Ce3Zr, the amount of paramagnetic Ce3+ ions decreased approximately twofold. Paramagnetic centers from Ce3+ have not been detected in the specimen with a low cerium content of 0.1 mol% after annealing which indicates the complete transition of Ce3+ to the Ce4+ state. We show that the forming cerium paramagnetic centers are bound by strong exchange interactions. No angular dependence of the EPR lines of the paramagnetic Ce3+ cations on the applied external magnetic field has been observed. Probable origin of the absence of angular dependence is that the impurity rare-earth ions are located close to one another, forming impurity clusters with an effective spin of Seff=1/2.

  1. Green Fluorescent Protein as a Model for Protein Crystal Growth Studies

    Science.gov (United States)

    Agena, Sabine; Smith, Lori; Karr, Laurel; Pusey, Marc

    1998-01-01

    Green fluorescent protein (GFP) from jellyfish Aequorea Victoria has become a popular marker for e.g. mutagenesis work. Its fluorescent property, which originates from a chromophore located in the center of the molecule, makes it widely applicable as a research too]. GFP clones have been produced with a variety of spectral properties, such as blue and yellow emitting species. The protein is a single chain of molecular weight 27 kDa and its structure has been determined at 1.9 Angstrom resolution. The combination of GFP's fluorescent property, the knowledge of its several crystallization conditions, and its increasing use in biophysical and biochemical studies, all led us to consider it as a model material for macromolecular crystal growth studies. Initial preparations of GFP were from E.coli with yields of approximately 5 mg/L of culture media. Current yields are now in the 50 - 120 mg/L range, and we hope to further increase this by expression of the GFP gene in the Pichia system. The results of these efforts and of preliminary crystal growth studies will be presented.

  2. Cyclic olefin homopolymer-based microfluidics for protein crystallization and in situ X-ray diffraction

    International Nuclear Information System (INIS)

    Emamzadah, Soheila; Petty, Tom J.; De Almeida, Victor; Nishimura, Taisuke; Joly, Jacques; Ferrer, Jean-Luc; Halazonetis, Thanos D.

    2009-01-01

    A cyclic olefin homopolymer-based microfluidics system has been established for protein crystallization and in situ X-ray diffraction. Microfluidics is a promising technology for the rapid identification of protein crystallization conditions. However, most of the existing systems utilize silicone elastomers as the chip material which, despite its many benefits, is highly permeable to water vapour. This limits the time available for protein crystallization to less than a week. Here, the use of a cyclic olefin homopolymer-based microfluidics system for protein crystallization and in situ X-ray diffraction is described. Liquid handling in this system is performed in 2 mm thin transparent cards which contain 500 chambers, each with a volume of 320 nl. Microbatch, vapour-diffusion and free-interface diffusion protocols for protein crystallization were implemented and crystals were obtained of a number of proteins, including chicken lysozyme, bovine trypsin, a human p53 protein containing both the DNA-binding and oligomerization domains bound to DNA and a functionally important domain of Arabidopsis Morpheus’ molecule 1 (MOM1). The latter two polypeptides have not been crystallized previously. For X-ray diffraction analysis, either the cards were opened to allow mounting of the crystals on loops or the crystals were exposed to X-rays in situ. For lysozyme, an entire X-ray diffraction data set at 1.5 Å resolution was collected without removing the crystal from the card. Thus, cyclic olefin homopolymer-based microfluidics systems have the potential to further automate protein crystallization and structural genomics efforts

  3. Cleaved thioredoxin fusion protein enables the crystallization of poorly soluble ERα in complex with synthetic ligands

    International Nuclear Information System (INIS)

    Cura, Vincent; Gangloff, Monique; Eiler, Sylvia; Moras, Dino; Ruff, Marc

    2007-01-01

    A new crystallization strategy: the presence of cleaved thioredoxin fusion is critical for crystallization of the estrogen nuclear receptor ligand binding domain in complex with synthetic ligands. This novel technique should be regarded as an interesting alternative for crystallization of difficult proteins. The ligand-binding domain (LBD) of human oestrogen receptor α was produced in Escherichia coli as a cleavable thioredoxin (Trx) fusion in order to improve solubility. Crystallization trials with either cleaved and purified LBD or with the purified fusion protein both failed to produce crystals. In another attempt, Trx was not removed from the LBD after endoproteolytic cleavage and its presence promoted nucleation and subsequent crystal growth, which allowed the structure determination of two different LBD–ligand–coactivator peptide complexes at 2.3 Å resolution. This technique is likely to be applicable to other low-solubility proteins

  4. Stability of halophilic proteins: from dipeptide attributes to discrimination classifier.

    Science.gov (United States)

    Zhang, Guangya; Huihua, Ge; Yi, Lin

    2013-02-01

    To investigate the molecular features responsible for protein halophilicity is of great significance for understanding the structure basis of protein halo-stability and would help to develop a practical strategy for designing halophilic proteins. In this work, we have systematically analyzed the dipeptide composition of the halophilic and non-halophilic protein sequences. We observed the halophilic proteins contained more DA, RA, AD, RR, AP, DD, PD, EA, VG and DV at the expense of LK, IL, II, IA, KK, IS, KA, GK, RK and AI. We identified some macromolecular signatures of halo-adaptation, and thought the dipeptide composition might contain more information than amino acid composition. Based on the dipeptide composition, we have developed a machine learning method for classifying halophilic and non-halophilic proteins for the first time. The accuracy of our method for the training dataset was 100.0%, and for the 10-fold cross-validation was 93.1%. We also discussed the influence of some specific dipeptides on prediction accuracy. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Comparative analysis of amino acids and amino-acid derivatives in protein crystallization

    International Nuclear Information System (INIS)

    Ito, Len; Shiraki, Kentaro; Yamaguchi, Hiroshi

    2010-01-01

    New types of aggregation suppressors, such as amino acids and their derivatives, were focused on as fourth-component additives. Data were obtained that indicated that the additives promote protein crystallization. Optimal conditions for protein crystallization are difficult to determine because proteins tend to aggregate in saturated solutions. This study comprehensively evaluates amino acids and amino-acid derivatives as additives for crystallization. This fourth component of the solution increases the probability of crystallization of hen egg-white lysozyme in various precipitants owing to a decrease in aggregation. These results suggest that the addition of certain types of amino acids and amino-acid derivatives, such as Arg, Lys and esterified and amidated amino acids, is a simple method of improving the success rate of protein crystallization

  6. Modification of calcite crystal growth by abalone shell proteins: an atomic force microscope study.

    OpenAIRE

    Walters, D A; Smith, B L; Belcher, A M; Paloczi, G T; Stucky, G D; Morse, D E; Hansma, P K

    1997-01-01

    A family of soluble proteins from the shell of Haliotis rufescens was introduced over a growing calcite crystal being scanned in situ by an atomic force microscope (AFM). Atomic step edges on the crystal surface were altered in shape and speed of growth by the proteins. Proteins attached nonuniformly to the surface, indicating different interactions with crystallographically different step edges. The observed changes were consistent with the habit modification induced by this family of protei...

  7. Effective stabilization of CLA by microencapsulation in pea protein.

    Science.gov (United States)

    Costa, A M M; Nunes, J C; Lima, B N B; Pedrosa, C; Calado, V; Torres, A G; Pierucci, A P T R

    2015-02-01

    CLA was microencapsulated by spray drying in ten varied wall systems (WS) consisting of pea protein isolate or pea protein concentrate (PPC) alone at varied core:WS ratios (1:2; 1:3 and 1:4), or blended with maltodextrin (M) and carboxymethylcellulose at a pea protein:carbohydrate ratio of 3:1. The physical-chemical properties of the CLA microparticles were characterised by core retention, microencapsulation efficiency (ME), particle size and moisture. CLA:M:PPC (1:1:3) showed the most promising results, thus we evaluated the effect of M addition in the WS on other physical-chemical characteristics and oxidative stability (CLA isomer profile, quantification of CLA and volatile compounds by SPME coupled with CG-MS) during two months of storage at room temperature, CLA:PPC (1:4) was selected for comparisons. CLA:M:PPC (1:1:3) microparticles demonstrated better morphology, solubility, dispersibility and higher glass-transition temperature values. M addition did not influence the oxidative stability of CLA, however its presence improved physical-chemical characteristics necessary for food applications. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Structure of anti-FLAG M2 Fab domain and its use in the stabilization of engineered membrane proteins

    International Nuclear Information System (INIS)

    Roosild, Tarmo P.; Castronovo, Samantha; Choe, Senyon

    2006-01-01

    The X-ray crystallographic analysis of anti-FLAG M2 Fab is reported and the implications of the structure on FLAG epitope binding are described as a first step in the development of a tool for the structural and biophysical study of membrane proteins. The inherent difficulties of stabilizing detergent-solubilized integral membrane proteins for biophysical or structural analysis demand the development of new methodologies to improve success rates. One proven strategy is the use of antibody fragments to increase the ‘soluble’ portion of any membrane protein, but this approach is limited by the difficulties and expense associated with producing monoclonal antibodies to an appropriate exposed epitope on the target protein. Here, the stabilization of a detergent-solubilized K + channel protein, KvPae, by engineering a FLAG-binding epitope into a known loop region of the protein and creating a complex with Fab fragments from commercially available anti-FLAG M2 monoclonal antibodies is reported. Although well diffracting crystals of the complex have not yet been obtained, during the course of crystallization trials the structure of the anti-FLAG M2 Fab domain was solved to 1.86 Å resolution. This structure, which should aid future structure-determination efforts using this approach by facilitating molecular-replacement phasing, reveals that the binding pocket appears to be specific only for the first four amino acids of the traditional FLAG epitope, namely DYKD. Thus, the use of antibody fragments for improving the stability of target proteins can be rapidly applied to the study of membrane-protein structure by placing the short DKYD motif within a predicted peripheral loop of that protein and utilizing commercially available anti-FLAG M2 antibody fragments

  9. Development of high-performance X-ray transparent crystallization plates for in situ protein crystal screening and analysis

    Energy Technology Data Exchange (ETDEWEB)

    Soliman, Ahmed S. M.; Warkentin, Matthew [Cornell University, Ithaca, New York (United States); Apker, Benjamin [MiTeGen LLC, Ithaca, New York (United States); Thorne, Robert E., E-mail: ret6@cornell.edu [Cornell University, Ithaca, New York (United States); MiTeGen LLC, Ithaca, New York (United States)

    2011-07-01

    An optically, UV and X-ray transparent crystallization plate suitable for in situ analysis has been developed. The plate uses contact line pinning rather than wells to confine the liquids. X-ray transparent crystallization plates based upon a novel drop-pinning technology provide a flexible, simple and inexpensive approach to protein crystallization and screening. The plates consist of open cells sealed top and bottom by thin optically, UV and X-ray transparent films. The plates do not need wells or depressions to contain liquids. Instead, protein drops and reservoir solution are held in place by rings with micrometre dimensions that are patterned onto the bottom film. These rings strongly pin the liquid contact lines, thereby improving drop shape and position uniformity, and thus crystallization reproducibility, and simplifying automated image analysis of drop contents. The same rings effectively pin solutions containing salts, proteins, cryoprotectants, oils, alcohols and detergents. Strong pinning by rings allows the plates to be rotated without liquid mixing to 90° for X-ray data collection or to be inverted for hanging-drop crystallization. The plates have the standard SBS format and are compatible with standard liquid-handling robots.

  10. Study of the effect of doping on the temperature stability of the optical properties of germanium single crystals

    Energy Technology Data Exchange (ETDEWEB)

    Podkopaev, O. I. [Joint-Stock Company “Germanium” (Russian Federation); Shimanskiy, A. F., E-mail: shimanaf@mail.ru [Siberian Federal University (Russian Federation); Kopytkova, S. A.; Filatov, R. A. [Joint-Stock Company “Germanium” (Russian Federation); Golubovskaya, N. O. [Siberian Federal University (Russian Federation)

    2016-10-15

    The effect of doping on the optical transmittance of germanium single crystals is studied by infrared Fourier spectroscopy. It is established that the introduction of silicon and tellurium additives into germanium doped with antimony provides a means for improving the temperature stability of the optical properties of the crystals.

  11. Study of the effect of doping on the temperature stability of the optical properties of germanium single crystals

    International Nuclear Information System (INIS)

    Podkopaev, O. I.; Shimanskiy, A. F.; Kopytkova, S. A.; Filatov, R. A.; Golubovskaya, N. O.

    2016-01-01

    The effect of doping on the optical transmittance of germanium single crystals is studied by infrared Fourier spectroscopy. It is established that the introduction of silicon and tellurium additives into germanium doped with antimony provides a means for improving the temperature stability of the optical properties of the crystals.

  12. Expression, purification and crystallization of the Cmi immunity protein from Escherichia coli

    International Nuclear Information System (INIS)

    Römer, Christin; Patzer, Silke I.; Albrecht, Reinhard; Zeth, Kornelius; Braun, Volkmar

    2011-01-01

    The colicin M immunity protein Cmi protects E. coli cells against killing by colicin M. The Cmi protein was produced for structure determination and crystals were obtained which diffracted to high resolution. Many bacteria kill related bacteria by secretion of bacteriocins. In Escherichia coli, the colicin M protein kills E. coli after uptake into the periplasm. Self-protection from destruction is provided by the co-expressed immunity protein. The colicin M immunity protein (Cmi) was cloned, overexpressed and purified to homogeneity. The correct fold of purified Cmi was analyzed by activity tests and circular-dichroism spectroscopy. Crystallization trials yielded crystals, one of which diffracted to a resolution of 1.9 Å in the orthorhombic space group C222 1 . The crystal packing, with unit-cell parameters a = 66.02, b = 83.47, c = 38.30 Å, indicated the presence of one monomer in the asymmetric unit with a solvent content of 53%

  13. Quantifying why urea is a protein denaturant, whereas glycine betaine is a protein stabilizer

    Science.gov (United States)

    Guinn, Emily J.; Pegram, Laurel M.; Capp, Michael W.; Pollock, Michelle N.; Record, M. Thomas

    2011-01-01

    To explain the large, opposite effects of urea and glycine betaine (GB) on stability of folded proteins and protein complexes, we quantify and interpret preferential interactions of urea with 45 model compounds displaying protein functional groups and compare with a previous analysis of GB. This information is needed to use urea as a probe of coupled folding in protein processes and to tune molecular dynamics force fields. Preferential interactions between urea and model compounds relative to their interactions with water are determined by osmometry or solubility and dissected using a unique coarse-grained analysis to obtain interaction potentials quantifying the interaction of urea with each significant type of protein surface (aliphatic, aromatic hydrocarbon (C); polar and charged N and O). Microscopic local-bulk partition coefficients Kp for the accumulation or exclusion of urea in the water of hydration of these surfaces relative to bulk water are obtained. Kp values reveal that urea accumulates moderately at amide O and weakly at aliphatic C, whereas GB is excluded from both. These results provide both thermodynamic and molecular explanations for the opposite effects of urea and glycine betaine on protein stability, as well as deductions about strengths of amide NH—amide O and amide NH—amide N hydrogen bonds relative to hydrogen bonds to water. Interestingly, urea, like GB, is moderately accumulated at aromatic C surface. Urea m-values for protein folding and other protein processes are quantitatively interpreted and predicted using these urea interaction potentials or Kp values. PMID:21930943

  14. Enhancement of nucleation during hanging drop protein crystallization using HF treatment of cover glasses

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Yun-Zhu; Yin, Da-Chuan; Lu, Qin-Qin; Wang, Xi-Kai; Liu, Jun [Key Laboratory for Space Bioscience and Biotechnology, Faculty of Life Sciences, Northwestern Polytechnical University, Xi' an 710072, Shaanxi (China)

    2010-02-15

    We examined a simple approach, i.e., etching cover glasses using hydrofluoric acid (HF), to determine whether cover glass treatment enhances nucleation in hanging drop protein crystallization. Hen egg white lysozyme and proteinase K were used as the model proteins. We found that the treatment increased the success rate of crystallization. The results indicated that the simple treatment, which is easy to adopt without changing much in the hanging drop method, can be utilized as an alternative method to enhance protein crystallization screens (copyright 2010 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  15. Can radiation damage to protein crystals be reduced using small-molecule compounds?

    Energy Technology Data Exchange (ETDEWEB)

    Kmetko, Jan [Kenyon College, Gambier, OH 43022 (United States); Warkentin, Matthew; Englich, Ulrich; Thorne, Robert E., E-mail: ret6@cornell.edu [Cornell University, Ithaca, NY 14853 (United States); Kenyon College, Gambier, OH 43022 (United States)

    2011-10-01

    Free-radical scavengers that are known to be effective protectors of proteins in solution are found to increase global radiation damage to protein crystals. Protective mechanisms may become deleterious in the protein-dense environment of a crystal. Recent studies have defined a data-collection protocol and a metric that provide a robust measure of global radiation damage to protein crystals. Using this protocol and metric, 19 small-molecule compounds (introduced either by cocrystallization or soaking) were evaluated for their ability to protect lysozyme crystals from radiation damage. The compounds were selected based upon their ability to interact with radiolytic products (e.g. hydrated electrons, hydrogen, hydroxyl and perhydroxyl radicals) and/or their efficacy in protecting biological molecules from radiation damage in dilute aqueous solutions. At room temperature, 12 compounds had no effect and six had a sensitizing effect on global damage. Only one compound, sodium nitrate, appeared to extend crystal lifetimes, but not in all proteins and only by a factor of two or less. No compound provided protection at T = 100 K. Scavengers are ineffective in protecting protein crystals from global damage because a large fraction of primary X-ray-induced excitations are generated in and/or directly attack the protein and because the ratio of scavenger molecules to protein molecules is too small to provide appreciable competitive protection. The same reactivity that makes some scavengers effective radioprotectors in protein solutions may explain their sensitizing effect in the protein-dense environment of a crystal. A more productive focus for future efforts may be to identify and eliminate sensitizing compounds from crystallization solutions.

  16. Can radiation damage to protein crystals be reduced using small-molecule compounds?

    International Nuclear Information System (INIS)

    Kmetko, Jan; Warkentin, Matthew; Englich, Ulrich; Thorne, Robert E.

    2011-01-01

    Free-radical scavengers that are known to be effective protectors of proteins in solution are found to increase global radiation damage to protein crystals. Protective mechanisms may become deleterious in the protein-dense environment of a crystal. Recent studies have defined a data-collection protocol and a metric that provide a robust measure of global radiation damage to protein crystals. Using this protocol and metric, 19 small-molecule compounds (introduced either by cocrystallization or soaking) were evaluated for their ability to protect lysozyme crystals from radiation damage. The compounds were selected based upon their ability to interact with radiolytic products (e.g. hydrated electrons, hydrogen, hydroxyl and perhydroxyl radicals) and/or their efficacy in protecting biological molecules from radiation damage in dilute aqueous solutions. At room temperature, 12 compounds had no effect and six had a sensitizing effect on global damage. Only one compound, sodium nitrate, appeared to extend crystal lifetimes, but not in all proteins and only by a factor of two or less. No compound provided protection at T = 100 K. Scavengers are ineffective in protecting protein crystals from global damage because a large fraction of primary X-ray-induced excitations are generated in and/or directly attack the protein and because the ratio of scavenger molecules to protein molecules is too small to provide appreciable competitive protection. The same reactivity that makes some scavengers effective radioprotectors in protein solutions may explain their sensitizing effect in the protein-dense environment of a crystal. A more productive focus for future efforts may be to identify and eliminate sensitizing compounds from crystallization solutions

  17. Crystallization and preliminary X-ray analysis of the RAD protein from Antirrhinum majus

    International Nuclear Information System (INIS)

    Stevenson, Clare E. M.; Burton, Nicolas; Costa, Manuela; Nath, Utpal; Dixon, Ray A.; Coen, Enrico S.; Lawson, David M.

    2005-01-01

    An 8 kDa proteolytic fragment of the A. majus RADIALIS protein was crystallized and X-ray data were collected to 2 Å resolution. Crystals of the RADIALIS protein from Antirrhinum majus were grown by vapour diffusion after limited proteolysis. Mass spectrometry indicated that an 8 kDa fragment had been crystallized corresponding to the predicted MYB DNA-binding domain. X-ray data collected at room temperature were consistent with tetragonal symmetry, whereas data collected at 100 K using crystals cryoprotected by supplementing the mother liquor with ethylene glycol conformed to orthorhombic symmetry. It was subsequently shown that crystals soaked in cryoprotectants that were ‘osmolality-matched’ to the mother liquor retained tetragonal symmetry. Using these crystals, X-ray data were collected in-house to a maximum resolution of 2 Å

  18. New directions towards structure formation and stability of protein-rich foods from globular proteins

    NARCIS (Netherlands)

    Purwanti, N.; Goot, van der A.J.; Boom, R.M.; Vereijken, J.M.

    2010-01-01

    Concentrated protein-rich foods have strong potential to be developed in terms of health and well-being roles. Unfortunately, limitations in creating products with the rights texture and stability hinder the use of those products by consumers. Main reason is that the formation of micro- and

  19. Humidity control and hydrophilic glue coating applied to mounted protein crystals improves X-ray diffraction experiments

    Science.gov (United States)

    Baba, Seiki; Hoshino, Takeshi; Ito, Len; Kumasaka, Takashi

    2013-01-01

    Protein crystals are fragile, and it is sometimes difficult to find conditions suitable for handling and cryocooling the crystals before conducting X-ray diffraction experiments. To overcome this issue, a protein crystal-mounting method has been developed that involves a water-soluble polymer and controlled humid air that can adjust the moisture content of a mounted crystal. By coating crystals with polymer glue and exposing them to controlled humid air, the crystals were stable at room temperature and were cryocooled under optimized humidity. Moreover, the glue-coated crystals reproducibly showed gradual transformations of their lattice constants in response to a change in humidity; thus, using this method, a series of isomorphous crystals can be prepared. This technique is valuable when working on fragile protein crystals, including membrane proteins, and will also be useful for multi-crystal data collection. PMID:23999307

  20. Photoelectrochemical Stability and Alteration Products of n-Type Single-Crystal ZnO Photoanodes

    Directory of Open Access Journals (Sweden)

    I. E. Paulauskas

    2011-01-01

    Full Text Available The photoelectrochemical stability and surface-alteration characteristics of doped and undoped n-type ZnO single-crystal photoanode electrodes were investigated. The single-crystal ZnO photoanode properties were analyzed using current-voltage measurements plus spectral and time-dependent quantum-yield methods. These measurements revealed a distinct anodic peak and an accompanying cathodic surface degradation process at negative potentials. The features of this peak depended on time and the NaOH concentration in the electrolyte, but were independent of the presence of electrode illumination. Current measurements performed at the peak indicate that charging and discharging effects are apparently taking place at the semiconductor/electrolyte interface. This result is consistent with the significant reactive degradation that takes place on the ZnO single crystal photoanode surface and that ultimately leads to the reduction of the ZnO surface to Zn metal. The resulting Zn-metal reaction products create unusual, dendrite-like, surface alteration structural features that were analyzed using x-ray diffraction, energy-dispersive analysis, and scanning electron microscopy. ZnO doping methods were found to be effective in increasing the n-type character of the crystals. Higher doping levels result in smaller depletion widths and lower quantum yields, since the minority carrier diffusion lengths are very short in these materials.

  1. Enhancement of crystal homogeneity of protein crystals under application of an external alternating current electric field

    Energy Technology Data Exchange (ETDEWEB)

    Koizumi, H.; Uda, S.; Fujiwara, K.; Nozawa, J. [Institute for Materials Research, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, 980-8577 (Japan); Tachibana, M. [Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama, 236-0027 (Japan); Kojima, K. [Department of Education, Yokohama Soei University, 1 Miho-tyou, Midori-ku, Yokohama, 226-0015 (Japan)

    2014-10-06

    X-ray diffraction rocking-curve measurements were performed on tetragonal hen egg white (HEW) lysozyme crystals grown with and without the application of an external alternating current (AC) electric field. The crystal quality was assessed by the full width at half maximum (FWHM) value for each rocking curve. For two-dimensional maps of the FWHMs measured on the 440 and the 12 12 0 reflection, the crystal homogeneity was improved under application of an external electric field at 1 MHz, compared with that without. In particular, the significant improvement of the crystal homogeneity was observed for the 12 12 0 reflection.

  2. Roles of electrostatics and conformation in protein-crystal interactions.

    Directory of Open Access Journals (Sweden)

    Paul V Azzopardi

    2010-02-01

    Full Text Available In vitro studies have shown that the phosphoprotein osteopontin (OPN inhibits the nucleation and growth of hydroxyapatite (HA and other biominerals. In vivo, OPN is believed to prevent the calcification of soft tissues. However, the nature of the interaction between OPN and HA is not understood. In the computational part of the present study, we used molecular dynamics simulations to predict the adsorption of 19 peptides, each 16 amino acids long and collectively covering the entire sequence of OPN, to the {100} face of HA. This analysis showed that there is an inverse relationship between predicted strength of adsorption and peptide isoelectric point (P<0.0001. Analysis of the OPN sequence by PONDR (Predictor of Naturally Disordered Regions indicated that OPN sequences predicted to adsorb well to HA are highly disordered. In the experimental part of the study, we synthesized phosphorylated and non-phosphorylated peptides corresponding to OPN sequences 65-80 (pSHDHMDDDDDDDDDGD and 220-235 (pSHEpSTEQSDAIDpSAEK. In agreement with the PONDR analysis, these were shown by circular dichroism spectroscopy to be largely disordered. A constant-composition/seeded growth assay was used to assess the HA-inhibiting potencies of the synthetic peptides. The phosphorylated versions of OPN65-80 (IC(50 = 1.93 microg/ml and OPN220-235 (IC(50 = 1.48 microg/ml are potent inhibitors of HA growth, as is the nonphosphorylated version of OPN65-80 (IC(50 = 2.97 microg/ml; the nonphosphorylated version of OPN220-235 has no measurable inhibitory activity. These findings suggest that the adsorption of acidic proteins to Ca2+-rich crystal faces of biominerals is governed by electrostatics and is facilitated by conformational flexibility of the polypeptide chain.

  3. Room-temperature serial crystallography using a kinetically optimized microfluidic device for protein crystallization and on-chip X-ray diffraction

    Directory of Open Access Journals (Sweden)

    Michael Heymann

    2014-09-01

    Full Text Available An emulsion-based serial crystallographic technology has been developed, in which nanolitre-sized droplets of protein solution are encapsulated in oil and stabilized by surfactant. Once the first crystal in a drop is nucleated, the small volume generates a negative feedback mechanism that lowers the supersaturation. This mechanism is exploited to produce one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room-temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93% complete data set is obtained by merging single diffraction frames taken from different unoriented crystals. As proof of concept, the structure of glucose isomerase was solved to 2.1 Å, demonstrating the feasibility of high-throughput serial X-ray crystallography using synchrotron radiation.

  4. Towards protein-crystal centering using second-harmonic generation (SHG) microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Kissick, David J.; Dettmar, Christopher M. [Purdue University, West Lafayette, IN 47907 (United States); Becker, Michael [Argonne National Laboratory, Argonne, IL 60439 (United States); Mulichak, Anne M. [Hauptman–Woodward Medical Research Institute, Argonne, IL 60439 (United States); Cherezov, Vadim [The Scripps Research Institute, La Jolla, CA 92037 (United States); Ginell, Stephan L. [Argonne National Laboratory, Argonne, IL 60439 (United States); Battaile, Kevin P.; Keefe, Lisa J. [Hauptman–Woodward Medical Research Institute, Argonne, IL 60439 (United States); Fischetti, Robert F. [Argonne National Laboratory, Argonne, IL 60439 (United States); Simpson, Garth J., E-mail: gsimpson@purdue.edu [Purdue University, West Lafayette, IN 47907 (United States)

    2013-05-01

    The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-ray diffraction of protein crystals has been explored. The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-ray diffraction of protein crystals was explored. These studies included (i) comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and (ii) X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging. In studies using β{sub 2} adrenergic receptor membrane-protein crystals prepared in lipidic mesophase, the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X-ray minibeam. SHG imaging was found to provide about 2 µm spatial resolution and shorter image-acquisition times. The general insensitivity of SHG images to optical scatter enabled the reliable identification of microcrystals within opaque cryocooled lipidic mesophases that were not identified by conventional bright-field imaging. The potential impact of extended exposure of protein crystals to five times a typical imaging dose from an ultrafast laser source was also assessed. Measurements of myoglobin and thaumatin crystals resulted in no statistically significant differences between structures obtained from diffraction data acquired from exposed and unexposed regions of single crystals. Practical constraints for integrating SHG imaging into an active beamline for routine automated crystal centering are discussed.

  5. Microseed matrix screening for optimization in protein crystallization: what have we learned?

    Science.gov (United States)

    D'Arcy, Allan; Bergfors, Terese; Cowan-Jacob, Sandra W; Marsh, May

    2014-09-01

    Protein crystals obtained in initial screens typically require optimization before they are of X-ray diffraction quality. Seeding is one such optimization method. In classical seeding experiments, the seed crystals are put into new, albeit similar, conditions. The past decade has seen the emergence of an alternative seeding strategy: microseed matrix screening (MMS). In this strategy, the seed crystals are transferred into conditions unrelated to the seed source. Examples of MMS applications from in-house projects and the literature include the generation of multiple crystal forms and different space groups, better diffracting crystals and crystallization of previously uncrystallizable targets. MMS can be implemented robotically, making it a viable option for drug-discovery programs. In conclusion, MMS is a simple, time- and cost-efficient optimization method that is applicable to many recalcitrant crystallization problems.

  6. Monoolein lipid phases as incorporation and enrichment materials for membrane protein crystallization.

    Directory of Open Access Journals (Sweden)

    Ellen Wallace

    Full Text Available The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive

  7. The liquid protein phase in crystallization: a case study—intact immunoglobulins

    Science.gov (United States)

    Kuznetsov, Yurii G.; Malkin, Alexander J.; McPherson, Alexander

    2001-11-01

    A common observation by protein chemists has been the appearance, for many proteins in aqueous solutions, of oil like droplets, or in more extreme cases the formation of a second oil like phase. These may accompany the formation of precipitate in "salting out" or "salting in' procedures, but more commonly appear in place of any precipitate. Such phase separations also occur, with even greater frequency, in the presence of polymeric precipitants such as polyethyleneglycol (PEG). In general the appearance of a second liquid phase has been taken as indicative of protein aggregation, though an aggregate state distinctly different from that characteristic of amorphous precipitate. While the latter is thought to be composed of linear and branched assemblies, polymers of a sort, the oil phase suggests a more compact, three-dimensional, but fluid state. An important property of an alternate, fluid phase is that it can mediate transitions between other states, for example, between protein molecules free in solution and protein molecules immobilized in amorphous precipitate or crystals. The "liquid protein" phase can be readily observed in many crystallization experiments either prior to the appearance of visible crystals, or directly participating in the crystal growth process. In some cases the relationship between the liquid phase and developing crystals is intimate. Crystals grow directly from the liquid phase, or appear only after the visible formation of the liquid phase. We describe here our experience with a class of macromolecules, immunoglobulins, and particularly IDEC-151, an IgG specific for CD4 on human lymphocytes. This protein has been crystallized from a Jeffamine-LiSO 4 mother liquor and, its crystallization illustrates many of the features associated with the liquid protein, or protein rich phase.

  8. Crystallization of the A-Domain of the Mannitol Transport Protein Enzyme IImtl

    NARCIS (Netherlands)

    Lammers, Leidy A.; Dijkstra, Bauke W.; Weeghel, Rob P. van; Pas, Hendri H.; Robillard, George T.

    1992-01-01

    The A-domain of the mannitol transport protein enzyme IImtl from Escherichia coli (relative molecular mass 16,300) was crystallized, both at room temperature and 4°C, from 40% polyethylene glycol 6000 (pH 8.5 to 9.0) using the hanging-drop method of vapour diffusion. The crystals have the monoclinic

  9. Expression, purification, crystallization and preliminary crystallographic analysis of the proliferation-associated protein Ebp1

    International Nuclear Information System (INIS)

    Kowalinski, Eva; Bange, Gert; Wild, Klemens; Sinning, Irmgard

    2007-01-01

    Preliminary X-ray analysis of the proliferation-associated protein Ebp1 from Homo sapiens is provided. ErbB-3-binding protein 1 (Ebp1) is a member of the family of proliferation-associated 2G4 proteins (PA2G4s) and plays a role in cellular growth and differentiation. Ligand-induced activation of the transmembrane receptor ErbB3 leads to dissociation of Ebp1 from the receptor in a phosphorylation-dependent manner. The non-associated protein is involved in transcriptional and translational regulation in the cell. Here, the overexpression, purification, crystallization and preliminary crystallographic studies of Ebp1 from Homo sapiens are reported. Initially observed crystals were improved by serial seeding to single crystals suitable for data collection. The optimized crystals belong to the tetragonal space group P4 1 2 1 2 or P4 3 2 1 2 and diffracted to a resolution of 1.6 Å

  10. Crystallization Process of Protein Rv0731c from Mycobacterium Tuberculosis for a Successful Atomic Resolution Crystal Structure at 1.2 Angstrom

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Liang Cong

    2009-06-08

    Proteins are bio-macromolecules consisting of basic 20 amino acids and have distinct three-dimensional folds. They are essential parts of organisms and participate in every process within cells. Proteins are crucial for human life, and each protein within the body has a specific function, such as antibodies, contractile proteins, enzymes, hormonal proteins, structural proteins, storage proteins and transport proteins. Determining three-dimensional structure of a protein can help researchers discover the remarkable protein folding, binding site, conformation and etc, in order to understand well of protein interaction and aid for possible drug design. The research on protein structure by X-ray protein crystallography carried by Li-Wei Hung's research group in the Physical Bioscience Division at Lawrence Berkeley National Laboratory (LBNL) is focusing on protein crystallography. The research in this lab is in the process of from crystallizing the proteins to determining the three dimensional crystal structures of proteins. Most protein targets are selected from Mycobacterium Tuberculosis. TB (Tuberculosis) is a possible fatal infectious disease. By studying TB target protein can help discover antituberculer drugs, and find treatment for TB. The high-throughput mode of crystallization, crystal harvesting, crystal screening and data collection are applied to the research pipeline (Figure 1). The X-ray diffraction data by protein crystals can be processed and analyzed to result in a three dimensional representation of electron density, producing a detailed model of protein structure. Rv0731c is a conserved hypothetical protein with unknown function from Mycobacterium Tuberculosis. This paper is going to report the crystallization process and brief structure information of Rv0731c.

  11. Fast high-pressure freezing of protein crystals in their mother liquor

    International Nuclear Information System (INIS)

    Burkhardt, Anja; Warmer, Martin; Panneerselvam, Saravanan; Wagner, Armin; Zouni, Athina; Glöckner, Carina; Reimer, Rudolph; Hohenberg, Heinrich; Meents, Alke

    2012-01-01

    Protein crystals were vitrified using high-pressure freezing in their mother liquor at 210 MPa and 77 K without cryoprotectants or oil coating. The method was successfully applied to photosystem II, which is representative of a membrane protein with a large unit cell and weak crystal contacts. High-pressure freezing (HPF) is a method which allows sample vitrification without cryoprotectants. In the present work, protein crystals were cooled to cryogenic temperatures at a pressure of 210 MPa. In contrast to other HPF methods published to date in the field of cryocrystallography, this protocol involves rapid sample cooling using a standard HPF device. The fast cooling rates allow HPF of protein crystals directly in their mother liquor without the need for cryoprotectants or external reagents. HPF was first attempted with hen egg-white lysozyme and cubic insulin crystals, yielding good to excellent diffraction quality. Non-cryoprotected crystals of the membrane protein photosystem II have been successfully cryocooled for the first time. This indicates that the presented HPF method is well suited to the vitrification of challenging systems with large unit cells and weak crystal contacts

  12. Effect of γ-(Fe,Ni) crystal-size stabilization in Fe-Ni-B amorphous ribbon

    Science.gov (United States)

    Gorshenkov, M. V.; Glezer, A. M.; Korchuganova, O. A.; Aleev, A. A.; Shurygina, N. A.

    2017-02-01

    The effect of stabilizing crystal size in a melt-quenched amorphous Fe50Ni33B17 ribbon is described upon crystallization in a temperature range of 360-400°C. The shape, size, volume fraction, and volume density have been investigated by transmission electron microscopy and X-ray diffraction methods. The formation of an amorphous layer of the Fe50Ni29B21 compound was found by means of atomic-probe tomography at the boundary of the crystallite-amorphous phase. The stabilization of crystal sizes during annealing is due to the formation of a barrier amorphous layer that has a crystallization temperature that exceeds the crystallization temperature of the matrix amorphous alloy.

  13. Stabilization of liquid crystal dispersion by nonionic surfactant/acrylamide copolymer containing hydrophobic moieties

    Energy Technology Data Exchange (ETDEWEB)

    Park, S.J.; Kim, M.H.; Lee, J.R. [Korea Research Institute of Chemical Technology, Taejon (Korea)

    1999-07-01

    The effect of nonionic surfactant (H(OCH){sub 2}-OC{sub 6}H{sub 4}-C{sub 9}H{sub 19}, NP-8) and acrylamide copolymer containing nonylphenyl groups as hydrophobic moieties on the stabilization of liquid crystal (LC)-in-water dispersion has been studied. According to cloud point and adsorption measurements, the hydrophobically strong interaction between NP-8 and the nonylphenol moieties is formed. And the addition of surfactant increases the stability of LC dispersion and improve the electrooptical properties of the nematic curvilinear aligned phase (NCAP) composite film. It is due to the presence of surfactant which allows the formation of nonpolar microenvironment in the round of LC droplet and finally reduces the anchoring effect between LC and the polymeric wall. 21 refs., 8 figs.

  14. Stabilization of crystal and magnetic structure of Gd2Al1-xInx

    International Nuclear Information System (INIS)

    Niazi, M.; Yazdani, A.

    2006-01-01

    A random mixture of two compounds with different type of crystal structure and magnetic characteristic which can lead to a new phase, is studied to find out the critical point at which both of them stabilized. such an interesting random system can be found by; 1) Gd 2 Al with two different magnetic phase transition on which T c is unstable 2) Gd 2 In which is also shows two magnetic phase transition, but completely different with Gd 2 Al on which T N is unstable. The stabilized point which is consider experimentally by X-Ray diffraction and theoretically by WIEN2K is suggested to be at x=0.3 where it is a good agreement between these two methods.

  15. Acid Stability of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity

    Energy Technology Data Exchange (ETDEWEB)

    DuBois, Rebecca M.; Zaraket, Hassan; Reddivari, Muralidhar; Heath, Richard J.; White, Stephen W.; Russell, Charles J. (Tennessee-HSC); (SJCH)

    2012-12-10

    Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulate the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 {angstrom} resolution and two structures of HP HA at 2.95 and 3.10 {angstrom} resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species.

  16. Distribution, transition and thermodynamic stability of protein conformations in the denaturant-induced unfolding of proteins.

    Science.gov (United States)

    Bian, Liujiao; Ji, Xu

    2014-01-01

    Extensive and intensive studies on the unfolding of proteins require appropriate theoretical model and parameter to clearly illustrate the feature and characteristic of the unfolding system. Over the past several decades, four approaches have been proposed to describe the interaction between proteins and denaturants, but some ambiguity and deviations usually occur in the explanation of the experimental data. In this work, a theoretical model was presented to show the dependency of the residual activity ratio of the proteins on the molar denaturant concentration. Through the characteristic unfolding parameters ki and Δmi in this model, the distribution, transition and thermodynamic stability of protein conformations during the unfolding process can be quantitatively described. This model was tested with the two-state unfolding of bovine heart cytochrome c and the three-state unfolding of hen egg white lysozyme induced by both guanidine hydrochloride and urea, the four-state unfolding of bovine carbonic anhydrase b induced by guanidine hydrochloride and the unfolding of some other proteins induced by denaturants. The results illustrated that this model could be used accurately to reveal the distribution and transition of protein conformations in the presence of different concentrations of denaturants and to evaluate the unfolding tendency and thermodynamic stability of different conformations. In most denaturant-induced unfolding of proteins, the unfolding became increasingly hard in next transition step and the proteins became more unstable as they attained next successive stable conformation. This work presents a useful method for people to study the unfolding of proteins and may be used to describe the unfolding and refolding of other biopolymers induced by denaturants, inducers, etc.

  17. Effects of monohydric alcohols and polyols on the thermal stability of a protein

    Energy Technology Data Exchange (ETDEWEB)

    Murakami, Shota [Graduate School of Energy Science, Kyoto University, Uji, Kyoto 611-0011 (Japan); Kinoshita, Masahiro, E-mail: kinoshit@iae.kyoto-u.ac.jp [Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2016-03-28

    The thermal stability of a protein is lowered by the addition of a monohydric alcohol, and this effect becomes larger as the size of hydrophobic group in an alcohol molecule increases. By contrast, it is enhanced by the addition of a polyol possessing two or more hydroxyl groups per molecule, and this effect becomes larger as the number of hydroxyl groups increases. Here, we show that all of these experimental observations can be reproduced even in a quantitative sense by rigid-body models focused on the entropic effect originating from the translational displacement of solvent molecules. The solvent is either pure water or water-cosolvent solution. Three monohydric alcohols and five polyols are considered as cosolvents. In the rigid-body models, a protein is a fused hard spheres accounting for the polyatomic structure in the atomic detail, and the solvent is formed by hard spheres or a binary mixture of hard spheres with different diameters. The effective diameter of cosolvent molecules and the packing fractions of water and cosolvent, which are crucially important parameters, are carefully estimated using the experimental data of properties such as the density of solid crystal of cosolvent, parameters in the pertinent cosolvent-cosolvent interaction potential, and density of water-cosolvent solution. We employ the morphometric approach combined with the integral equation theory, which is best suited to the physical interpretation of the calculation result. It is argued that the degree of solvent crowding in the bulk is the key factor. When it is made more serious by the cosolvent addition, the solvent-entropy gain upon protein folding is magnified, leading to the enhanced thermal stability. When it is made less serious, the opposite is true. The mechanism of the effects of monohydric alcohols and polyols is physically the same as that of sugars. However, when the rigid-body models are employed for the effect of urea, its addition is predicted to enhance the

  18. Crystal structure of secretory protein Hcp3 from Pseudomonas aeruginosa

    OpenAIRE

    Osipiuk, Jerzy; Xu, Xiaohui; Cui, Hong; Savchenko, Alexei; Edwards, Aled; Joachimiak, Andrzej

    2011-01-01

    The Type VI secretion pathway transports proteins across the cell envelope of Gram-negative bacteria. Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen infecting humans, uses the type VI secretion pathway to export specific effector proteins crucial for its pathogenesis. The HSI-I virulence locus encodes for several proteins that has been proposed to participate in protein transport including the Hcp1 protein, which forms hexameric rings that assemble into nanotubes in...

  19. Improved success of sparse matrix protein crystallization screening with heterogeneous nucleating agents.

    Directory of Open Access Journals (Sweden)

    Anil S Thakur

    2007-10-01

    Full Text Available Crystallization is a major bottleneck in the process of macromolecular structure determination by X-ray crystallography. Successful crystallization requires the formation of nuclei and their subsequent growth to crystals of suitable size. Crystal growth generally occurs spontaneously in a supersaturated solution as a result of homogenous nucleation. However, in a typical sparse matrix screening experiment, precipitant and protein concentration are not sampled extensively, and supersaturation conditions suitable for nucleation are often missed.We tested the effect of nine potential heterogenous nucleating agents on crystallization of ten test proteins in a sparse matrix screen. Several nucleating agents induced crystal formation under conditions where no crystallization occurred in the absence of the nucleating agent. Four nucleating agents: dried seaweed; horse hair; cellulose and hydroxyapatite, had a considerable overall positive effect on crystallization success. This effect was further enhanced when these nucleating agents were used in combination with each other.Our results suggest that the addition of heterogeneous nucleating agents increases the chances of crystal formation when using sparse matrix screens.

  20. In Vitro Calcite Crystal Morphology Is Modulated by Otoconial Proteins Otolin-1 and Otoconin-90

    Science.gov (United States)

    Moreland, K. Trent; Hong, Mina; Lu, Wenfu; Rowley, Christopher W.; Ornitz, David M.; De Yoreo, James J.; Thalmann, Ruediger

    2014-01-01

    Otoconia are formed embryonically and are instrumental in detecting linear acceleration and gravity. Degeneration and fragmentation of otoconia in elderly patients leads to imbalance resulting in higher frequency of falls that are positively correlated with the incidence of bone fractures and death. In this work we investigate the roles otoconial proteins Otolin-1 and Otoconin 90 (OC90) perform in the formation of otoconia. We demonstrate by rotary shadowing and atomic force microscopy (AFM) experiments that Otolin-1 forms homomeric protein complexes and self-assembled networks supporting the hypothesis that Otolin-1 serves as a scaffold protein of otoconia. Our calcium carbonate crystal growth data demonstrate that Otolin-1 and OC90 modulate in vitro calcite crystal morphology but neither protein is sufficient to produce the shape of otoconia. Coadministration of these proteins produces synergistic effects on crystal morphology that contribute to morphology resembling otoconia. PMID:24748133

  1. Time-dependent deformation of polymer network in polymer-stabilized cholesteric liquid crystals (Conference Presentation)

    Science.gov (United States)

    Lee, Kyung Min; Tondiglia, Vincent P.; Bunning, Timothy J.; White, Timothy J.

    2017-02-01

    Recently, we reported direct current (DC) field controllable electro-optic (EO) responses of negative dielectric anisotropy polymer stabilized cholesteric liquid crystals (PSCLCs). A potential mechanism is: Ions in the liquid crystal mixtures are trapped in/on the polymer network during the fast photopolymerization process, and the movement of ions by the application of the DC field distorts polymer network toward the negative electrode, inducing pitch variation through the cell thickness, i.e., pitch compression on the negative electrode side and pitch expansion on positive electrode side. As the DC voltage is directly applied to a target voltage, charged polymer network is deformed and the reflection band is tuned. Interestingly, the polymer network deforms further (red shift of reflection band) with time when constantly applied DC voltage, illustrating DC field induced time dependent deformation of polymer network (creep-like behavior). This time dependent reflection band changes in PSCLCs are investigated by varying the several factors, such as type and concentration of photoinitiators, liquid crystal monomer content, and curing condition (UV intensity and curing time). In addition, simple linear viscoelastic spring-dashpot models, such as 2-parameter Kelvin and 3-parameter linear models, are used to investigate the time-dependent viscoelastic behaviors of polymer networks in PSCLC.

  2. Influence of iron on crystallization behavior and thermal stability of the insulating materials - porous calcium silicates

    DEFF Research Database (Denmark)

    Haastrup, Sonja; Yu, Donghong; Yue, Yuanzheng

    2017-01-01

    The properties of porous calcium silicate for high temperature insulation are strongly influenced by impurities. In this work we determine the influence of Fe3+ on the crystallization behavior and thermal stability of hydrothermally derived calcium silicate. We synthesize porous calcium silicate...... with Ca/Si molar ratio of 1, to which Fe2O3 is added with Fe/Si molar ratios of 0.1, 0.5, 0.7, 1.0, and 1.3%. Structure and morphology of the porous calcium silicate, with different iron concentrations, are investigated using Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). FTIR...... measurements reveal a pronounced decrease in the number of Q3 sites in the calcium silicate with an increase of Fe3+, and thereby lower the crystal fraction of xonotlite (Ca6Si6O17(OH)2) phase, and increase the crystal fractions of tobermorite(Ca5Si6O16(OH)2·4H2O) and calcite (CaCO3) phases, as confirmed...

  3. Interaction of Tenebrio Molitor Antifreeze Protein with Ice Crystal: Insights from Molecular Dynamics Simulations.

    Science.gov (United States)

    Ramya, L; Ramakrishnan, Vigneshwar

    2016-07-01

    Antifreeze proteins (AFP) observed in cold-adapting organisms bind to ice crystals and prevent further ice growth. However, the molecular mechanism of AFP-ice binding and AFP-inhibited ice growth remains unclear. Here we report the interaction of the insect antifreeze protein (Tenebrio molitor, TmAFP) with ice crystal by molecular dynamics simulation studies. Two sets of simulations were carried out at 263 K by placing the protein near the primary prism plane (PP) and basal plane (BL) of the ice crystal. To delineate the effect of temperatures, both the PP and BL simulations were carried out at 253 K as well. The analyses revealed that the protein interacts strongly with the ice crystal in BL simulation than in PP simulation both at 263 K and 253 K. Further, it was observed that the interactions are primarily mediated through the interface waters. We also observed that as the temperature decreases, the interaction between the protein and the ice increases which can be attributed to the decreased flexibility and the increased structuring of the protein at low temperature. In essence, our study has shed light on the interaction mechanism between the TmAFP antifreeze protein and the ice crystal. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. SUMOylation Confers Posttranslational Stability on NPM-ALK Oncogenic Protein

    Directory of Open Access Journals (Sweden)

    Deeksha Vishwamitra

    2015-09-01

    Full Text Available Nucleophosmin-anaplastic lymphoma kinase–expressing (NPM-ALK+ T-cell lymphoma is an aggressive form of cancer that commonly affects children and adolescents. The expression of NPM-ALK chimeric oncogene results from the chromosomal translocation t(2;5(p23;q35 that causes the fusion of the ALK and NPM genes. This translocation generates the NPM-ALK protein tyrosine kinase that forms the constitutively activated NPM-ALK/NPM-ALK homodimers. In addition, NPM-ALK is structurally associated with wild-type NPM to form NPM/NPM-ALK heterodimers, which can translocate to the nucleus. The mechanisms that sustain the stability of NPM-ALK are not fully understood. SUMOylation is a posttranslational modification that is characterized by the reversible conjugation of small ubiquitin-like modifiers (SUMOs with target proteins. SUMO competes with ubiquitin for substrate binding and therefore, SUMOylation is believed to protect target proteins from proteasomal degradation. Moreover, SUMOylation contributes to the subcellular distribution of target proteins. Herein, we found that the SUMOylation pathway is deregulated in NPM-ALK+ T-cell lymphoma cell lines and primary lymphoma tumors from patients. We also identified Lys24 and Lys32 within the NPM domain as the sites where NPM-ALK conjugates with SUMO-1 and SUMO-3. Importantly, antagonizing SUMOylation by the SENP1 protease decreased the accumulation of NPM-ALK and suppressed lymphoma cell viability, proliferation, and anchorage-independent colony formation. One possible mechanism for the SENP1-mediated decrease in NPM-ALK levels was the increase in NPM-ALK association with ubiquitin, which facilitates its degradation. Our findings propose a model in which aberrancies in SUMOylation contribute to the pathogenesis of NPM-ALK+ T-cell lymphoma. Unraveling such pathogenic mechanisms may lead to devising novel strategies to eliminate this aggressive neoplasm.

  5. Innovative aspects of protein stability in ionic liquid mixtures.

    Science.gov (United States)

    Kumar, Awanish; Venkatesu, Pannuru

    2018-06-01

    Mixtures of ionic liquids (ILs) have attracted our attention because of their extraordinary performances in extraction technologies and in absorbing large amount of CO 2 gas. It has been observed that when two or more ILs are mixed in different proportions, a new solvent is obtained which is much better than that of each component of ILs from which the mixture is obtained. Within a mixture of ILs, several unidentified interactions occur among several ions which give rise to unique solvent properties to the mixture. Herein, in this review, we have highlighted the utilization of the advantageous properties of the IL mixtures in protein stability studies. This approach is exceptional and opens new directions to the use of ILs in biotechnology.

  6. Crystallization and preliminary crystallographic analysis of recombinant immunoglobulin G-binding protein from Streptococcus suis

    Energy Technology Data Exchange (ETDEWEB)

    Khan, Abdul Hamid; Chu, Fuliang; Feng, Youjun; Zhang, Qinagmin [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); Qi, Jianxun [Institute of Physics, Chinese Academy of Sciences, Beijing 100080 (China); Gao, George Fu, E-mail: gaof@im.ac.cn [Center for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China)

    2008-08-01

    Crystallization of recombinant IgG-binding protein expressed in Escherichia coli using the hanging-drop vapour-diffusion method is described. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.98, b = 43.94, c = 78.17 Å. Streptococcus suis, an important zoonotic pathogen, expresses immunoglobulin G-binding protein, which is thought to be helpful to the organism in eluding the host defence system. Recombinant IgG-binding protein expressed in Escherichia coli has been crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 38.98, b = 43.94, c = 78.17 Å and one molecule in the asymmetric unit. Diffraction data were collected to 2.60 Å resolution.

  7. Characterisation of protein stability in rod-insert vaginal rings.

    Science.gov (United States)

    Pattani, Aditya; Lowry, Deborah; Curran, Rhonda M; McGrath, Stephanie; Kett, Vicky L; Andrews, Gavin P; Malcolm, R Karl

    2012-07-01

    A major goal in vaccine development is elimination of the 'cold chain', the transport and storage system for maintenance and distribution of the vaccine product. This is particularly pertinent to liquid formulation of vaccines. We have previously described the rod-insert vaginal ring (RiR) device, comprising an elastomeric body into which are inserted lyophilised, rod-shaped, solid drug dosage forms, and having potential for sustained mucosal delivery of biomacromolecules, such as HIV envelope protein-based vaccine candidates. Given the solid, lyophilised nature of these insert dosage forms, we hypothesised that antigen stability may be significantly increased compared with more conventional solubilised vaginal gel format. In this study, we prepared and tested vaginal ring devices fitted with lyophilised rod inserts containing the model antigen bovine serum albumin (BSA). Both the RiRs and the gels that were freeze-dried to prepare the inserts were evaluated for BSA stability using PAGE, turbidimetry, microbial load, MALDI-TOF and qualitative precipitate solubility measurements. When stored at 4 °C, but not when stored at 40 °C/75% RH, the RiR formulation offered protection against structural and conformational changes to BSA. The insert also retained matrix integrity and release characteristics. The results demonstrate that lypophilised gels can provide relative protection against degradation at lower temperatures compared to semi-solid gels. The major mechanism of degradation at 40 °C/75% RH was shown to be protein aggregation. Finally, in a preliminary study, we found that addition of trehalose to the formulation significantly reduces the rate of BSA degradation compared to the original formulation when stored at 40 °C/75% RH. Establishing the mechanism of degradation, and finding that degradation is decelerated in the presence of trehalose, will help inform further development of RiRs specifically and polymer based freeze-dried systems in general. Copyright

  8. Formulating food protein-stabilized indomethacin nanosuspensions into pellets by fluid-bed coating technology: physical characterization, redispersibility, and dissolution.

    Science.gov (United States)

    He, Wei; Lu, Yi; Qi, Jianping; Chen, Lingyun; Yin, Lifang; Wu, Wei

    2013-01-01

    Drug nanosuspensions are very promising for enhancing the dissolution and bioavailability of drugs that are poorly soluble in water. However, the poor stability of nanosuspensions, reflected in particle growth, aggregation/agglomeration, and change in crystallinity state greatly limits their applications. Solidification of nanosuspensions is an ideal strategy for addressing this problem. Hence, the present work aimed to convert drug nanosuspensions into pellets using fluid-bed coating technology. Indomethacin nanosuspensions were prepared by the precipitation-ultrasonication method using food proteins (soybean protein isolate, whey protein isolate, β-lactoglobulin) as stabilizers. Dried nanosuspensions were prepared by coating the nanosuspensions onto pellets. The redispersibility, drug dissolution, solid-state forms, and morphology of the dried nanosuspensions were evaluated. The mean particle size for the nanosuspensions stabilized using soybean protein isolate, whey protein isolate, and β-lactoglobulin was 588 nm, 320 nm, and 243 nm, respectively. The nanosuspensions could be successfully layered onto pellets with high coating efficiency. Both the dried nanosuspensions and nanosuspensions in their original amorphous state and not influenced by the fluid-bed coating drying process could be redispersed in water, maintaining their original particle size and size distribution. Both the dried nanosuspensions and the original drug nanosuspensions showed similar dissolution profiles, which were both much faster than that of the raw crystals. Fluid-bed coating technology has potential for use in the solidification of drug nanosuspensions.

  9. Some Aspects of Crystal Centering During X-ray High-throughput Protein Crystallography Experiment

    Science.gov (United States)

    Gaponov, Yu. A.; Matsugaki, N.; Sasajima, K.; Igarashi, N.; Wakatsuki, S.

    A set of algorithms and procedures of a crystal loop centering during X-ray high-throughput protein crystallography experiment has been designed and developed. A simple algorithm of the crystal loop detection and preliminary recognition has been designed and developed. The crystal loop detection algorithm is based on finding out the crystal loop ending point (opposite to the crystal loop pin) using image cross section (digital image column) profile analysis. The crystal loop preliminary recognition procedure is based on finding out the crystal loop sizes and position using image cross section profile analysis. The crystal loop fine recognition procedure based on Hooke-Jeeves pattern search method with an ellipse as a fitting pattern has been designed and developed. The procedure of restoring missing coordinate of the crystal loop is described. Based on developed algorithms and procedures the optimal auto-centering procedure has been designed and developed. A procedure of optimal manual crystal centering (Two Clicks Procedure) has been designed and developed. Developed procedures have been integrated into control software system PCCS installed at crystallography beamlines Photon Factory BL5A and PF-AR NW12, KEK.

  10. Crystal Structure of a Plant Multidrug and Toxic Compound Extrusion Family Protein.

    Science.gov (United States)

    Tanaka, Yoshiki; Iwaki, Shigehiro; Tsukazaki, Tomoya

    2017-09-05

    The multidrug and toxic compound extrusion (MATE) family of proteins consists of transporters responsible for multidrug resistance in prokaryotes. In plants, a number of MATE proteins were identified by recent genomic and functional studies, which imply that the proteins have substrate-specific transport functions instead of multidrug extrusion. The three-dimensional structure of eukaryotic MATE proteins, including those of plants, has not been reported, preventing a better understanding of the molecular mechanism of these proteins. Here, we describe the crystal structure of a MATE protein from the plant Camelina sativa at 2.9 Å resolution. Two sets of six transmembrane α helices, assembled pseudo-symmetrically, possess a negatively charged internal pocket with an outward-facing shape. The crystal structure provides insight into the diversity of plant MATE proteins and their substrate recognition and transport through the membrane. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. StaRProtein, A Web Server for Prediction of the Stability of Repeat Proteins

    Science.gov (United States)

    Xu, Yongtao; Zhou, Xu; Huang, Meilan

    2015-01-01

    Repeat proteins have become increasingly important due to their capability to bind to almost any proteins and the potential as alternative therapy to monoclonal antibodies. In the past decade repeat proteins have been designed to mediate specific protein-protein interactions. The tetratricopeptide and ankyrin repeat proteins are two classes of helical repeat proteins that form different binding pockets to accommodate various partners. It is important to understand the factors that define folding and stability of repeat proteins in order to prioritize the most stable designed repeat proteins to further explore their potential binding affinities. Here we developed distance-dependant statistical potentials using two classes of alpha-helical repeat proteins, tetratricopeptide and ankyrin repeat proteins respectively, and evaluated their efficiency in predicting the stability of repeat proteins. We demonstrated that the repeat-specific statistical potentials based on these two classes of repeat proteins showed paramount accuracy compared with non-specific statistical potentials in: 1) discriminate correct vs. incorrect models 2) rank the stability of designed repeat proteins. In particular, the statistical scores correlate closely with the equilibrium unfolding free energies of repeat proteins and therefore would serve as a novel tool in quickly prioritizing the designed repeat proteins with high stability. StaRProtein web server was developed for predicting the stability of repeat proteins. PMID:25807112

  12. Membrane's Eleven: heavy-atom derivatives of membrane-protein crystals

    DEFF Research Database (Denmark)

    Morth, Jens Preben; Sørensen, Thomas Lykke-Møller; Nissen, Poul

    2006-01-01

    A database has been assembled of heavy-atom derivatives used in the structure determination of membrane proteins. The database can serve as a guide to the design of experiments in the search for heavy-atom derivatives of new membrane-protein crystals. The database pinpoints organomercurials...

  13. Blocking rapid ice crystal growth through nonbasal plane adsorption of antifreeze proteins

    NARCIS (Netherlands)

    Olijve, L.L.C.; Meister, K.; DeVries, A.L.; Duman, J.G.; Guo, S.; Bakker, H.J.; Voets, I.K.

    2016-01-01

    Antifreeze proteins (AFPs) are a unique class of proteins that bind to growing ice crystal surfaces and arrest further ice growth. AFPs have gained a large interest for their use in antifreeze formulations for water-based materials, such as foods, waterborne paints, and organ transplants. Instead of

  14. In vacuo X-ray data collection from graphene-wrapped protein crystals

    International Nuclear Information System (INIS)

    Warren, Anna J.; Crawshaw, Adam D.; Trincao, Jose; Aller, Pierre; Alcock, Simon; Nistea, Ioana; Salgado, Paula S.; Evans, Gwyndaf

    2015-01-01

    A method is reported for collecting room-temperature data from protein crystals under vacuum by protecting them with a thin graphene layer. The measurement of diffraction data from macromolecular crystal samples held in vacuo holds the promise of a very low X-ray background and zero absorption of incident and scattered beams, leading to better data and the potential for accessing very long X-ray wavelengths (>3 Å) for native sulfur phasing. Maintaining the hydration of protein crystals under vacuum is achieved by the use of liquid jets, as with serial data collection at free-electron lasers, or is side-stepped by cryocooling the samples, as implemented at new synchrotron beamlines. Graphene has been shown to protect crystals from dehydration by creating an extremely thin layer that is impermeable to any exchanges with the environment. Furthermore, owing to its hydrophobicity, most of the aqueous solution surrounding the crystal is excluded during sample preparation, thus eliminating most of the background caused by liquid. Here, it is shown that high-quality data can be recorded at room temperature from graphene-wrapped protein crystals in a rough vacuum. Furthermore, it was observed that graphene protects crystals exposed to different relative humidities and a chemically harsh environment

  15. Protein crystal growth results from the United States Microgravity Laboratory-1 mission

    Science.gov (United States)

    Delucas, Lawrence J.; Moore, K. M.; Vanderwoerd, M.; Bray, T. L.; Smith, C.; Carson, M.; Narayana, S. V. L.; Rosenblum, W. M.; Carter, D.; Clark, A. D, Jr.

    1994-01-01

    Protein crystal growth experiments have been performed by this laboratory on 18 Space Shuttle missions since April, 1985. In addition, a number of microgravity experiments also have been performed and reported by other investigators. These Space Shuttle missions have been used to grow crystals of a variety of proteins using vapor diffusion, liquid diffusion, and temperature-induced crystallization techniques. The United States Microgravity Laboratory - 1 mission (USML-1, June 25 - July 9, 1992) was a Spacelab mission dedicated to experiments involved in materials processing. New protein crystal growth hardware was developed to allow in orbit examination of initial crystal growth results, the knowledge from which was used on subsequent days to prepare new crystal growth experiments. In addition, new seeding hardware and techniques were tested as well as techniques that would prepare crystals for analysis by x-ray diffraction, a capability projected for the planned Space Station. Hardware that was specifically developed for the USML-1 mission will be discussed along with the experimental results from this mission.

  16. In vacuo X-ray data collection from graphene-wrapped protein crystals

    Energy Technology Data Exchange (ETDEWEB)

    Warren, Anna J. [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Crawshaw, Adam D. [Newcastle University, Newcastle upon Tyne NE2 4HH (United Kingdom); Trincao, Jose; Aller, Pierre; Alcock, Simon; Nistea, Ioana [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Salgado, Paula S. [Newcastle University, Newcastle upon Tyne NE2 4HH (United Kingdom); Evans, Gwyndaf, E-mail: gwyndaf.evans@diamond.ac.uk [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom)

    2015-09-26

    A method is reported for collecting room-temperature data from protein crystals under vacuum by protecting them with a thin graphene layer. The measurement of diffraction data from macromolecular crystal samples held in vacuo holds the promise of a very low X-ray background and zero absorption of incident and scattered beams, leading to better data and the potential for accessing very long X-ray wavelengths (>3 Å) for native sulfur phasing. Maintaining the hydration of protein crystals under vacuum is achieved by the use of liquid jets, as with serial data collection at free-electron lasers, or is side-stepped by cryocooling the samples, as implemented at new synchrotron beamlines. Graphene has been shown to protect crystals from dehydration by creating an extremely thin layer that is impermeable to any exchanges with the environment. Furthermore, owing to its hydrophobicity, most of the aqueous solution surrounding the crystal is excluded during sample preparation, thus eliminating most of the background caused by liquid. Here, it is shown that high-quality data can be recorded at room temperature from graphene-wrapped protein crystals in a rough vacuum. Furthermore, it was observed that graphene protects crystals exposed to different relative humidities and a chemically harsh environment.

  17. Crystallization and preliminary crystallographic analysis of molybdenum-cofactor biosynthesis protein C from Thermus thermophilus

    International Nuclear Information System (INIS)

    Kanaujia, Shankar Prasad; Ranjani, Chellamuthu Vasuki; Jeyakanthan, Jeyaraman; Baba, Seiki; Chen, Lirong; Liu, Zhi-Jie; Wang, Bi-Cheng; Nishida, Masami; Ebihara, Akio; Shinkai, Akeo; Kuramitsu, Seiki; Shiro, Yoshitsugu; Sekar, Kanagaraj; Yokoyama, Shigeyuki

    2006-01-01

    The molybdenum-cofactor biosynthesis protein C from T. thermophilus has been crystallized in two different space groups, P2 1 and R32; the crystals diffracted to 1.9 and 1.75 Å resolution, respectively. The Gram-negative aerobic eubacterium Thermus thermophilus is an extremely important thermophilic microorganism that was originally isolated from a thermal vent environment in Japan. The molybdenum cofactor in this organism is considered to be an essential component required by enzymes that catalyze diverse key reactions in the global metabolism of carbon, nitrogen and sulfur. The molybdenum-cofactor biosynthesis protein C derived from T. thermophilus was crystallized in two different space groups. Crystals obtained using the first crystallization condition belong to the monoclinic space group P2 1 , with unit-cell parameters a = 64.81, b = 109.84, c = 115.19 Å, β = 104.9°; the crystal diffracted to a resolution of 1.9 Å. The other crystal form belonged to space group R32, with unit-cell parameters a = b = 106.57, c = 59.25 Å, and diffracted to 1.75 Å resolution. Preliminary calculations reveal that the asymmetric unit contains 12 monomers and one monomer for the crystals belonging to space group P2 1 and R32, respectively

  18. Purification, crystallization and preliminary crystallographic analysis of SMU.1108c protein from Streptococcus mutans

    International Nuclear Information System (INIS)

    Feng, Ming-Jing; Fu, Tian-Min; Liu, Xiang; Li, Lan-Fen

    2010-01-01

    SMU.1108c, a putative uncharacterized protein from S. mutans, was crystallized and X-ray diffraction data were collected to a resolution of 2.2 Å. Streptococcus mutans SMU.1108c (KEGG database) encodes a functionally uncharacterized protein consisting of 270 amino-acid residues. This protein is predicted to have a haloacid dehalogenase hydrolase-like domain and is a homologue of haloacid dehalogenase phosphatases that catalyze phosphoryl-transfer reactions. In this work, SMU.1108c was cloned into the pET28a vector and overexpressed in Escherichia coli strain BL21 (DE3). The protein was purified to homogeneity and crystallized using the sitting-drop vapour-diffusion method. The best crystal diffracted to 2.0 Å resolution and belonged to space group C2, with unit-cell parameters a = 77.1, b = 80.2, c = 47.9 Å, β = 99.5°

  19. Humidity control and hydrophilic glue coating applied to mounted protein crystals improves X-ray diffraction experiments

    International Nuclear Information System (INIS)

    Baba, Seiki; Hoshino, Takeshi; Ito, Len; Kumasaka, Takashi

    2013-01-01

    A new crystal-mounting method has been developed that involves a combination of controlled humid air and polymer glue for crystal coating. This method is particularly useful when applied to fragile protein crystals that are known to be sensitive to subtle changes in their physicochemical environment. Protein crystals are fragile, and it is sometimes difficult to find conditions suitable for handling and cryocooling the crystals before conducting X-ray diffraction experiments. To overcome this issue, a protein crystal-mounting method has been developed that involves a water-soluble polymer and controlled humid air that can adjust the moisture content of a mounted crystal. By coating crystals with polymer glue and exposing them to controlled humid air, the crystals were stable at room temperature and were cryocooled under optimized humidity. Moreover, the glue-coated crystals reproducibly showed gradual transformations of their lattice constants in response to a change in humidity; thus, using this method, a series of isomorphous crystals can be prepared. This technique is valuable when working on fragile protein crystals, including membrane proteins, and will also be useful for multi-crystal data collection

  20. Humidity control and hydrophilic glue coating applied to mounted protein crystals improves X-ray diffraction experiments

    Energy Technology Data Exchange (ETDEWEB)

    Baba, Seiki; Hoshino, Takeshi; Ito, Len; Kumasaka, Takashi, E-mail: kumasaka@spring8.or.jp [Japan Synchrotron Radiation Research Institute (JASRI/SPring-8), 1-1-1 Kouto, Sayo, Hyogo 679-5198 (Japan)

    2013-09-01

    A new crystal-mounting method has been developed that involves a combination of controlled humid air and polymer glue for crystal coating. This method is particularly useful when applied to fragile protein crystals that are known to be sensitive to subtle changes in their physicochemical environment. Protein crystals are fragile, and it is sometimes difficult to find conditions suitable for handling and cryocooling the crystals before conducting X-ray diffraction experiments. To overcome this issue, a protein crystal-mounting method has been developed that involves a water-soluble polymer and controlled humid air that can adjust the moisture content of a mounted crystal. By coating crystals with polymer glue and exposing them to controlled humid air, the crystals were stable at room temperature and were cryocooled under optimized humidity. Moreover, the glue-coated crystals reproducibly showed gradual transformations of their lattice constants in response to a change in humidity; thus, using this method, a series of isomorphous crystals can be prepared. This technique is valuable when working on fragile protein crystals, including membrane proteins, and will also be useful for multi-crystal data collection.

  1. Analysis of protein stability and ligand interactions by thermal shift assay.

    Science.gov (United States)

    Huynh, Kathy; Partch, Carrie L

    2015-02-02

    Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye. Copyright © 2015 John Wiley & Sons, Inc.

  2. The Escherichia coli antiterminator protein BglG stabilizes the 5 ...

    Indian Academy of Sciences (India)

    Unknown

    Keywords. Antitermination; mRNA stability; RNA binding protein ... factor, Rho, and the pBR322 copy number protein, Rop, have been .... Transcription analysis using the oligo- ..... Retarded RNA turnover in Escherichia coli a means of main-.

  3. Image processing of small protein-crystals in electron microscopy

    International Nuclear Information System (INIS)

    Feinberg, D.A.

    1978-11-01

    This electron microscope study was undertaken to determine whether high resolution reconstructed images could be obtained from statistically noisy micrographs by the super-position of several small areas of images of well-ordered crystals of biological macromolecules. Methods of rotational and translational alignment which use Fourier space data were demonstrated to be superior to methods which use Real space image data. After alignment, the addition of the diffraction patterns of four small areas did not produce higher resolution because of unexpected image distortion effects. A method was developed to determine the location of the distortion origin and the coefficients of spiral distortion and pincushion/barrel distortion in order to make future correction of distortions in electron microscope images of large area crystals

  4. Improvement of an automated protein crystal exchange system PAM for high-throughput data collection

    International Nuclear Information System (INIS)

    Hiraki, Masahiko; Yamada, Yusuke; Chavas, Leonard M. G.; Wakatsuki, Soichi; Matsugaki, Naohiro

    2013-01-01

    A special liquid-nitrogen Dewar with double capacity for the sample-exchange robot has been created at AR-NE3A at the Photon Factory, allowing continuous fully automated data collection. In this work, this new system is described and the stability of its calibration is discussed. Photon Factory Automated Mounting system (PAM) protein crystal exchange systems are available at the following Photon Factory macromolecular beamlines: BL-1A, BL-5A, BL-17A, AR-NW12A and AR-NE3A. The beamline AR-NE3A has been constructed for high-throughput macromolecular crystallography and is dedicated to structure-based drug design. The PAM liquid-nitrogen Dewar can store a maximum of three SSRL cassettes. Therefore, users have to interrupt their experiments and replace the cassettes when using four or more of them during their beam time. As a result of investigation, four or more cassettes were used in AR-NE3A alone. For continuous automated data collection, the size of the liquid-nitrogen Dewar for the AR-NE3A PAM was increased, doubling the capacity. In order to check the calibration with the new Dewar and the cassette stand, calibration experiments were repeatedly performed. Compared with the current system, the parameters of the novel system are shown to be stable

  5. Improvement of an automated protein crystal exchange system PAM for high-throughput data collection

    Energy Technology Data Exchange (ETDEWEB)

    Hiraki, Masahiko, E-mail: masahiko.hiraki@kek.jp; Yamada, Yusuke; Chavas, Leonard M. G. [High Energy Accelerator Research Organization, 1-1 Oho, Tsukuba, Ibaraki 305-0801 (Japan); Wakatsuki, Soichi [High Energy Accelerator Research Organization, 1-1 Oho, Tsukuba, Ibaraki 305-0801 (Japan); SLAC National Accelerator Laboratory, 2575 Sand Hill Road, MS 69, Menlo Park, CA 94025-7015 (United States); Stanford University, Beckman Center B105, Stanford, CA 94305-5126 (United States); Matsugaki, Naohiro [High Energy Accelerator Research Organization, 1-1 Oho, Tsukuba, Ibaraki 305-0801 (Japan)

    2013-11-01

    A special liquid-nitrogen Dewar with double capacity for the sample-exchange robot has been created at AR-NE3A at the Photon Factory, allowing continuous fully automated data collection. In this work, this new system is described and the stability of its calibration is discussed. Photon Factory Automated Mounting system (PAM) protein crystal exchange systems are available at the following Photon Factory macromolecular beamlines: BL-1A, BL-5A, BL-17A, AR-NW12A and AR-NE3A. The beamline AR-NE3A has been constructed for high-throughput macromolecular crystallography and is dedicated to structure-based drug design. The PAM liquid-nitrogen Dewar can store a maximum of three SSRL cassettes. Therefore, users have to interrupt their experiments and replace the cassettes when using four or more of them during their beam time. As a result of investigation, four or more cassettes were used in AR-NE3A alone. For continuous automated data collection, the size of the liquid-nitrogen Dewar for the AR-NE3A PAM was increased, doubling the capacity. In order to check the calibration with the new Dewar and the cassette stand, calibration experiments were repeatedly performed. Compared with the current system, the parameters of the novel system are shown to be stable.

  6. Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging

    OpenAIRE

    Warren, Anna J.; Armour, Wes; Axford, Danny; Basham, Mark; Connolley, Thomas; Hall, David R.; Horrell, Sam; McAuley, Katherine E.; Mykhaylyk, Vitaliy; Wagner, Armin; Evans, Gwyndaf

    2013-01-01

    The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolec...

  7. Formation, stability and crystal structure of the {sigma} phase in Mo-Re-Si alloys

    Energy Technology Data Exchange (ETDEWEB)

    Bei, H., E-mail: beih@ornl.gov [Oak Ridge National Laboratory, Materials Science and Technology Division, Oak Ridge, TN 37831 (United States); Yang, Y., E-mail: ying.yang@computherm.com [CompuTherm LLC, Madison, WI 53719 (United States); Viswanathan, G.B. [Air Force Research Laboratory, Wright-Patterson AFB, OH 45433 (United States); Rawn, C.J.; George, E.P. [Oak Ridge National Laboratory, Materials Science and Technology Division, Oak Ridge, TN 37831 (United States)] [University of Tennessee, Department of Materials Science and Engineering, Knoxville, TN 37996 (United States); Tiley, J. [Air Force Research Laboratory, Wright-Patterson AFB, OH 45433 (United States); Chang, Y.A. [CompuTherm LLC, Madison, WI 53719 (United States)] [University of Wisconsin-Madison, Madison, WI 53705 (United States)

    2010-10-15

    The formation, stability and crystal structure of the {sigma} phase in Mo-Re-Si alloys were investigated. Guided by thermodynamic calculations, six critically selected alloys were arc melted and annealed at 1600 deg. C for 150 h. Their as-cast and annealed microstructures, including phase fractions and distributions, the compositions of the constituent phases and the crystal structure of the {sigma} phase were analyzed by thermodynamic modeling coupled with experimental characterization by scanning electron microscopy, electron probe microanalysis, X-ray diffraction and transmission electron microscopy. Two key findings resulted from this work. One is the large homogeneity range of the {sigma} phase region, extending from binary Mo-Re to ternary Mo-Re-Si. The other is the formation of a {sigma} phase in Mo-rich alloys either through the peritectic reaction of liquid + Mo{sub ss} {yields} {sigma} or primary solidification. These findings are important in understanding the effects of Re on the microstructure and providing guidance on the design of Mo-Re-Si alloys.

  8. Thermoelectric properties and thermal stability of Bi-doped PbTe single crystal

    Science.gov (United States)

    Chen, Zhong; Li, Decong; Deng, Shuping; Tang, Yu; Sun, Luqi; Liu, Wenting; Shen, Lanxian; Yang, Peizhi; Deng, Shukang

    2018-06-01

    In this study, n-type Bi-doped single-crystal PbTe thermoelectric materials were prepared by melting and slow cooling method according to the stoichiometric ratio of Pb:Bi:Te = 1-x:x:1 (x = 0, 0.1, 0.15, 0.2, 0.25). The X-ray diffraction patterns of Pb1-xBixTe samples show that all main diffraction peaks are well matched with the PbTe matrix, which has a face-centered cubic structure with the space group Fm 3 bar m . Electron probe microanalysis reveals that Pb content decreases gradually, and Te content remains invariant basically with the increase of Bi content, indicating that Bi atoms are more likely to replace Pb atoms. Thermal analysis shows that the prepared samples possess relatively high thermal stability. Simultaneously, transmission electron microscopy and selected area electron diffraction pattern indicate that the prepared samples have typical single-crystal structures with good mechanical properties. Moreover, the electrical conductivity of the prepared samples improved significantly compared with that of the pure sample, and the maximum ZT value of 0.84 was obtained at 600 K by the sample with x = 0.2.

  9. Comparison of NMR and crystal structures highlights conformational isomerism in protein active sites

    International Nuclear Information System (INIS)

    Serrano, Pedro; Pedrini, Bill; Geralt, Michael; Jaudzems, Kristaps; Mohanty, Biswaranjan; Horst, Reto; Herrmann, Torsten; Elsliger, Marc-André; Wilson, Ian A.; Wüthrich, Kurt

    2010-01-01

    Tools for systematic comparisons of NMR and crystal structures developed by the JCSG were applied to two proteins with known functions: the T. maritima anti-σ factor antagonist TM1081 and the mouse γ-glutamylamine cyclotransferase A2LD1 (gi:13879369). In an attempt to exploit the complementarity of crystal and NMR data, the combined use of the two structure-determination techniques was explored for the initial steps in the challenge of searching proteins of unknown functions for putative active sites. The JCSG has recently developed a protocol for systematic comparisons of high-quality crystal and NMR structures of proteins. In this paper, the extent to which this approach can provide function-related information on the two functionally annotated proteins TM1081, a Thermotoga maritima anti-σ factor antagonist, and A2LD1 (gi:13879369), a mouse γ-glutamylamine cyclotransferase, is explored. The NMR structures of the two proteins have been determined in solution at 313 and 298 K, respectively, using the current JCSG protocol based on the software package UNIO for extensive automation. The corresponding crystal structures were solved by the JCSG at 100 K and 1.6 Å resolution and at 100 K and 1.9 Å resolution, respectively. The NMR and crystal structures of the two proteins share the same overall molecular architectures. However, the precision of the structure determination along the amino-acid sequence varies over a significantly wider range in the NMR structures than in the crystal structures. Thereby, in each of the two NMR structures about 65% of the residues have displacements below the average and in both proteins the less well ordered residues include large parts of the active sites, in addition to some highly solvent-exposed surface areas. Whereas the latter show increased disorder in the crystal and in solution, the active-site regions display increased displacements only in the NMR structures, where they undergo local conformational exchange on the

  10. Science Issues Associated with the Use of a Microfluidic Chip Designed Specifically for Protein Crystallization

    Science.gov (United States)

    Holmes, Anna M.; Monaco, Lisa; Barnes, Cindy; Spearing, Scott; Jenkins, Andy; Johnson, Todd; Mayer, Derek; Cole, Helen

    2003-01-01

    The Iterative Biological Crystallization team in partnership with Caliper Technologies has produced a prototype microfluidic chip for batch crystallization that has been designed and tested. The chip is designed for the mixing and dispensing of up to five solutions with possible variation of the recipe being delivered to two growth wells. Developments that have led to the successful on-chip crystallization of a few model proteins have required investigative insight into many different areas, including fluid mixing dynamics, surface treatments, quantification and fidelity of reagent delivery. This presentation will encompass the ongoing studies and data accumulated toward these efforts.

  11. Matrix Gla Protein is Involved in Crystal Formation in Kidney of Hyperoxaluric Rats

    Directory of Open Access Journals (Sweden)

    Xiuli Lu

    2013-02-01

    Full Text Available Background: Matrix Gla protein (MGP is a molecular determinant regulating vascular calcification of the extracellular matrix. However, it is still unclear how MGP may be invovled in crystal formation in the kidney of hyperoxaluric rats. Methods: Male Sprague-Dawley rats were divided into the hyperoxaluric group and control group. Hyperoxaluric rats were administrated by 0.75% ethylene glycol (EG for up to 8 weeks. Renal MGP expression was detected by the standard avidin-biotin complex (ABC method. Renal crystal deposition was observed by a polarizing microscope. Total RNA and protein from the rat kidney tissue were extracted. The levels of MGP mRNA and protein expression were analyzed by the real-time polymerase chain reaction (RT-PCR and Western blot. Results: Hyperoxaluria was induced successfully in rats. The MGP was polarly distributed, on the apical membrane of renal tubular epithelial cells, and was found in the ascending thick limbs of Henle's loop (cTAL and the distal convoluted tubule (DCT in hyperoxaluric rats, its expression however, was present in the medullary collecting duct (MCD in stone-forming rats. Crystals with multilaminated structure formed in the injurious renal tubules with lack of MGP expression.MGP mRNA expression was significantly upregulated by the crystals' stimulations. Conclusion: Our results suggested that the MGP was involved in crystals formation by the continuous expression, distributing it polarly in the renal tubular cells and binding directly to the crystals.

  12. Protein capped nanosilver free radical oxidation: role of biomolecule capping on nanoparticle colloidal stability and protein oxidation.

    Science.gov (United States)

    Ahumada, Manuel; Bohne, Cornelia; Oake, Jessy; Alarcon, Emilio I

    2018-05-03

    We studied the effect of human serum albumin protein capped spherical nanosilver on the nanoparticle stability upon peroxyl radical oxidation. The nanoparticle-protein composite is less prone to oxidation compared to the individual components. However, higher concentrations of hydrogen peroxide were formed in the nanoparticle-protein system.

  13. Crystal structure of secretory protein Hcp3 from Pseudomonas aeruginosa.

    Science.gov (United States)

    Osipiuk, Jerzy; Xu, Xiaohui; Cui, Hong; Savchenko, Alexei; Edwards, Aled; Joachimiak, Andrzej

    2011-03-01

    The Type VI secretion pathway transports proteins across the cell envelope of Gram-negative bacteria. Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen infecting humans, uses the type VI secretion pathway to export specific effector proteins crucial for its pathogenesis. The HSI-I virulence locus encodes for several proteins that has been proposed to participate in protein transport including the Hcp1 protein, which forms hexameric rings that assemble into nanotubes in vitro. Two Hcp1 paralogues have been identified in the P. aeruginosa genome, Hsp2 and Hcp3. Here, we present the structure of the Hcp3 protein from P. aeruginosa. The overall structure of the monomer resembles Hcp1 despite the lack of amino-acid sequence similarity between the two proteins. The monomers assemble into hexamers similar to Hcp1. However, instead of forming nanotubes in head-to-tail mode like Hcp1, Hcp3 stacks its rings in head-to-head mode forming double-ring structures.

  14. Purification, crystallization and preliminary crystallographic analysis of biotin protein ligase from Staphylococcus aureus

    International Nuclear Information System (INIS)

    Pendini, Nicole R.; Polyak, Steve W.; Booker, Grant W.; Wallace, John C.; Wilce, Matthew C. J.

    2008-01-01

    The biotin protein ligase from S. aureus has been overexpressed in E. coli, purified, crystallized by the hanging-drop vapour-diffusion method and analysed using X-ray diffraction. Biotin protein ligase from Staphylococcus aureus catalyses the biotinylation of acetyl-CoA carboxylase and pyruvate carboxylase. Recombinant biotin protein ligase from S. aureus has been cloned, expressed and purified. Crystals were grown using the hanging-drop vapour-diffusion method using PEG 8000 as the precipitant at 295 K. X-ray diffraction data were collected to 2.3 Å resolution from crystals using synchrotron X-ray radiation at 100 K. The diffraction was consistent with the tetragonal space group P4 2 2 1 2, with unit-cell parameters a = b = 93.665, c = 131.95

  15. Expression, purification, crystallization and structure of human adipocyte lipid-binding protein (aP2)

    International Nuclear Information System (INIS)

    Marr, Eric; Tardie, Mark; Carty, Maynard; Brown Phillips, Tracy; Wang, Ing-Kae; Soeller, Walt; Qiu, Xiayang; Karam, George

    2006-01-01

    The crystal structure of human adipocyte lipid-binding protein (aP2) with a bound palmitate is reported at 1.5 Å resolution. Human adipocyte lipid-binding protein (aP2) belongs to a family of intracellular lipid-binding proteins involved in the transport and storage of lipids. Here, the crystal structure of human aP2 with a bound palmitate is described at 1.5 Å resolution. Unlike the known crystal structure of murine aP2 in complex with palmitate, this structure shows that the fatty acid is in a folded conformation and that the loop containing Phe57 acts as a lid to regulate ligand binding by excluding solvent exposure to the central binding cavity

  16. Biochemical, immunological and toxicological characteristics of the crystal proteins of Bacillus thuringiensis subsp. medellin

    Directory of Open Access Journals (Sweden)

    Sergio Orduz

    1996-04-01

    Full Text Available Characterization of the insecticidal and hemolytic activity of solubilized crystal proteins of Bacillus thuringiensis (Bt subsp. medellin (Btmed was performed and compared to solubilized crystal proteins of isolates 1884 of B. thuringiensis subsp. israelensis (Bti and isolate PG-14 of B. thuringiensis subsp. morrisoni (Btm. In general, at acid pH values solubilization of the Bt crystalline parasporal inclusions (CPI was lower than at alkaline pH. The larvicidal activity demonstrated by the CPI of Btmed indicated that optimal solubilization of CPI takes place at a pH value of 11.3, in Bti at pH values from 5.03 to 11.3 and in Btm at pH values from 9.05 to 11.3. Hemolytic activity against sheep red blood cells was mainly found following extraction at pH 11.3 in all Bt strains tested. Polyacrylamide gel electrophoresis under denaturing conditions revealed that optimal solubilization of the CPI in all Bt strains takes place at the alkaline pH values from 9.05 to 11.3. An enriched preparation of Btmed crystals was obtained, solubilized and crystal proteins were separated on a size exclusion column (Sephacryl S-200. Three main protein peaks were observed on the chromatogram. The first peak had two main proteins that migrate between 90 to 100 kDa. These proteins are apparently not common to other Bt strains isolated to date. The second and third peaks obtained from the size exclusion column yielded polypeptides of 68 and 28-30 kDa, respectively. Each peak independently, showed toxicity against 1st instar Culex quinquefasciatus larvae. Interestingly, combinations of the fractions corresponding to the 68 and 30 kDa protein showed an increased toxicity. These results suggest that the 94 kDa protein is an important component of the Btmed toxins with the highest potency to kill mosquito larvae. When crystal proteins of Bti were probed with antisera raised independently against the three main protein fractions of Btmed, the only crystal protein that showed

  17. Femtosecond X-ray diffraction from two-dimensional protein crystals

    Directory of Open Access Journals (Sweden)

    Matthias Frank

    2014-03-01

    Full Text Available X-ray diffraction patterns from two-dimensional (2-D protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Å resolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.

  18. A convenient tool for gas derivatization using fine-needle capillary mounting for protein crystals

    International Nuclear Information System (INIS)

    Mizuno, Nobuhiro; Makino, Masatomo; Kumasaka, Takashi

    2013-01-01

    A convenient gas-derivatization tool for protein crystals is presented in combination with a fine-needle capillary and a gas-pressure regulator. Gas derivatization of protein crystals is useful not only to analyse gas-binding proteins but also to solve the phase problem of X-ray crystallography by using noble gases. However, the gas pressurization tools for these experiments are often elaborate and need to release the gas before flash-cooling. To simplify this step, a procedure using a fine-needle capillary to mount and flash-cool protein crystals under the pressurization of gases has been developed. After the crystals are picked up with the capillary, the capillary is sealed with an adhesive and then connected directly to a gas regulator. The quality of the diffraction data using this method is comparable with that of data from conventional pressurization procedures. The preparation of xenon-derivatives of hen egg-white lysozyme using this method was a success. In the derivatives, two new xenon binding sites were found and one of their sites vanished by releasing the gas. This observation shows the availability of flash-cooling under gas pressurization. This procedure is simple and useful for preparing gas-derivative crystals

  19. Stabilization of primary mobile radiation defects in MgF{sub 2} crystals

    Energy Technology Data Exchange (ETDEWEB)

    Lisitsyn, V.M. [National Research Tomsk Polytechnic University, pr. Lenina 30, Tomsk 634050 (Russian Federation); Lisitsyna, L.A. [State University of Architecture and Building, pl. Solyanaya 2, Tomsk 634003 (Russian Federation); Popov, A.I., E-mail: popov@ill.fr [Institute of Solid State Physics, University of Latvia, 8 Kengaraga Str., LV-1063 Riga (Latvia); Kotomin, E.A. [Institute of Solid State Physics, University of Latvia, 8 Kengaraga Str., LV-1063 Riga (Latvia); Max Planck Institute for Solid State Research, Heisenbergstr. 1, 70569 Stuttgart (Germany); Abuova, F.U.; Akilbekov, A. [L.N. Gumilyov Eurasian National University, 3 Munaitpasova Str., Astana (Kazakhstan); Maier, J. [Max Planck Institute for Solid State Research, Heisenbergstr. 1, 70569 Stuttgart (Germany)

    2016-05-01

    Non-radiative decay of the electronic excitations (excitons) into point defects (F–H pairs of Frenkel defects) is main radiation damage mechanism in many ionic (halide) solids. Typical time scale of the relaxation of the electronic excitation into a primary, short-lived defect pair is about 1–50 ps with the quantum yield up to 0.2–0.8. However, only a small fraction of these primary defects are spatially separated and survive after transformation into stable, long-lived defects. The survival probability (or stable defect accumulation efficiency) can differ by orders of magnitude, dependent on the material type; e.g. ∼10% in alkali halides with f.c.c. or b.c.c. structure, 0.1% in rutile MgF{sub 2} and <0.001% in fluorides MeF{sub 2} (Me: Ca, Sr, Ba). The key factor determining accumulation of stable radiation defects is stabilization of primary defects, first of all, highly mobile hole H centers, through their transformation into more complex immobile defects. In this talk, we present the results of theoretical calculations of the migration energies of the F and H centers in poorely studied MgF{sub 2} crystals with a focus on the H center stabilization in the form of the interstitial F{sub 2} molecules which is supported by presented experimental data.

  20. Purification, crystallization and preliminary crystallographic analysis of biotin protein ligase from Staphylococcus aureus.

    Science.gov (United States)

    Pendini, Nicole R; Polyak, Steve W; Booker, Grant W; Wallace, John C; Wilce, Matthew C J

    2008-06-01

    Biotin protein ligase from Staphylococcus aureus catalyses the biotinylation of acetyl-CoA carboxylase and pyruvate carboxylase. Recombinant biotin protein ligase from S. aureus has been cloned, expressed and purified. Crystals were grown using the hanging-drop vapour-diffusion method using PEG 8000 as the precipitant at 295 K. X-ray diffraction data were collected to 2.3 A resolution from crystals using synchrotron X-ray radiation at 100 K. The diffraction was consistent with the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 93.665, c = 131.95.

  1. Crystallization and preliminary X-ray analysis of stationary phase survival protein E (SurE) from Xylella fastidiosa in two crystal forms

    International Nuclear Information System (INIS)

    Reis, Marcelo Augusto dos; Saraiva, Antonio Marcos; Santos, Marcelo Leite dos; Souza, Anete Pereira de; Aparicio, Ricardo

    2012-01-01

    The crystallization and preliminary analysis of two crystal forms of survival protein E from X. fastidiosa are reported. The bacterium Xylella fastidiosa is a phytopathogenic organism that causes citrus variegated chlorosis, a disease which attacks economically important crops, mainly oranges. In this communication, the crystallization and preliminary X-ray crystallographic analysis of XfSurE, a survival protein E from X. fastidiosa, are reported. Data were collected for two crystal forms, I and II, to 1.93 and 2.9 Å resolution, respectively. Crystal form I belonged to space group C2, with unit-cell parameters a = 172.36, b = 84.18, c = 87.24 Å, α = γ = 90, β = 96.59°, whereas crystal form II belonged to space group C2, with unit-cell parameters a = 88.05, b = 81.26, c = 72.84 Å, α = γ = 90, β = 94.76°

  2. Time-resolved Laue diffraction from protein crystals: Instrumental considerations

    International Nuclear Information System (INIS)

    Bilderback, D.H.; Cornell Univ., Ithaca, NY; Moffat, K.; Szebenyi, D.M.E.

    1984-01-01

    A serious limitation of macromolecular crystallography has been its inability to determine changes in structure on a biochemical time scale of milliseconds or less. Recently, we have shown that X-ray exposures on single crystals of macromolecules may be obtained in the millisecond time range through the use of intense, polychromatic radiation with Δlambda/lambda approx.= 0.2 derived from the Cornell High Energy Synchrotron Source, CHESS. Such radiation falling on a stationary crystal yields a Laue diffraction pattern, in which almost all Laue reflections arise from a unique set of Miller indices and where their intensities are automatically integrated over wavelength. This Laue technique requires wide band pass optics, which may be obtained by a combination of reflection and transmission mirrors, filters or layered synthetic microstructures. Time-resolved macromolecular crystallography may be achieved by several data collection schemes: 'one-shot' recording coupled to a simple streak camera, repetitive sample perturbation coupled to a detector with temporal resolution and repetitive perturbation which uses the synchrotron pulses for stroboscopic triggering and detection. These schemes are appropriate for different time scales, roughly the milli-, micro- and nanosecond regimes. It appears that time-resolved crystallography is entirely feasible, with an ultimate time resolution limited only by the length of a synchrotron light pulse, some 150 ps at CHESS. (orig.)

  3. Crystal structure analysis, overexpression and refolding behaviour of a DING protein with single mutation

    International Nuclear Information System (INIS)

    Gai, Zuoqi; Nakamura, Akiyoshi; Tanaka, Yoshikazu; Hirano, Nagisa; Tanaka, Isao; Yao, Min

    2013-01-01

    Crystals of a member of the DING protein family (HPBP) were obtained accidentally, and the structure was determined at 1.35 Å resolution. For further analysis, a system for preparation of HPBP was constructed and the structure of a prepared sample was confirmed by X-ray crystal structure analysis at 1.03 Å resolution. After crystallization of a certain protein–RNA complex, well diffracting crystals were obtained. However, the asymmetric unit of the crystal was too small to locate any components. Mass spectrometry and X-ray crystal structure analysis showed that it was a member of the DING protein family (HPBP). Surprisingly, the structure of HPBP reported previously was also determined accidentally as a contaminant, suggesting that HPBP has a strong tendency to crystallize. Furthermore, DING proteins were reported to relate in disease. These observations suggest that DING has potential for application in a wide range of research fields. To enable further analyses, a system for preparation of HPBP was constructed. As HPBP was expressed in insoluble form in Escherichia coli, it was unfolded chemically and refolded. Finally, a very high yield preparation method was constructed, in which 43 mg of HPBP was obtained from 1 L of culture. Furthermore, to evaluate the validity of refolding, its crystal structure was determined at 1.03 Å resolution. The determined structure was identical to the native structure, in which two disulfide bonds were recovered correctly and a phosphate ion was captured. Based on these results, it was concluded that the refolded HPBP recovers its structure correctly

  4. Crystal structure analysis, overexpression and refolding behaviour of a DING protein with single mutation

    Energy Technology Data Exchange (ETDEWEB)

    Gai, Zuoqi; Nakamura, Akiyoshi [Hokkaido University, Sapporo 060-0810 (Japan); Tanaka, Yoshikazu, E-mail: tanaka@sci.hokudai.ac.jp [Hokkaido University, Sapporo 060-0810 (Japan); Hokkaido University, Sapporo 060-0810 (Japan); Hirano, Nagisa [Hokkaido University, Sapporo 060-0810 (Japan); Tanaka, Isao; Yao, Min [Hokkaido University, Sapporo 060-0810 (Japan); Hokkaido University, Sapporo 060-0810 (Japan)

    2013-11-01

    Crystals of a member of the DING protein family (HPBP) were obtained accidentally, and the structure was determined at 1.35 Å resolution. For further analysis, a system for preparation of HPBP was constructed and the structure of a prepared sample was confirmed by X-ray crystal structure analysis at 1.03 Å resolution. After crystallization of a certain protein–RNA complex, well diffracting crystals were obtained. However, the asymmetric unit of the crystal was too small to locate any components. Mass spectrometry and X-ray crystal structure analysis showed that it was a member of the DING protein family (HPBP). Surprisingly, the structure of HPBP reported previously was also determined accidentally as a contaminant, suggesting that HPBP has a strong tendency to crystallize. Furthermore, DING proteins were reported to relate in disease. These observations suggest that DING has potential for application in a wide range of research fields. To enable further analyses, a system for preparation of HPBP was constructed. As HPBP was expressed in insoluble form in Escherichia coli, it was unfolded chemically and refolded. Finally, a very high yield preparation method was constructed, in which 43 mg of HPBP was obtained from 1 L of culture. Furthermore, to evaluate the validity of refolding, its crystal structure was determined at 1.03 Å resolution. The determined structure was identical to the native structure, in which two disulfide bonds were recovered correctly and a phosphate ion was captured. Based on these results, it was concluded that the refolded HPBP recovers its structure correctly.

  5. Purification, crystallization and preliminary X-ray diffraction of fluorescence recovery protein from Synechocystis PCC 6803

    International Nuclear Information System (INIS)

    Liu, Ting; Shuai, Yingli; Zhou, Honggang

    2011-01-01

    Fluorescence recovery protein from Synechocystis PCC 6803 plays a key role in the orange carotenoid protein-related photoprotective mechanism in cyanobacteria. The full-length form and a truncated form were overexpressed, purified and crystallized, and diffraction was observed to 2.75 Å resolution. Fluorescence recovery protein (FRP), which is encoded by the slr1964 gene in Synechocystis PCC 6803, plays a key role in the orange carotenoid protein-related photoprotective mechanism in cyanobacteria. As the crystal structure of FRP may provide information about the biological functions and mechanism of action of the protein, recombinant full-length FRP and a truncated form were overexpressed, purified and crystallized at 291 K using ethylene imine polymer as the precipitant. An FRP data set was collected to a resolution of 2.75 Å at low temperature (100 K). The crystal belonged to space group P4 1 2 1 2, with unit-cell parameters a = b = 61.9, c = 160.7 Å, α = β = γ = 90°. Assuming that the asymmetric unit contains three molecules, the Matthews coefficient was calculated to be 2.1 Å 3 Da −1

  6. Crystallization, diffraction data collection and preliminary crystallographic analysis of DING protein from Pseudomonas fluorescens

    International Nuclear Information System (INIS)

    Moniot, Sebastien; Elias, Mikael; Kim, Donghyo; Scott, Ken; Chabriere, Eric

    2007-01-01

    Crystallization of DING protein from P. fluorescens is reported. A complete data set was collected to 1.43 Å resolution. PfluDING is a phosphate-binding protein expressed in Pseudomonas fluorescens. This protein is clearly distinct from the bacterial ABC transporter soluble phosphate-binding protein PstS and is more homologous to eukaryotic DING proteins. Interestingly, bacterial DING proteins have only been detected in certain Pseudomonas species. Although DING proteins seem to be ubiquitous in eukaryotes, they are systematically absent from eukaryotic genomic databases and thus are still quite mysterious and poorly characterized. PfluDING displays mitogenic activity towards human cells and binds various ligands such as inorganic phosphate, pyrophosphate, nucleotide triphosphates and cotinine. Here, the crystallization of PfluDING is reported in a monoclinic space group (P2 1 ), with typical unit-cell parameters a = 36.7, b = 123.7, c = 40.8 Å, α = 90, β = 116.7, γ = 90°. Preliminary crystallographic analysis reveals good diffraction quality for these crystals and a 1.43 Å resolution data set has been collected

  7. Ultra-High Pressure Homogenization improves oxidative stability and interfacial properties of soy protein isolate-stabilized emulsions.

    Science.gov (United States)

    Fernandez-Avila, C; Trujillo, A J

    2016-10-15

    Ultra-High Pressure Homogenization (100-300MPa) has great potential for technological, microbiological and nutritional aspects of fluid processing. Its effect on the oxidative stability and interfacial properties of oil-in-water emulsions prepared with 4% (w/v) of soy protein isolate and soybean oil (10 and 20%, v/v) were studied and compared to emulsions treated by conventional homogenization (15MPa). Emulsions were characterized by particle size, emulsifying activity index, surface protein concentration at the interface and by transmission electron microscopy. Primary and secondary lipid oxidation products were evaluated in emulsions upon storage. Emulsions with 20% oil treated at 100 and 200MPa exhibited the most oxidative stability due to higher amount of oil and protein surface load at the interface. This manuscript addresses the improvement in oxidative stability in emulsions treated by UHPH when compared to conventional emulsions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Protein preparation, crystallization and preliminary X-ray crystallographic analysis of SMU.961 protein from the caries pathogen Streptococcus mutans

    International Nuclear Information System (INIS)

    Gao, Xiong-Zhuo; Li, Lan-Fen; Su, Xiao-Dong; Zhao, XiaoJun; Liang, Yu-He

    2007-01-01

    The SMU.961 protein from S. mutans was crystallized and preliminary characterization of the crystals, which diffracted to 2.9 Å resolution, shows them to belong to space group C2. The smu.961 gene encodes a putative protein of 183 residues in Streptococcus mutans, a major pathogen in human dental caries. The gene was cloned into expression vector pET28a and expressed in a substantial quantity in Escherichia coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.961 was purified to homogeneity in a two-step procedure consisting of Ni 2+ -chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.9 Å resolution at beamline I911-3, MAX-II-lab, Sweden. The crystal belonged to space group C2, with unit-cell parameters a = 98.62, b = 73.73, c = 184.73 Å, β = 98.82°

  9. Protein preparation, crystallization and preliminary X-ray crystallographic analysis of SMU.961 protein from the caries pathogen Streptococcus mutans

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Xiong-Zhuo [Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China); National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Li, Lan-Fen; Su, Xiao-Dong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Zhao, XiaoJun, E-mail: zhaoxj@scu.edu.cn [Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China); Liang, Yu-He, E-mail: zhaoxj@scu.edu.cn [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu 610065, Sichuan (China)

    2007-10-01

    The SMU.961 protein from S. mutans was crystallized and preliminary characterization of the crystals, which diffracted to 2.9 Å resolution, shows them to belong to space group C2. The smu.961 gene encodes a putative protein of 183 residues in Streptococcus mutans, a major pathogen in human dental caries. The gene was cloned into expression vector pET28a and expressed in a substantial quantity in Escherichia coli strain BL21 (DE3) with a His tag at its N-terminus. The recombinant protein SMU.961 was purified to homogeneity in a two-step procedure consisting of Ni{sup 2+}-chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained by the hanging-drop vapour-diffusion method and diffracted to 2.9 Å resolution at beamline I911-3, MAX-II-lab, Sweden. The crystal belonged to space group C2, with unit-cell parameters a = 98.62, b = 73.73, c = 184.73 Å, β = 98.82°.

  10. Regulation of PCNA-protein interactions for genome stability

    DEFF Research Database (Denmark)

    Mailand, Niels; Gibbs-Seymour, Ian; Bekker-Jensen, Simon

    2013-01-01

    Proliferating cell nuclear antigen (PCNA) has a central role in promoting faithful DNA replication, providing a molecular platform that facilitates the myriad protein-protein and protein-DNA interactions that occur at the replication fork. Numerous PCNA-associated proteins compete for binding...

  11. Ultrafast crystallization and thermal stability of In-Ge doped eutectic Sb70Te30 phase change material

    International Nuclear Information System (INIS)

    Lee Meiling; Miao Xiangshui; Ting Leehou; Shi Luping

    2008-01-01

    Effect of In and Ge doping in the form of In 2 Ge 8 Sb 85 Te 5 on optical and thermal properties of eutectic Sb 70 Te 30 alloys was investigated. Crystalline structure of In 2 Ge 8 Sb 85 Te 5 phase change material consists of a mixture of phases. Thermal analysis shows higher crystallization temperature and activation energy for crystallization. Isothermal reflectivity-time measurement shows a growth-dominated crystallization mechanism. Ultrafast crystallization speed of 30 ns is realized upon irradiation by blue laser beam. The use of ultrafast and thermally stable In 2 Ge 8 Sb 85 Te 5 phase change material as mask layer in aperture-type super-resolution near-field phase change disk is realized to increase the carrier-to-noise ratio and thermal stability

  12. Thermal stability of radiation-induced free radicals in γ-irradiated l-alanine single crystals

    International Nuclear Information System (INIS)

    Maltar-Strmecki, N.; Rakvin, B.

    2005-01-01

    Decay of the radiation-induced stable free radicals in l-alanine single crystals and powders at the temperatures from 379 to 476K was examined by electron paramagnetic resonance. For single crystals, the calculated activation energy of the radical decay is 104.3±1.7kJ/mol (i.e. 12 538+/-202K) and the frequency factor lnν 0 is 24.1±0.4min -1 . The lifetime of the radical in single crystals at 296K is 162 years. The results confirm the long-term stability of the radicals, but the decay was found to be faster in large crystals than in powders

  13. Cloning and characterization of an insecticidal crystal protein gene ...

    Indian Academy of Sciences (India)

    A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed in E. coli. The recombinant protein produced 92% mortality in first-instar larvae of Spodoptera litura and 86% inhibition of adult emergence in Phthorimaea operculella, but showed very low toxicity against ...

  14. Understanding water equilibration fundamentals as a step for rational protein crystallization.

    Directory of Open Access Journals (Sweden)

    Pedro M Martins

    Full Text Available BACKGROUND: Vapor diffusion is the most widely used technique for protein crystallization and the rate of water evaporation plays a key role on the quality of the crystals. Attempts have been made in the past to solve the mass transfer problem governing the evaporation process, either analytically or by employing numerical methods. Despite these efforts, the methods used for protein crystallization remain based on trial and error techniques rather than on fundamental principles. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a new theoretical model which describes the hanging drop method as a function of the different variables that are known to influence the evaporation process. The model is extensively tested against experimental data published by other authors and considering different crystallizing conditions. Aspects responsible for the discrepancies between the existing theories and the measured evaporation kinetics are especially discussed; they include the characterization of vapor-liquid equilibrium, the role of mass transfer within the evaporating droplet, and the influence of the droplet-reservoir distance. CONCLUSIONS/SIGNIFICANCE: The validation tests show that the proposed model can be used to predict the water evaporation rates under a wide range of experimental conditions used in the hanging drop vapor-diffusion method, with no parameter fitting or computational requirements. This model combined with protein solubility data is expected to become a useful tool for a priori screening of crystallization conditions.

  15. Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging

    International Nuclear Information System (INIS)

    Warren, Anna J.; Armour, Wes; Axford, Danny; Basham, Mark; Connolley, Thomas; Hall, David R.; Horrell, Sam; McAuley, Katherine E.; Mykhaylyk, Vitaliy; Wagner, Armin; Evans, Gwyndaf

    2013-01-01

    A comparison of X-ray diffraction and radiographic techniques for the location and characterization of protein crystals is demonstrated on membrane protein crystals mounted within lipid cubic phase material. The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required

  16. Modeling of protein electrophoresis in silica colloidal crystals having brush layers of polyacrylamide

    Science.gov (United States)

    Birdsall, Robert E.; Koshel, Brooke M.; Hua, Yimin; Ratnayaka, Saliya N.; Wirth, Mary J.

    2013-01-01

    Sieving of proteins in silica colloidal crystals of mm dimensions is characterized for particle diameters of nominally 350 and 500 nm, where the colloidal crystals are chemically modified with a brush layer of polyacrylamide. A model is developed that relates the reduced electrophoretic mobility to the experimentally measurable porosity. The model fits the data with no adjustable parameters for the case of silica colloidal crystals packed in capillaries, for which independent measurements of the pore radii were made from flow data. The model also fits the data for electrophoresis in a highly ordered colloidal crystal formed in a channel, where the unknown pore radius was used as a fitting parameter. Plate heights as small as 0.4 μm point to the potential for miniaturized separations. Band broadening increases as the pore radius approaches the protein radius, indicating that the main contribution to broadening is the spatial heterogeneity of the pore radius. The results quantitatively support the notion that sieving occurs for proteins in silica colloidal crystals, and facilitate design of new separations that would benefit from miniaturization. PMID:23229163

  17. Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging

    Energy Technology Data Exchange (ETDEWEB)

    Warren, Anna J. [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Armour, Wes [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Oxford e-Research Centre, 7 Keble Road, Oxford OX1 3QG (United Kingdom); Axford, Danny; Basham, Mark; Connolley, Thomas; Hall, David R. [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Horrell, Sam [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); University of Liverpool, Liverpool L69 3BX (United Kingdom); McAuley, Katherine E.; Mykhaylyk, Vitaliy; Wagner, Armin; Evans, Gwyndaf, E-mail: gwyndaf.evans@diamond.ac.uk [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom)

    2013-07-01

    A comparison of X-ray diffraction and radiographic techniques for the location and characterization of protein crystals is demonstrated on membrane protein crystals mounted within lipid cubic phase material. The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required.

  18. Role of Tamm-Horsfall protein and uromodulin in calcium oxalate crystallization

    Directory of Open Access Journals (Sweden)

    Carvalho M.

    2002-01-01

    Full Text Available One of the defenses against nephrolithiasis is provided by macromolecules that modulate the nucleation, growth, aggregation and retention of crystals in the kidneys. The aim of the present study was to determine the behavior of two of these proteins, Tamm-Horsfall and uromodulin, in calcium oxalate crystallization in vitro. We studied a group of 10 male stone formers who had formed at least one kidney stone composed of calcium oxalate. They were classified as having idiopathic nephrolithiasis and had no well-known metabolic risk factors involved in kidney stone pathogenesis. Ten normal men were used as controls, as was a group consisting of five normal women and another consisting of five pregnant women. Crystallization was induced by a fixed supersaturation of calcium oxalate and measured with a Coulter Counter. All findings were confirmed by light and scanning electron microscopy. The number of particulate material deposited from patients with Tamm-Horsfall protein was higher than that of the controls (P<0.001. However, Tamm-Horsfall protein decreased the particle diameter of the stone formers when analyzed by the mode of the volume distribution curve (P<0.002 (5.64 ± 0.55 µm compared to 11.41 ± 0.48 µm of uromodulin; 15.94 ± 3.93 µm and 12.45 ± 0.97 µm of normal men Tamm-Horsfall protein and uromodulin, respectively; 8.17 ± 1.57 µm and 9.82 ± 0.95 µm of normal women Tamm-Horsfall protein and uromodulin, respectively; 12.17 ± 1.41 µm and 12.99 ± 0.51 µm of pregnant Tamm-Horsfall protein and uromodulin, respectively. Uromodulin produced fewer particles than Tamm-Horsfall protein in all groups. Nonetheless, the total volume of the crystals produced by uromodulin was higher than that produced by Tamm-Horsfall protein. Our results indicate a different effect of Tamm-Horsfall protein and uromodulin. This dual behavior suggests different functions. Tamm-Horsfall protein may act on nucleation and inhibit crystal aggregation, while

  19. Cloning, Expression, Purification, Crystallization and Preliminary X-ray Analysis of Mycoplasma Genitalium Protein MG289

    Energy Technology Data Exchange (ETDEWEB)

    Sippel, K.; Boehlein, S; Sakai, Y; Quirit, J; Agbandje-McKenna, M; Rosser, C; McKenna, R

    2009-01-01

    Mycoplasma genitalium is a human pathogen that is associated with nongonococcal urethritis in men and cervicitis in women. The cloning, expression, purification and crystallization of the protein MG289 from M. genitalium strain G37 are reported here. Crystals of MG289 diffracted X-rays to 2.8 {angstrom} resolution. The crystals belonged to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 49.7, b = 90.9, c = 176.1 {angstrom}. The diffraction data after processing had an overall R{sub merge} of 8.7%. The crystal structure of Cypl, the ortholog of MG289 from M. hyorhinis, has recently been determined, providing a reasonable phasing model; molecular replacement is currently under way.

  20. Precise Manipulation and Patterning of Protein Crystals for Macromolecular Crystallography Using Surface Acoustic Waves.

    Science.gov (United States)

    Guo, Feng; Zhou, Weijie; Li, Peng; Mao, Zhangming; Yennawar, Neela H; French, Jarrod B; Huang, Tony Jun

    2015-06-01

    Advances in modern X-ray sources and detector technology have made it possible for crystallographers to collect usable data on crystals of only a few micrometers or less in size. Despite these developments, sample handling techniques have significantly lagged behind and often prevent the full realization of current beamline capabilities. In order to address this shortcoming, a surface acoustic wave-based method for manipulating and patterning crystals is developed. This method, which does not damage the fragile protein crystals, can precisely manipulate and pattern micrometer and submicrometer-sized crystals for data collection and screening. The technique is robust, inexpensive, and easy to implement. This method not only promises to significantly increase efficiency and throughput of both conventional and serial crystallography experiments, but will also make it possible to collect data on samples that were previously intractable. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Electrostatic contribution of surface charge residues to the stability of a thermophilic protein: benchmarking experimental and predicted pKa values.

    Directory of Open Access Journals (Sweden)

    Chi-Ho Chan

    Full Text Available Optimization of the surface charges is a promising strategy for increasing thermostability of proteins. Electrostatic contribution of ionizable groups to the protein stability can be estimated from the differences between the pKa values in the folded and unfolded states of a protein. Using this pKa-shift approach, we experimentally measured the electrostatic contribution of all aspartate and glutamate residues to the stability of a thermophilic ribosomal protein L30e from Thermococcus celer. The pKa values in the unfolded state were found to be similar to model compound pKas. The pKa values in both the folded and unfolded states obtained at 298 and 333 K were similar, suggesting that electrostatic contribution of ionizable groups to the protein stability were insensitive to temperature changes. The experimental pKa values for the L30e protein in the folded state were used as a benchmark to test the robustness of pKa prediction by various computational methods such as H++, MCCE, MEAD, pKD, PropKa, and UHBD. Although the predicted pKa values were affected by crystal contacts that may alter the side-chain conformation of surface charged residues, most computational methods performed well, with correlation coefficients between experimental and calculated pKa values ranging from 0.49 to 0.91 (p<0.01. The changes in protein stability derived from the experimental pKa-shift approach correlate well (r = 0.81 with those obtained from stability measurements of charge-to-alanine substituted variants of the L30e protein. Our results demonstrate that the knowledge of the pKa values in the folded state provides sufficient rationale for the redesign of protein surface charges leading to improved protein stability.

  2. The Stanford Automated Mounter: Enabling High-Throughput Protein Crystal Screening at SSRL

    International Nuclear Information System (INIS)

    Smith, C.A.; Cohen, A.E.

    2009-01-01

    The macromolecular crystallography experiment lends itself perfectly to high-throughput technologies. The initial steps including the expression, purification, and crystallization of protein crystals, along with some of the later steps involving data processing and structure determination have all been automated to the point where some of the last remaining bottlenecks in the process have been crystal mounting, crystal screening, and data collection. At the Stanford Synchrotron Radiation Laboratory, a National User Facility that provides extremely brilliant X-ray photon beams for use in materials science, environmental science, and structural biology research, the incorporation of advanced robotics has enabled crystals to be screened in a true high-throughput fashion, thus dramatically accelerating the final steps. Up to 288 frozen crystals can be mounted by the beamline robot (the Stanford Auto-Mounting System) and screened for diffraction quality in a matter of hours without intervention. The best quality crystals can then be remounted for the collection of complete X-ray diffraction data sets. Furthermore, the entire screening and data collection experiment can be controlled from the experimenter's home laboratory by means of advanced software tools that enable network-based control of the highly automated beamlines.

  3. Comparison of the colloidal stability, bioaccessibility and antioxidant activity of corn protein hydrolysate and sodium caseinate stabilized curcumin nanoparticles.

    Science.gov (United States)

    Wang, Yong-Hui; Yuan, Yang; Yang, Xiao-Quan; Wang, Jin-Mei; Guo, Jian; Lin, Yuan

    2016-07-01

    The aims of this work were to construct corn protein hydrolysate (CPH)-based curcumin nanoparticles (Cur NPs) and to compare the colloidal stability, bioaccessibility and antioxidant activity of the Cur NPs stabilized CPH and sodium caseinate (NaCas) respectively. The results indicated that Cur solubility could be considerably improved after the Cur NPs fabrication. The spectroscopy results demonstrated that the solubilization of Cur should be attributed to its complexation with CPH or NaCas. The Cur NPs exhibited good colloidal stability after 1 week's storage but showed smaller (40 nm) size in CPH than in NaCas (100 nm). After lyophilization, the Cur NPs powders showed good rehydration properties and chemical stability, and compared with NaCas, the size of Cur NPs stabilized by CPH was still smaller. Additionally, the Cur NPs exhibited higher chemical stability against the temperature compared with free Cur, and the CPH could protect Cur from degradation more efficiently. Comparing with NaCas, the Cur NPs stabilized by CPH exhibited better bioaccessibility and antioxidant activity. This study demonstrated that CPH may be better than NaCas in Cur NPs fabrication and it opens up the possibility of using hydrophobic protein hydrolysate to construct the NPs delivery system.

  4. High-resolution diffraction from crystals of a membrane-protein complex: bacterial outer membrane protein OmpC complexed with the antibacterial eukaryotic protein lactoferrin

    International Nuclear Information System (INIS)

    Sundara Baalaji, N.; Acharya, K. Ravi; Singh, T. P.; Krishnaswamy, S.

    2005-01-01

    Crystals of the complex formed between the bacterial membrane protein OmpC and the antibacterial protein lactoferrin suitable for high-resolution structure determination have been obtained. The crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å. Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å, α = β = 90, γ = 120°. This indicated a Matthews coefficient (V M ) of 3.3 Å 3 Da −1 , corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 Å resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin

  5. Anchoring protein crystals to mounting loops with hydrogel using inkjet technology.

    Science.gov (United States)

    Shinoda, Akira; Tanaka, Yoshikazu; Yao, Min; Tanaka, Isao

    2014-11-01

    X-ray crystallography is an important technique for structure-based drug discovery, mainly because it is the only technique that can reveal whether a ligand binds to the target protein as well as where and how it binds. However, ligand screening by X-ray crystallography involves a crystal-soaking experiment, which is usually performed manually. Thus, the throughput is not satisfactory for screening large numbers of candidate ligands. In this study, a technique to anchor protein crystals to mounting loops by using gel and inkjet technology has been developed; the method allows soaking of the mounted crystals in ligand-containing solution. This new technique may assist in the design of a fully automated drug-screening pipeline.

  6. Expression, purification, crystallization and preliminary crystallographic analysis of laminin-binding protein (Lmb) from Streptococcus agalactiae

    International Nuclear Information System (INIS)

    Ragunathan, Preethi; Spellerberg, Barbara; Ponnuraj, Karthe

    2009-01-01

    Laminin-binding protein from S. agalactiae was expressed, purified and crystallized and X-ray diffraction data were collected to 2.5 Å resolution. Laminin-binding protein (Lmb), a surface-exposed lipoprotein from Streptococcus agalactiae (group B streptococcus), mediates attachment to human laminin and plays a crucial role in the adhesion/invasion of eukaryotic host cells. However, the structural basis of laminin binding still remains unclear. In the context of detailed structural analysis, the lmb gene has been cloned, expressed in Escherichia coli, purified and crystallized. The crystals diffracted to a resolution of 2.5 Å and belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 56.63, b = 70.60, c = 75.37 Å, β = 96.77°

  7. Purification, crystallization and preliminary crystallographic analysis of Streptococcus pyogenes laminin-binding protein Lbp

    International Nuclear Information System (INIS)

    Linke, Christian; Caradoc-Davies, Tom T.; Proft, Thomas; Baker, Edward N.

    2008-01-01

    The S. pyogenes laminin-binding protein Lbp, which is essential for adhesion to human laminin, has been expressed, purified and crystallized. The laminin-binding protein Lbp (Spy2007) from Streptococcus pyogenes (a group A streptococcus) mediates adhesion to the human basal lamina glycoprotein laminin. Accordingly, Lbp is essential in in vitro models of cell adhesion and invasion. However, the molecular and structural basis of laminin binding by bacteria remains unknown. Therefore, the lbp gene has been cloned for recombinant expression in Escherichia coli. Lbp has been purified and crystallized from 30%(w/v) PEG 1500 by the sitting-drop vapour-diffusion method. The crystals belonged to the monoclinic space group P2 1 , with unit-cell parameters a = 42.62, b = 92.16, c = 70.61 Å, β = 106.27°, and diffracted to 2.5 Å resolution

  8. Purification, crystallization and preliminary crystallographic analysis of Streptococcus pyogenes laminin-binding protein Lbp

    Energy Technology Data Exchange (ETDEWEB)

    Linke, Christian, E-mail: clin180@ec.auckland.ac.nz [School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland (New Zealand); Caradoc-Davies, Tom T. [School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland (New Zealand); Australian Synchrotron, Clayton, Victoria 3168 (Australia); Proft, Thomas [School of Medical Sciences, University of Auckland, Private Bag 92019, Auckland (New Zealand); Baker, Edward N. [School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland (New Zealand)

    2008-02-01

    The S. pyogenes laminin-binding protein Lbp, which is essential for adhesion to human laminin, has been expressed, purified and crystallized. The laminin-binding protein Lbp (Spy2007) from Streptococcus pyogenes (a group A streptococcus) mediates adhesion to the human basal lamina glycoprotein laminin. Accordingly, Lbp is essential in in vitro models of cell adhesion and invasion. However, the molecular and structural basis of laminin binding by bacteria remains unknown. Therefore, the lbp gene has been cloned for recombinant expression in Escherichia coli. Lbp has been purified and crystallized from 30%(w/v) PEG 1500 by the sitting-drop vapour-diffusion method. The crystals belonged to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 42.62, b = 92.16, c = 70.61 Å, β = 106.27°, and diffracted to 2.5 Å resolution.

  9. Expression, purification and crystallization of two major envelope proteins from white spot syndrome virus

    International Nuclear Information System (INIS)

    Tang, Xuhua; Hew, Choy Leong

    2007-01-01

    The crystallization of the N-terminal transmembrane region-truncated VP26 and VP28 of white spot syndrome virus is described. White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals of SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 M sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 Å resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 Å. SeMet-labelled VP28 crystallizes in space group P2 1 2 1 2 1 , with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 Å, and diffracts to 2.0 Å resolution

  10. Expression, purification and crystallization of two major envelope proteins from white spot syndrome virus

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Xuhua; Hew, Choy Leong, E-mail: dbshewcl@nus.edu.sg [Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543 (Singapore)

    2007-07-01

    The crystallization of the N-terminal transmembrane region-truncated VP26 and VP28 of white spot syndrome virus is described. White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals of SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 M sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 Å resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 Å. SeMet-labelled VP28 crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 Å, and diffracts to 2.0 Å resolution.

  11. Imaging transport phenomena during lysozyme protein crystal growth by the hanging drop technique

    Science.gov (United States)

    Sethia Gupta, Anamika; Gupta, Rajive; Panigrahi, P. K.; Muralidhar, K.

    2013-06-01

    The present study reports the transport process that occurs during the growth of lysozyme protein crystals by the hanging drop technique. A rainbow schlieren technique has been employed for imaging changes in salt concentration. A one dimensional color filter is used to record the deflection of the light beam. An optical microscope and an X-ray crystallography unit are used to characterize the size, tetragonal shape and Bravais lattice constants of the grown crystals. A parametric study on the effect of drop composition, drop size, reservoir height and number of drops on the crystal size and quality is reported. Changes in refractive index are not large enough to create a meaningful schlieren image in the air gap between the drop and the reservoir. However, condensation of fresh water over the reservoir solution creates large changes in the concentration of NaCl, giving rise to clear color patterns in the schlieren images. These have been analyzed to obtain salt concentration profiles near the free surface of the reservoir solution as a function of time. The diffusion of fresh water into the reservoir solution at the early stages of crystal growth followed by the mass flux of salt from the bulk solution towards the free surface has been recorded. The overall crystal growth process can be classified into two regimes, as demarcated by the changes in slope of salt concentration within the reservoir. The salt concentration in the reservoir equilibrates at long times when the crystallization process is complete. Thus, transport processes in the reservoir emerge as the route to monitor protein crystal growth in the hanging drop configuration. Results show that crystal growth rate is faster for a higher lysozyme concentration, smaller drops, and larger reservoir heights.

  12. EDM-DEDM and protein crystal structure solution.

    Science.gov (United States)

    Caliandro, Rocco; Carrozzini, Benedetta; Cascarano, Giovanni Luca; Giacovazzo, Carmelo; Mazzone, Anna Maria; Siliqi, Dritan

    2009-05-01

    Electron-density modification (EDM) procedures are the classical tool for driving model phases closer to those of the target structure. They are often combined with automated model-building programs to provide a correct protein model. The task is not always performed, mostly because of the large initial phase error. A recently proposed procedure combined EDM with DEDM (difference electron-density modification); the method was applied to the refinement of phases obtained by molecular replacement, ab initio or SAD phasing [Caliandro, Carrozzini, Cascarano, Giacovazzo, Mazzone & Siliqi (2009), Acta Cryst. D65, 249-256] and was more effective in improving phases than EDM alone. In this paper, a novel fully automated protocol for protein structure refinement based on the iterative application of automated model-building programs combined with the additional power derived from the EDM-DEDM algorithm is presented. The cyclic procedure was successfully tested on challenging cases for which all other approaches had failed.

  13. The Nucleation of Protein Aggregates - From Crystals to Amyloid Fibrils.

    Science.gov (United States)

    Buell, Alexander K

    2017-01-01

    The condensation and aggregation of individual protein molecules into dense insoluble phases is of relevance in such diverse fields as materials science, medicine, structural biology and pharmacology. A common feature of these condensation phenomena is that they usually are nucleated processes, i.e. the first piece of the condensed phase is energetically costly to create and hence forms slowly compared to its subsequent growth. Here we give a compact overview of the differences and similarities of various protein nucleation phenomena, their theoretical description in the framework of colloid and polymer science and their experimental study. Particular emphasis is put on the nucleation of a specific type of filamentous protein aggregates, amyloid fibrils. The current experimentally derived knowledge on amyloid fibril nucleation is critically assessed, and we argue that it is less advanced than is generally believed. This is due to (I) the lack of emphasis that has been put on the distinction between homogeneous and heterogeneous nucleation in experimental studies (II) the use of oversimplifying and/or inappropriate theoretical frameworks for the analysis of kinetic data of amyloid fibril nucleation. A strategy is outlined and advocated of how our understanding of this important class of processes can be improved in the future. © 2017 Elsevier Inc. All rights reserved.

  14. Resistance: a threat to the insecticidal crystal proteins of Bacillus thuringiensis

    Science.gov (United States)

    Leah S. Bauer

    1995-01-01

    Insecticidal crystal proteins (also known as d-endotoxins) synthesized by the bacterium Bacillus thuringiensis Berliner (Bt) are the active ingredient of various environmentally friendly insecticides that are 1) highly compatible with natural enemies and other nontarget organisms due to narrow host specificity, 2) harmless to vertebrates, 3) biodegradable in the...

  15. Overexpression, purification and crystallization of a choline-binding protein CbpI from Streptococcus pneumoniae

    International Nuclear Information System (INIS)

    Paterson, Neil G.; Riboldi-Tunicliffe, Alan; Mitchell, Timothy J.; Isaacs, Neil W.

    2006-01-01

    The choline-binding protein CbpI from S. pneumoniae has been purified and crystallized and diffraction data have been collected to 3.5 Å resolution. The choline-binding protein CbpI from Streptococcus pneumoniae is a 23.4 kDa protein with no known function. The protein has been successfully purified initially using Ni–NTA chromatography and to homogeneity using Q-Sepharose ion-exchange resin as an affinity column. CbpI was crystallized using PEG 3350 as a precipitant and X-ray crystallographic analysis showed that the crystals belonged to the tetragonal space group P4, with unit-cell parameters a = b = 83.31, c = 80.29 Å, α = β = γ = 90°. The crystal contains two molecules in the asymmetric unit with a solvent content of 55.7% (V M = 2.77 Å 3 Da −1 ) and shows a diffraction limit of 3.5 Å

  16. Crystal structure of the β2 adrenergic receptor-Gs protein complex

    DEFF Research Database (Denmark)

    Rasmussen, Søren Gøgsig Faarup; DeVree, Brian T; Zou, Yaozhong

    2011-01-01

    -occupied receptor. The β(2) adrenergic receptor (β(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β(2)AR and nucleotide-free Gs...

  17. Fluid-crystal coexistence for proteins and inorganic nanocolloids : Dependence on ionic strength

    NARCIS (Netherlands)

    Prinsen, P.; Odijk, T.

    2006-01-01

    We investigate theoretically the fluid-crystal coexistence of solutions of globular charged nanoparticles such as proteins and inorganic colloids. The thermodynamic properties of the fluid phase are computed via the optimized Baxter model P. Prinsen and T. Odijk [J. Chem. Phys. 121, 6525 (2004)].

  18. Expression, purification, crystallization and preliminary crystallographic analysis of the proliferation-associated protein Ebp1

    Energy Technology Data Exchange (ETDEWEB)

    Kowalinski, Eva; Bange, Gert; Wild, Klemens; Sinning, Irmgard, E-mail: irmi.sinning@bzh.uni-heidelberg.de [Heidelberg University Biochemistry Center, INF 328, D-69120 Heidelberg (Germany)

    2007-09-01

    Preliminary X-ray analysis of the proliferation-associated protein Ebp1 from Homo sapiens is provided. ErbB-3-binding protein 1 (Ebp1) is a member of the family of proliferation-associated 2G4 proteins (PA2G4s) and plays a role in cellular growth and differentiation. Ligand-induced activation of the transmembrane receptor ErbB3 leads to dissociation of Ebp1 from the receptor in a phosphorylation-dependent manner. The non-associated protein is involved in transcriptional and translational regulation in the cell. Here, the overexpression, purification, crystallization and preliminary crystallographic studies of Ebp1 from Homo sapiens are reported. Initially observed crystals were improved by serial seeding to single crystals suitable for data collection. The optimized crystals belong to the tetragonal space group P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2 and diffracted to a resolution of 1.6 Å.

  19. Computer simulations of radiation damage in protein crystals; Simulationsrechnungen zu Strahlenschaeden an Proteinkristallen

    Energy Technology Data Exchange (ETDEWEB)

    Zehnder, M

    2007-03-15

    The achievable resolution and the quality of the dataset of an intensity data collection for structure analysis of protein crystals with X-rays is limited among other factors by radiation damage. The aim of this work is to obtain a better quantitative understanding of the radiation damage process in proteins. Since radiation damage is unavoidable it was intended to look for the optimum ratio between elastically scattered intensity and radiation damage. Using a Monte Carlo algorithm physical processes after an inelastic photon interaction are studied. The main radiation damage consists of ionizations of the atoms through the electron cascade following any inelastic photon interaction. Results of the method introduced in this investigation and results of an earlier theoretical studies of the influence of Auger-electron transport in diamond are in a good agreement. The dependence of the radiation damage as a function of the energy of the incident photon was studied by computer-aided simulations. The optimum energy range for diffraction experiments on the protein myoglobin is 10-40 keV. Studies of radiation damage as a function of crystal volume and shape revealed that very small plate or rod shaped crystals suffer less damage than crystals formed like a cube with the same volume. Furthermore the influence of a few heavy atoms in the protein molecule on radiation damage was examined. Already two iron atoms in the unit cell of myoglobin increase radiation damage significantly. (orig.)

  20. Three-Dimentional Structures of Autophosphorylation Complexes in Crystals of Protein Kinases

    KAUST Repository

    Dumbrack, Roland

    2016-01-26

    Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Several autophosphorylation complexes have been identified in crystals of protein kinases, with a known serine, threonine, or tyrosine autophosphorylation site of one kinase monomer sitting in the active site of another monomer of the same protein in the crystal. We utilized a structural bioinformatics method to identify all such autophosphorylation complexes in X-ray crystallographic structures in the Protein Data Bank (PDB) by generating all unique kinase/kinase interfaces within and between asymmetric units of each crystal and measuring the distance between the hydroxyl oxygen of potential autophosphorylation sites and the oxygen atoms of the active site aspartic acid residue side chain. We have identified 15 unique autophosphorylation complexes in the PDB, of which 5 complexes have not previously been described in the relevant publications on the crystal structures (N-terminal juxtamembrane regions of CSF1R and EPHA2, activation loop tyrosines of LCK and IGF1R, and a serine in a nuclear localization signal region of CLK2. Mutation of residues in the autophosphorylation complex interface of LCK either severely impaired autophosphorylation or increased it. Taking the autophosphorylation complexes as a whole and comparing them with peptide-substrate/kinase complexes, we observe a number of important features among them. The novel and previously observed autophosphorylation sites are conserved in many kinases, indicating that by homology we can extend the relevance of these complexes to many other clinically relevant drug targets.

  1. Characterization of Baculovirus Insecticides Expressing Tailored Bacillus thuringiensis CryIA(b) Crystal Proteins

    NARCIS (Netherlands)

    Martens, John W M; Knoester, Marga; Weijts, Franci; Groffen, Sander J A; Hu, Zhihong; Bosch, Dirk; Vlak, Just M.

    1995-01-01

    Full-length, truncated, and mature forms of the CryIA(b) insecticidal crystal protein gene of Bacillus thuringiensis were engineered into the p10 locus of Autographa californica nuclear polyhedrosis virus (AcNPV). A signal sequence of Heliothis virescens juvenile hormone esterase was introduced at

  2. Overexpression, purification and crystallization of a choline-binding protein CbpI from Streptococcus pneumoniae

    Energy Technology Data Exchange (ETDEWEB)

    Paterson, Neil G., E-mail: neison@chem.gla.ac.uk; Riboldi-Tunicliffe, Alan [Department of Chemistry and WestCHEM, Glasgow Biomedical Research Centre (GBRC), University of Glasgow, 120 University Place, Glasgow G12 8TA,Scotland (United Kingdom); Mitchell, Timothy J. [Division of Infection and Immunity (IBLS), Glasgow Biomedical Research Centre (GBRC), University of Glasgow, 120 University Place, Glasgow G12 8TA,Scotland (United Kingdom); Isaacs, Neil W. [Department of Chemistry and WestCHEM, Glasgow Biomedical Research Centre (GBRC), University of Glasgow, 120 University Place, Glasgow G12 8TA,Scotland (United Kingdom)

    2006-07-01

    The choline-binding protein CbpI from S. pneumoniae has been purified and crystallized and diffraction data have been collected to 3.5 Å resolution. The choline-binding protein CbpI from Streptococcus pneumoniae is a 23.4 kDa protein with no known function. The protein has been successfully purified initially using Ni–NTA chromatography and to homogeneity using Q-Sepharose ion-exchange resin as an affinity column. CbpI was crystallized using PEG 3350 as a precipitant and X-ray crystallographic analysis showed that the crystals belonged to the tetragonal space group P4, with unit-cell parameters a = b = 83.31, c = 80.29 Å, α = β = γ = 90°. The crystal contains two molecules in the asymmetric unit with a solvent content of 55.7% (V{sub M} = 2.77 Å{sup 3} Da{sup −1}) and shows a diffraction limit of 3.5 Å.

  3. Purification, crystallization and X-ray diffraction analysis of human dynamin-related protein 1 GTPase-GED fusion protein

    International Nuclear Information System (INIS)

    Klinglmayr, Eva; Wenger, Julia; Mayr, Sandra; Bossy-Wetzel, Ella; Puehringer, Sandra

    2012-01-01

    The crystallization and initial diffraction analysis of human Drp1 GTPase-GED fusion protein are reported. The mechano-enzyme dynamin-related protein 1 plays an important role in mitochondrial fission and is implicated in cell physiology. Dysregulation of Drp1 is associated with abnormal mitochondrial dynamics and neuronal damage. Drp1 shares structural and functional similarities with dynamin 1 with respect to domain organization, ability to self-assemble into spiral-like oligomers and GTP-cycle-dependent membrane scission. Structural studies of human dynamin-1 have greatly improved the understanding of this prototypical member of the dynamin superfamily. However, high-resolution structural information for full-length human Drp1 covering the GTPase domain, the middle domain and the GTPase effector domain (GED) is still lacking. In order to obtain mechanistic insights into the catalytic activity, a nucleotide-free GTPase-GED fusion protein of human Drp1 was expressed, purified and crystallized. Initial X-ray diffraction experiments yielded data to 2.67 Å resolution. The hexagonal-shaped crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 53.59, b = 151.65, c = 43.53 Å, one molecule per asymmetric unit and a solvent content of 42%. Expression of selenomethionine-labelled protein is currently in progress. Here, the expression, purification, crystallization and X-ray diffraction analysis of the Drp1 GTPase-GED fusion protein are presented, which form a basis for more detailed structural and biophysical analysis

  4. Trade-off between positive and negative design of protein stability: from lattice models to real proteins.

    Directory of Open Access Journals (Sweden)

    Orly Noivirt-Brik

    2009-12-01

    Full Text Available Two different strategies for stabilizing proteins are (i positive design in which the native state is stabilized and (ii negative design in which competing non-native conformations are destabilized. Here, the circumstances under which one strategy might be favored over the other are explored in the case of lattice models of proteins and then generalized and discussed with regard to real proteins. The balance between positive and negative design of proteins is found to be determined by their average "contact-frequency", a property that corresponds to the fraction of states in the conformational ensemble of the sequence in which a pair of residues is in contact. Lattice model proteins with a high average contact-frequency are found to use negative design more than model proteins with a low average contact-frequency. A mathematical derivation of this result indicates that it is general and likely to hold also for real proteins. Comparison of the results of correlated mutation analysis for real proteins with typical contact-frequencies to those of proteins likely to have high contact-frequencies (such as disordered proteins and proteins that are dependent on chaperonins for their folding indicates that the latter tend to have stronger interactions between residues that are not in contact in their native conformation. Hence, our work indicates that negative design is employed when insufficient stabilization is achieved via positive design owing to high contact-frequencies.

  5. Recent advances in the applications of ionic liquids in protein stability and activity: a review.

    Science.gov (United States)

    Patel, Rajan; Kumari, Meena; Khan, Abbul Bashar

    2014-04-01

    Room temperatures ionic liquids are considered as miraculous solvents for biological system. Due to their inimitable properties and large variety of applications, they have been widely used in enzyme catalysis and protein stability and separation. The related information present in the current review is helpful to the researchers working in the field of biotechnology and biochemistry to design or choose an ionic liquid that can serve as a noble and selective solvent for any particular enzymatic reaction, protein preservation and other protein based applications. We have extensively analyzed the methods used for studying the protein-IL interaction which is useful in providing information about structural and conformational dynamics of protein. This can be helpful to develop and understanding about the effect of ionic liquids on stability and activity of proteins. In addition, the affect of physico-chemical properties of ionic liquids, viz. hydrogen bond capacity and hydrophobicity on protein stability are discussed.

  6. Mutation of exposed hydrophobic amino acids to arginine to increase protein stability

    Directory of Open Access Journals (Sweden)

    Czaplicki Jerzy

    2004-07-01

    Full Text Available Abstract Background One strategy to increase the stability of proteins is to reduce the area of water-accessible hydrophobic surface. Results In order to test it, we replaced 14 solvent-exposed hydrophobic residues of acetylcholinesterase by arginine. The stabilities of the resulting proteins were tested using denaturation by high temperature, organic solvents, urea and by proteolytic digestion. Conclusion Altough the mutational effects were rather small, this strategy proved to be successful since half of the mutants showed an increased stability. This stability may originate from the suppression of unfavorable interactions of nonpolar residues with water or from addition of new hydrogen bonds with the solvent. Other mechanisms may also contribute to the increased stability observed with some mutants. For example, introduction of a charge at the surface of the protein may provide a new coulombic interaction on the protein surface.

  7. Crystallization and preliminary electron diffraction study to 3. 7 A of DNA helix-destabilizing protein gp32*I

    Energy Technology Data Exchange (ETDEWEB)

    Chiu, W; Hosoda, J

    1978-01-01

    A two-dimensionally large and thin crystal has been obtained from gp32*I, a proteolytically digested product of a DNA helix-destabilizing protein coded by gene 32 in bacteriophage T4. High-resolution electron diffraction patterns (approx. 3.7 A) are recorded from both unstained and stained protein crystals embedded in glucose. The crystal is of orthorhombic space group with a = 62.9 A and b = 47.3 A.

  8. Model for calculation of electrostatic contribution into protein stability

    Science.gov (United States)

    Kundrotas, Petras; Karshikoff, Andrey

    2003-03-01

    Existing models of the denatured state of proteins consider only one possible spatial distribution of protein charges and therefore are applicable to a limited number of cases. In this presentation a more general framework for the modeling of the denatured state is proposed. It is based on the assumption that the titratable groups of an unfolded protein can adopt a quasi-random distribution, restricted by the protein sequence. The model was tested on two proteins, barnase and N-terminal domain of the ribosomal protein L9. The calculated free energy of denaturation, Δ G( pH), reproduces the experimental data essentially better than the commonly used null approximation (NA). It was demonstrated that the seemingly good agreement with experimental data obtained by NA originates from the compensatory effect between the pair-wise electrostatic interactions and the desolvation energy of the individual sites. It was also found that the ionization properties of denatured proteins are influenced by the protein sequence.

  9. A rapid, ensemble and free energy based method for engineering protein stabilities.

    Science.gov (United States)

    Naganathan, Athi N

    2013-05-02

    Engineering the conformational stabilities of proteins through mutations has immense potential in biotechnological applications. It is, however, an inherently challenging problem given the weak noncovalent nature of the stabilizing interactions. In this regard, we present here a robust and fast strategy to engineer protein stabilities through mutations involving charged residues using a structure-based statistical mechanical model that accounts for the ensemble nature of folding. We validate the method by predicting the absolute changes in stability for 138 experimental mutations from 16 different proteins and enzymes with a correlation of 0.65 and importantly with a success rate of 81%. Multiple point mutants are predicted with a higher success rate (90%) that is validated further by comparing meosphile-thermophile protein pairs. In parallel, we devise a methodology to rapidly engineer mutations in silico which we benchmark against experimental mutations of ubiquitin (correlation of 0.95) and check for its feasibility on a larger therapeutic protein DNase I. We expect the method to be of importance as a first and rapid step to screen for protein mutants with specific stability in the biotechnology industry, in the construction of stability maps at the residue level (i.e., hot spots), and as a robust tool to probe for mutations that enhance the stability of protein-based drugs.

  10. The Kinetics of Crystallization of Colloids and Proteins: A Light Scattering Study

    Science.gov (United States)

    McClymer, Jim

    2002-01-01

    Hard-sphere colloidal systems serve as model systems for aggregation, nucleation, crystallization and gelation as well as interesting systems in their own right.There is strong current interest in using colloidal systems to form photonic crystals. A major scientific thrust of NASA's microgravity research is the crystallization of proteins for structural determination. The crystallization of proteins is a complicated process that requires a great deal of trial and error experimentation. In spite of a great deal of work, "better" protein crystals cannot always be grown in microgravity and conditions for crystallization are not well understood. Crystallization of colloidal systems interacting as hard spheres and with an attractive potential induced by entropic forces have been studied in a series of static light scattering experiments. Additionally, aggregation of a protein as a function of pH has been studied using dynamic light scattering. For our experiments we used PMMA (polymethylacrylate) spherical particles interacting as hard spheres, with no attractive potential. These particles have a radius of 304 nanometers, a density of 1.22 gm/ml and an index of refraction of 1.52. A PMMA colloidal sample at a volume fraction of approximately 54% was index matched in a solution of cycloheptyl bromide (CHB) and cis-decalin. The sample is in a glass cylindrical vial that is placed in an ALV static and dynamic light scattering goniometer system. The vial is immersed in a toluene bath for index matching to minimize flair. Vigorous shaking melts any colloidal crystals initially present. The sample is illuminated with diverging laser light (632.8 nanometers) from a 4x microscope objective placed so that the beam is approximately 1 cm in diameter at the sample location. The sample is rotated about its long axis at approximately 3.5 revolutions per minute (highest speed) as the colloidal crystal system is non-ergodic. The scattered light is detected at various angles using the

  11. Matrix metalloproteinase-20 mediates dental enamel biomineralization by preventing protein occlusion inside apatite crystals.

    Science.gov (United States)

    Prajapati, Saumya; Tao, Jinhui; Ruan, Qichao; De Yoreo, James J; Moradian-Oldak, Janet

    2016-01-01

    Reconstruction of enamel-like materials is a central topic of research in dentistry and material sciences. The importance of precise proteolytic mechanisms in amelogenesis to form a hard tissue with more than 95% mineral content has already been reported. A mutation in the Matrix Metalloproteinase-20 (MMP-20) gene results in hypomineralized enamel that is thin, disorganized and breaks from the underlying dentin. We hypothesized that the absence of MMP-20 during amelogenesis results in the occlusion of amelogenin in the enamel hydroxyapatite crystals. We used spectroscopy and electron microscopy techniques to qualitatively and quantitatively analyze occluded proteins within the isolated enamel crystals from MMP-20 null and Wild type (WT) mice. Our results showed that the isolated enamel crystals of MMP-20 null mice had more organic macromolecules occluded inside them than enamel crystals from the WT. The crystal lattice arrangements of MMP-20 null enamel crystals analyzed by High Resolution Transmission Electron Microscopy (HRTEM) were found to be significantly different from those of the WT. Raman studies indicated that the crystallinity of the MMP-20 null enamel crystals was lower than that of the WT. In conclusion, we present a novel functional mechanism of MMP-20, specifically prevention of unwanted organic material entrapped in the forming enamel crystals, which occurs as the result of precise amelogenin cleavage. MMP-20 action guides the growth morphology of the forming hydroxyapatite crystals and enhances their crystallinity. Elucidating such molecular mechanisms can be applied in the design of novel biomaterials for future clinical applications in dental restoration or repair. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Protein modification by acrolein: Formation and stability of cysteine adducts

    OpenAIRE

    Cai, Jian; Bhatnagar, Aruni; Pierce, William M.

    2009-01-01

    The toxicity of the ubiquitous pollutant and endogenous metabolite, acrolein, is due in part to covalent protein modifications. Acrolein reacts readily with protein nucleophiles via Michael addition and Schiff base formation. Potential acrolein targets in protein include the nucleophilic side chains of cysteine, histidine, and lysine residues as well as the free amino terminus of proteins. Although cysteine is the most acrolein-reactive residue, cysteine-acrolein adducts are difficult to iden...

  13. Contribution of buried aspartic acid to the stability of the PDZ2 protein

    International Nuclear Information System (INIS)

    Jayasimha, Pruthvi; Shanmuganathan, Aranganathan; Suladze, Saba; Makhatadze, George I.

    2012-01-01

    Highlights: ► Buried Asp residues on average form 2.5 to 3 hydrogen bonds and/or 0.8 salt bridges. ► Contribution of buried Asp to stability was estimated using model protein PDZ2. ► The energetic contribution of Asp56 to PDZ2 stability estimated to be 18 kJ · mol −1 . ► Findings are discussed in terms of contribution of Asp residues to protein stability. - Abstract: Statistical analysis of protein structures shows that buried aspartic acid residues on average form 2.5 to 3 hydrogen bonds and/or 0.8 potential ionic interactions with other protein groups. To estimate the energetic contribution of such buried groups to the Gibbs free energy of proteins, we measured the effects of amino acid substitutions of D56 in a model protein PDZ2 on its stability. We used temperature-induced unfolding monitored by DSC and denaturant-induced unfolding monitored by the changes in fluorescence intensity. We find that all substitutions of D56 lead to protein unfolding, thus suggesting that this buried hydrogen bonded aspartic acid has a significant contribution to the stability. To quantify the changes in the Gibbs free energy, one of the variants, D56N was stabilized by addition of the protective osmolyte TMAO. Comparison of the stability of the D56N variant with the wild-type PDZ2 in the presence and absence of TMAO allowed us to estimate the contribution of D56 to the protein stability to be 18 kJ · mol −1 . These findings are discussed in terms of contribution of buried ionizable groups to protein stability.

  14. The stability and formation of native proteins from unfolded monomers is increased through interactions with unrelated proteins.

    Directory of Open Access Journals (Sweden)

    Claudia Rodríguez-Almazán

    Full Text Available The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins.

  15. Optical characterization of Pr3+-doped yttria-stabilized zirconia single crystals

    International Nuclear Information System (INIS)

    Savoini, B.; Munoz Santiuste, J.E.; Gonzalez, R.

    1997-01-01

    The optical absorption and fluorescence of Pr 3+ ions in yttria-stabilized zirconia single crystals are investigated. Fluorescence emissions from the 1 D 2 level are clearly dominant and low intensity emission lines from the 3 P 0 and 1 G 4 states are also observed. Analysis with the Judd-Ofelt theory of the absorption intensities has been made assuming that only ∼40% of the praseodymium ions contribute to the optical absorption bands. Quantum efficiency values of η( 3 P 0 )∼0.2 and η( 1 D 2 )∼ 1 are obtained at room temperature. 1 D 2 fluorescence quenching has been observed in heavily-doped samples due to cross relaxation processes among neighboring Pr 3+ ions. Analysis using the Inokuti-Hirayama model shows that electric dipole-dipole interactions are mainly responsible for the quenching effect. Pr 3+ ions are present in seven and sixfold configurations with a statistical distribution. The energy position of the 4f5d configuration is very different for each center. The fluorescence dynamics is explained by a mechanism involving thermally assisted population of the 3 P 1,2 + 1 I 6 upper levels and fast relaxation to the 1 D 2 level via states of the excited 4f5d configuration. copyright 1997 The American Physical Society

  16. Correlation between mechanical behavior of protein films at the air/water interface and intrinsic stability of protein molecules

    NARCIS (Netherlands)

    Martin, A.H.; Cohen Stuart, M.A.; Bos, M.A.; Vliet, T. van

    2005-01-01

    The relation between mechanical film properties of various adsorbed protein layers at the air/water interface and intrinsic stability of the corresponding proteins is discussed. Mechanical film properties were determined by surface deformation in shear and dilation. In shear, fracture stress, σf,

  17. Sulfatide Preserves Insulin Crystals Not by Being Integrated in the Lattice but by Stabilizing Their Surface

    DEFF Research Database (Denmark)

    Buschard, Karsten; Bracey, Austin W.; McElroy, Daniel L.

    2016-01-01

    Background. Sulfatide is known to chaperone insulin crystallization within the pancreatic beta cell, but it is not known if this results from sulfatide being integrated inside the crystal structure or by binding the surface of the crystal. With this study, we aimed to characterize the molecular m...

  18. High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

    DEFF Research Database (Denmark)

    Cherezov, Vadim; Rosenbaum, Daniel M; Hanson, Michael A

    2007-01-01

    Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to t...

  19. Bi2(Sr, Ln)2CuOz (Ln = Nd, Sm) phases: stability, crystal growth and superconducting properties

    International Nuclear Information System (INIS)

    Faqir, H.; Kikuchi, M.; Syono, Y.; Mansori, M.; Satre, P.; Sebaoun, A.; Vacquier, G.

    2000-01-01

    Bi 2 (Sr,Ln) 2 CuO z (Ln = Nd, Sm) single crystals were successfully grown by a self-flux method from stoichiometric and (Bi, Cu)-rich melts. Thermal analysis and thermogravimetry were used to determine stability and the melting sequence of Bi 2 (Sr,Ln) 2 CuO z phases in air. As-grown crystals of the ideal Bi 2 (Sr,Ln) 2 CuO z phase, of dimensions 1x0.5x0.03 mm 3 , exhibit superconducting behaviour with critical temperature T c = 21 K for the Bi 1.9 Sr 1.6 Nd 0.6 CuO z crystal and Tc = 14 K for the Bi 1.8 Sr 1.6 Sm 0.6 CuO z crystal. The compositions of these crystals were homogeneous and close to the stoichiometric composition. We report on the growth of Bi 2 Sr 2-x Sm x CuO z single crystals of large dimensions 9x3x0.03 mm 3 using Bi 2 Sr 1.5 Sm 0.5 CuO z as precursor and Bi 2 CuO 4 as flux. (author)

  20. Effects of insecticidal crystal proteins (Cry proteins) produced by genetically modified maize (Bt maize) on the nematode Caenorhabditis elegans

    International Nuclear Information System (INIS)

    Höss, Sebastian; Menzel, Ralph; Gessler, Frank; Nguyen, Hang T.; Jehle, Johannes A.; Traunspurger, Walter

    2013-01-01

    The genetically modified maize MON89034 × MON88017 expresses different crystal (Cry) proteins with pesticidal activity against the European corn borer (Cry1.105; Cry2Ab2) and the Western corn root worm (Cry3Bb1). Non-target organisms, such as soil nematodes, might be exposed to the Cry proteins that enter the soil in course of crop growing. Therefore, the risk of those proteins for nematodes was assessed by testing their toxic effects on Caenorhabditis elegans. All three insecticidal Cry proteins showed dose-dependent inhibitory effects on C. elegans reproduction (EC50: 0.12–0.38 μmol L −1 ), however, at concentrations that were far above the expected soil concentrations. Moreover, a reduced toxicity was observed when Cry proteins were added jointly. A C. elegans mutant strain deficient for receptors for the nematicidal Cry5B was also resistant against Cry1.105 and Cry2Ab2, suggesting that these Cry proteins bound to the same or similar receptors as nematicidal Cry proteins and thereby affect the reproduction of C. elegans. -- Highlights: •Insecticidal Cry proteins dose-dependently inhibited the reproduction of C. elegans. •Mixture toxicity was lower than expected from concentration-additive single effects. •Genes for MAPK-defense-pathway were up-regulated in presence of Cry protein mixture. •Knock-out strains deficient for Cry5B-receptors showed lower susceptibility to insecticidal Cry proteins. •Toxicity of insecticidal Cry-proteins on C. elegans occurred at concentrations far above expected field concentrations. -- Insecticidal Cry proteins expressed by genetically modified maize act on nematodes via a similar mode of action as nematicidal Cry proteins, however, at concentrations far above expected soil levels

  1. Development of an X-ray fluorescence holographic measurement system for protein crystals

    Energy Technology Data Exchange (ETDEWEB)

    Sato-Tomita, Ayana, E-mail: ayana.sato@jichi.ac.jp, E-mail: shibayam@jichi.ac.jp, E-mail: hayashi.koichi@nitech.ac.jp; Shibayama, Naoya, E-mail: ayana.sato@jichi.ac.jp, E-mail: shibayam@jichi.ac.jp, E-mail: hayashi.koichi@nitech.ac.jp; Okabe, Takahiro [Division of Biophysics, Department of Physiology, Jichi Medical University, Yakushiji, Shimotsuke 329-0498 (Japan); Happo, Naohisa [Department of Computer and Network Engineering, Graduate School of Information Sciences, Hiroshima City University, Asa-Minami-Ku, Hiroshima 731-3194 (Japan); Kimura, Koji [Department of Physical Science and Engineering, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555 (Japan); Matsushita, Tomohiro [Japan Synchrotron Radiation Research Institute (JASRI), SPring-8, Sayo, Hyogo 679-5198 (Japan); Park, Sam-Yong [Drug Design Laboratory, Department of Medical Life Science, Yokohama City University, Suehiro, Tsurumi, Yokohama 230-0045 (Japan); Sasaki, Yuji C. [Department of Advanced Material Science, Graduate School of Frontier Science, The University of Tokyo, Kashiwanoha, Kashiwa 277-8561 (Japan); Hayashi, Kouichi, E-mail: ayana.sato@jichi.ac.jp, E-mail: shibayam@jichi.ac.jp, E-mail: hayashi.koichi@nitech.ac.jp [Department of Physical Science and Engineering, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555 (Japan); Frontier Research Institute for Materials Science, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555 (Japan)

    2016-06-15

    Experimental procedure and setup for obtaining X-ray fluorescence hologram of crystalline metalloprotein samples are described. Human hemoglobin, an α{sub 2}β{sub 2} tetrameric metalloprotein containing the Fe(II) heme active-site in each chain, was chosen for this study because of its wealth of crystallographic data. A cold gas flow system was introduced to reduce X-ray radiation damage of protein crystals that are usually fragile and susceptible to damage. A χ-stage was installed to rotate the sample while avoiding intersection between the X-ray beam and the sample loop or holder, which is needed for supporting fragile protein crystals. Huge hemoglobin crystals (with a maximum size of 8 × 6 × 3 mm{sup 3}) were prepared and used to keep the footprint of the incident X-ray beam smaller than the sample size during the entire course of the measurement with the incident angle of 0°-70°. Under these experimental and data acquisition conditions, we achieved the first observation of the X-ray fluorescence hologram pattern from the protein crystals with minimal radiation damage, opening up a new and potential method for investigating the stereochemistry of the metal active-sites in biomacromolecules.

  2. Development of an X-ray fluorescence holographic measurement system for protein crystals

    International Nuclear Information System (INIS)

    Sato-Tomita, Ayana; Shibayama, Naoya; Okabe, Takahiro; Happo, Naohisa; Kimura, Koji; Matsushita, Tomohiro; Park, Sam-Yong; Sasaki, Yuji C.; Hayashi, Kouichi

    2016-01-01

    Experimental procedure and setup for obtaining X-ray fluorescence hologram of crystalline metalloprotein samples are described. Human hemoglobin, an α_2β_2 tetrameric metalloprotein containing the Fe(II) heme active-site in each chain, was chosen for this study because of its wealth of crystallographic data. A cold gas flow system was introduced to reduce X-ray radiation damage of protein crystals that are usually fragile and susceptible to damage. A χ-stage was installed to rotate the sample while avoiding intersection between the X-ray beam and the sample loop or holder, which is needed for supporting fragile protein crystals. Huge hemoglobin crystals (with a maximum size of 8 × 6 × 3 mm"3) were prepared and used to keep the footprint of the incident X-ray beam smaller than the sample size during the entire course of the measurement with the incident angle of 0°-70°. Under these experimental and data acquisition conditions, we achieved the first observation of the X-ray fluorescence hologram pattern from the protein crystals with minimal radiation damage, opening up a new and potential method for investigating the stereochemistry of the metal active-sites in biomacromolecules.

  3. crystal

    Science.gov (United States)

    Yu, Yi; Huang, Yisheng; Zhang, Lizhen; Lin, Zhoubin; Sun, Shijia; Wang, Guofu

    2014-07-01

    A Nd3+:Na2La4(WO4)7 crystal with dimensions of ϕ 17 × 30 mm3 was grown by the Czochralski method. The thermal expansion coefficients of Nd3+:Na2La4(WO4)7 crystal are 1.32 × 10-5 K-1 along c-axis and 1.23 × 10-5 K-1 along a-axis, respectively. The spectroscopic characteristics of Nd3+:Na2La4(WO4)7 crystal were investigated. The Judd-Ofelt theory was applied to calculate the spectral parameters. The absorption cross sections at 805 nm are 2.17 × 10-20 cm2 with a full width at half maximum (FWHM) of 15 nm for π-polarization, and 2.29 × 10-20 cm2 with a FWHM of 14 nm for σ-polarization. The emission cross sections are 3.19 × 10-20 cm2 for σ-polarization and 2.67 × 10-20 cm2 for π-polarization at 1,064 nm. The fluorescence quantum efficiency is 67 %. The quasi-cw laser of Nd3+:Na2La4(WO4)7 crystal was performed. The maximum output power is 80 mW. The slope efficiency is 7.12 %. The results suggest Nd3+:Na2La4(WO4)7 crystal as a promising laser crystal fit for laser diode pumping.

  4. Crystal structure of an orthomyxovirus matrix protein reveals mechanisms for self-polymerization and membrane association.

    Science.gov (United States)

    Zhang, Wenting; Zheng, Wenjie; Toh, Yukimatsu; Betancourt-Solis, Miguel A; Tu, Jiagang; Fan, Yanlin; Vakharia, Vikram N; Liu, Jun; McNew, James A; Jin, Meilin; Tao, Yizhi J

    2017-08-08

    Many enveloped viruses encode a matrix protein. In the influenza A virus, the matrix protein M1 polymerizes into a rigid protein layer underneath the viral envelope to help enforce the shape and structural integrity of intact viruses. The influenza virus M1 is also known to mediate virus budding as well as the nuclear export of the viral nucleocapsids and their subsequent packaging into nascent viral particles. Despite extensive studies on the influenza A virus M1 (FLUA-M1), only crystal structures of its N-terminal domain are available. Here we report the crystal structure of the full-length M1 from another orthomyxovirus that infects fish, the infectious salmon anemia virus (ISAV). The structure of ISAV-M1 assumes the shape of an elbow, with its N domain closely resembling that of the FLUA-M1. The C domain, which is connected to the N domain through a flexible linker, is made of four α-helices packed as a tight bundle. In the crystal, ISAV-M1 monomers form infinite 2D arrays with a network of interactions involving both the N and C domains. Results from liposome flotation assays indicated that ISAV-M1 binds membrane via electrostatic interactions that are primarily mediated by a positively charged surface loop from the N domain. Cryoelectron tomography reconstruction of intact ISA virions identified a matrix protein layer adjacent to the inner leaflet of the viral membrane. The physical dimensions of the virion-associated matrix layer are consistent with the 2D ISAV-M1 crystal lattice, suggesting that the crystal lattice is a valid model for studying M1-M1, M1-membrane, and M1-RNP interactions in the virion.

  5. Stabilizing salt-bridge enhances protein thermostability by reducing the heat capacity change of unfolding.

    Directory of Open Access Journals (Sweden)

    Chi-Ho Chan

    Full Text Available Most thermophilic proteins tend to have more salt bridges, and achieve higher thermostability by up-shifting and broadening their protein stability curves. While the stabilizing effect of salt-bridge has been extensively studied, experimental data on how salt-bridge influences protein stability curves are scarce. Here, we used double mutant cycles to determine the temperature-dependency of the pair-wise interaction energy and the contribution of salt-bridges to ΔC(p in a thermophilic ribosomal protein L30e. Our results showed that the pair-wise interaction energies for the salt-bridges E6/R92 and E62/K46 were stabilizing and insensitive to temperature changes from 298 to 348 K. On the other hand, the pair-wise interaction energies between the control long-range ion-pair of E90/R92 were negligible. The ΔC(p of all single and double mutants were determined by Gibbs-Helmholtz and Kirchhoff analyses. We showed that the two stabilizing salt-bridges contributed to a reduction of ΔC(p by 0.8-1.0 kJ mol⁻¹ K⁻¹. Taken together, our results suggest that the extra salt-bridges found in thermophilic proteins enhance the thermostability of proteins by reducing ΔC(p, leading to the up-shifting and broadening of the protein stability curves.

  6. Membrane Protein Stability Analyses by Means of Protein Energy Profiles in Case of Nephrogenic Diabetes Insipidus

    Directory of Open Access Journals (Sweden)

    Florian Heinke

    2012-01-01

    Full Text Available Diabetes insipidus (DI is a rare endocrine, inheritable disorder with low incidences in an estimated one per 25,000–30,000 live births. This disease is characterized by polyuria and compensatory polydypsia. The diverse underlying causes of DI can be central defects, in which no functional arginine vasopressin (AVP is released from the pituitary or can be a result of defects in the kidney (nephrogenic DI, NDI. NDI is a disorder in which patients are unable to concentrate their urine despite the presence of AVP. This antidiuretic hormone regulates the process of water reabsorption from the prourine that is formed in the kidney. It binds to its type-2 receptor (V2R in the kidney induces a cAMP-driven cascade, which leads to the insertion of aquaporin-2 water channels into the apical membrane. Mutations in the genes of V2R and aquaporin-2 often lead to NDI. We investigated a structure model of V2R in its bound and unbound state regarding protein stability using a novel protein energy profile approach. Furthermore, these techniques were applied to the wild-type and selected mutations of aquaporin-2. We show that our results correspond well to experimental water ux analysis, which confirms the applicability of our theoretical approach to equivalent problems.

  7. Hydrophobic environment is a key factor for the stability of thermophilic proteins.

    Science.gov (United States)

    Gromiha, M Michael; Pathak, Manish C; Saraboji, Kadhirvel; Ortlund, Eric A; Gaucher, Eric A

    2013-04-01

    The stability of thermophilic proteins has been viewed from different perspectives and there is yet no unified principle to understand this stability. It would be valuable to reveal the most important interactions for designing thermostable proteins for such applications as industrial protein engineering. In this work, we have systematically analyzed the importance of various interactions by computing different parameters such as surrounding hydrophobicity, inter-residue interactions, ion-pairs and hydrogen bonds. The importance of each interaction has been determined by its predicted relative contribution in thermophiles versus the same contribution in mesophilic homologues based on a dataset of 373 protein families. We predict that hydrophobic environment is the major factor for the stability of thermophilic proteins and found that 80% of thermophilic proteins analyzed showed higher hydrophobicity than their mesophilic counterparts. Ion pairs, hydrogen bonds, and interaction energy are also important and favored in 68%, 50%, and 62% of thermophilic proteins, respectively. Interestingly, thermophilic proteins with decreased hydrophobic environments display a greater number of hydrogen bonds and/or ion pairs. The systematic elimination of mesophilic proteins based on surrounding hydrophobicity, interaction energy, and ion pairs/hydrogen bonds, led to correctly identifying 95% of the thermophilic proteins in our analyses. Our analysis was also applied to another, more refined set of 102 thermophilic-mesophilic pairs, which again identified hydrophobicity as a dominant property in 71% of the thermophilic proteins. Further, the notion of surrounding hydrophobicity, which characterizes the hydrophobic behavior of residues in a protein environment, has been applied to the three-dimensional structures of elongation factor-Tu proteins and we found that the thermophilic proteins are enriched with a hydrophobic environment. The results obtained in this work highlight the

  8. Stability enhancement of an electrically tunable colloidal photonic crystal using modified electrodes with a large electrochemical potential window

    Energy Technology Data Exchange (ETDEWEB)

    Shim, HongShik [Samsung Advanced Institute of Technology, Yongin-Si, Gyeonggi-do (Korea, Republic of); Department of Chemistry, Seoul National University, Seoul (Korea, Republic of); Gyun Shin, Chang; Heo, Chul-Joon; Jeon, Seog-Jin; Jin, Haishun; Woo Kim, Jung; Jin, YongWan; Lee, SangYoon; Gyu Han, Moon, E-mail: moongyu.han@samsung.com, E-mail: jinklee@snu.ac.kr [Samsung Advanced Institute of Technology, Yongin-Si, Gyeonggi-do (Korea, Republic of); Lim, Joohyun; Lee, Jin-Kyu, E-mail: moongyu.han@samsung.com, E-mail: jinklee@snu.ac.kr [Department of Chemistry, Seoul National University, Seoul (Korea, Republic of)

    2014-02-03

    The color tuning behavior and switching stability of an electrically tunable colloidal photonic crystal system were studied with particular focus on the electrochemical aspects. Photonic color tuning of the colloidal arrays composed of monodisperse particles dispersed in water was achieved using external electric field through lattice constant manipulation. However, the number of effective color tuning cycle was limited due to generation of unwanted ions by electrolysis of the water medium during electrical switching. By introducing larger electrochemical potential window electrodes, such as conductive diamond-like carbon or boron-doped diamond, the switching stability was appreciably enhanced through reducing the number of ions generated.

  9. Dispersive heterodyne probing method for laser frequency stabilization based on spectral hole burning in rare-earth doped crystals

    DEFF Research Database (Denmark)

    Gobron, Olivier; Jung, K.; Galland, N.

    2017-01-01

    Frequency-locking a laser to a spectral hole in rare-earth doped crystals at cryogenic temperature has been shown to be a promising alternative to the use of high finesse Fabry-Perot cavities when seeking a very high short term stability laser (M. J. Thorpe et al., Nature Photonics 5, 688 (2011......)). We demonstrate here a novel technique for achieving such stabilization, based on generating a heterodyne beat-note between a master laser and a slave laser whose dephasing caused by propagation near a spectral hole generate the error signal of the frequency lock. The master laser is far detuned from...

  10. Light assisted drying (LAD) for protein stabilization: optical characterization of samples

    Science.gov (United States)

    Young, Madison A.; McKinnon, Madison E.; Elliott, Gloria D.; Trammell, Susan R.

    2018-02-01

    Light-Assisted Drying (LAD) is a novel biopreservation technique which allows proteins to be immobilized in a dry, amorphous solid at room temperature. Indicator proteins are used in a variety of diagnostic assays ranging from highthroughput 96-well plates to new microfluidic devices. A challenge in the development of protein-based assays is preserving the structure of the protein during production and storage of the assay, as the structure of the protein is responsible for its functional activity. Freeze-drying or freezing are currently the standard for the preservation of proteins, but these methods are expensive and can be challenging in some environments due to a lack of available infrastructure. An inexpensive, simple processing method that enables supra-zero temperature storage of proteins used in assays is needed. Light-assisted drying offers a relatively inexpensive method for drying samples. Proteins suspended in a trehalose solution are dehydrated using near-infrared laser light. The laser radiation speeds drying and as water is removed the sugar forms a protective matrix. The goal of this study is optically characterize samples processed with LAD. We use polarized light imaging (PLI) to look at crystallization kinetics of samples and determine optimal humidity. PLI shows a 62.5% chance of crystallization during LAD processing and negligible crystallization during low RH storage.

  11. Interdependence of laforin and malin proteins for their stability and ...

    Indian Academy of Sciences (India)

    https://www.ias.ac.in/article/fulltext/jbsc/040/05/0863-0871. Keywords. Epilepsy; locus heterogeneity; post-translational modification; protein-protein interaction. Abstract. Lafora disease (LD), an autosomal recessive and fatal form of neurodegenerative disorder, is characterized by the presence of polyglucosan inclusions in ...

  12. Crystallization and preliminary X-ray diffraction analysis of rat protein tyrosine phosphatase η

    Energy Technology Data Exchange (ETDEWEB)

    Matozo, Huita C.; Nascimento, Alessandro S.; Santos, Maria A. M. [Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, Avenida Trabalhador São Carlense 400, CEP 13566-590 São Carlos, SP (Brazil); Iuliano, Rodolfo [Dipartimento di Medicina Sperimentale e Clinica, Facoltà di Medicina e Chirurgia, Università di Catanzaro, 88100 Catanzaro (Italy); Fusco, Alfredo [Dipartimento di Biologia e Patologia Cellulare e Molecolare, c/o Instituto di Endocrinologia ed Oncologia Sperimentale del CNR, Facolta di Medicina e Chirurgia, Università degli Studi di Napoli ‘Federico II’, Via Pansini 5, 80131 Naples (Italy); NOGEC (Naples Oncogenomocs Center)-CEINGE, Biotecnologie Avanzate, Via Comunale Margherita 482, 80145 Naples (Italy); Polikarpov, Igor, E-mail: ipolikarpov@if.sc.usp.br [Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, Avenida Trabalhador São Carlense 400, CEP 13566-590 São Carlos, SP (Brazil); Laboratório Nacional de Luz Síncrotron, Campinas, SP (Brazil)

    2006-09-01

    In this study, the catalytic domain of rat protein tyrosine phosphatase η was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. The rat protein tyrosine phosphatase η (rPTPη) is a cysteine-dependent phosphatase which hydrolyzes phosphoester bonds in proteins and other molecules. rPTPη and its human homologue DEP-1 are involved in neoplastic transformations. Thus, expression of the protein is reduced in all oncogene-transformed thyroid cell lines and is absent in highly malignant thyroid cells. Moreover, consistent with the suggested tumour suppression role of PTPη, inhibition of the tumorigenic process occurs after its exogenous reconstitution, suggesting that PTPη might be important for gene therapy of cancers. In this study, the catalytic domain of rPTPη was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.87 Å resolution. The crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 46.46, b = 63.07, c = 111.64 Å, and contains one molecule per asymmetric unit.

  13. Crystallization and preliminary X-ray diffraction analysis of rat protein tyrosine phosphatase η

    International Nuclear Information System (INIS)

    Matozo, Huita C.; Nascimento, Alessandro S.; Santos, Maria A. M.; Iuliano, Rodolfo; Fusco, Alfredo; Polikarpov, Igor

    2006-01-01

    In this study, the catalytic domain of rat protein tyrosine phosphatase η was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. The rat protein tyrosine phosphatase η (rPTPη) is a cysteine-dependent phosphatase which hydrolyzes phosphoester bonds in proteins and other molecules. rPTPη and its human homologue DEP-1 are involved in neoplastic transformations. Thus, expression of the protein is reduced in all oncogene-transformed thyroid cell lines and is absent in highly malignant thyroid cells. Moreover, consistent with the suggested tumour suppression role of PTPη, inhibition of the tumorigenic process occurs after its exogenous reconstitution, suggesting that PTPη might be important for gene therapy of cancers. In this study, the catalytic domain of rPTPη was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.87 Å resolution. The crystal belongs to space group P2 1 2 1 2 1 , with unit-cell parameters a = 46.46, b = 63.07, c = 111.64 Å, and contains one molecule per asymmetric unit

  14. Clinical application for the preservation of phospho-proteins through in-situ tissue stabilization

    Directory of Open Access Journals (Sweden)

    Ding Wei

    2010-11-01

    Full Text Available Abstract Background Protein biomarkers will play a pivotal role in the future of personalized medicine for both diagnosis and treatment decision-making. While the results of several pre-clinical and small-scale clinical studies have demonstrated the value of protein biomarkers, there have been significant challenges to translating these findings into routine clinical care. Challenges to the use of protein biomarkers include inter-sample variability introduced by differences in post-collection handling and ex vivo degradation of proteins and protein modifications. Results In this report, we re-create laboratory and clinical scenarios for sample collection and test the utility of a new tissue stabilization technique in preserving proteins and protein modifications. In the laboratory setting, tissue stabilization with the Denator Stabilizor T1 resulted in a significantly higher yield of phospho-protein when compared to standard snap freeze preservation. Furthermore, in a clinical scenario, tissue stabilization at collection resulted in a higher yield of total phospho-protein, total phospho-tyrosine, pErkT202/Y204 and pAktS473 when compared to standard methods. Tissue stabilization did not have a significant effect on other post-translational modifications such as acetylation and glycosylation, which are more stable ex-vivo. Tissue stabilization did decrease total RNA quantity and quality. Conclusion Stabilization at the time of collection offers the potential to better preserve tissue protein and protein modification levels, as well as reduce the variability related to tissue processing delays that are often associated with clinical samples.

  15. Mutation of exposed hydrophobic amino acids to arginine to increase protein stability

    OpenAIRE

    Strub, Caroline; Alies, Carole; Lougarre, Andrée; Ladurantie, Caroline; Czaplicki, Jerzy; Fournier, Didier

    2004-01-01

    Abstract Background One strategy to increase the stability of proteins is to reduce the area of water-accessible hydrophobic surface. Results In order to test it, we replaced 14 solvent-exposed hydrophobic residues of acetylcholinesterase by arginine. The stabilities of the resulting proteins were tested using denaturation by high temperature, organic solvents, urea and by proteolytic digestion. Conclusion Altough the mutational effects were rather small, this strategy proved to be successful...

  16. Analysis of polytype stability in PVT grown silicon carbide single crystal using competitive lattice model Monte Carlo simulations

    Directory of Open Access Journals (Sweden)

    Hui-Jun Guo

    2014-09-01

    Full Text Available Polytype stability is very important for high quality SiC single crystal growth. However, the growth conditions for the 4H, 6H and 15R polytypes are similar, and the mechanism of polytype stability is not clear. The kinetics aspects, such as surface-step nucleation, are important. The kinetic Monte Carlo method is a common tool to study surface kinetics in crystal growth. However, the present lattice models for kinetic Monte Carlo simulations cannot solve the problem of the competitive growth of two or more lattice structures. In this study, a competitive lattice model was developed for kinetic Monte Carlo simulation of the competition growth of the 4H and 6H polytypes of SiC. The site positions are fixed at the perfect crystal lattice positions without any adjustment of the site positions. Surface steps on seeds and large ratios of diffusion/deposition have positive effects on the 4H polytype stability. The 3D polytype distribution in a physical vapor transport method grown SiC ingot showed that the facet preserved the 4H polytype even if the 6H polytype dominated the growth surface. The theoretical and experimental results of polytype growth in SiC suggest that retaining the step growth mode is an important factor to maintain a stable single 4H polytype during SiC growth.

  17. Comparison of NMR and crystal structures for the proteins TM1112 and TM1367

    International Nuclear Information System (INIS)

    Mohanty, Biswaranjan; Serrano, Pedro; Pedrini, Bill; Jaudzems, Kristaps; Geralt, Michael; Horst, Reto; Herrmann, Torsten; Elsliger, Marc-André; Wilson, Ian A.; Wüthrich, Kurt

    2010-01-01

    NMR structures of the proteins TM1112 and TM1367 solved by the JCSG in solution at 298 K could be superimposed with the corresponding crystal structures at 100 K with r.m.s.d. values of <1.0 Å for the backbone heavy atoms. For both proteins the structural differences between multiple molecules in the asymmetric unit of the crystals correlated with structural variations within the bundles of conformers used to represent the NMR solution structures. A recently introduced JCSG NMR structure-determination protocol, which makes use of the software package UNIO for extensive automation, was further evaluated by comparison of the TM1112 structure obtained using these automated methods with another NMR structure that was independently solved in another PSI center, where a largely interactive approach was applied. The NMR structures of the TM1112 and TM1367 proteins from Thermotoga maritima in solution at 298 K were determined following a new protocol which uses the software package UNIO for extensive automation. The results obtained with this novel procedure were evaluated by comparison with the crystal structures solved by the JCSG at 100 K to 1.83 and 1.90 Å resolution, respectively. In addition, the TM1112 solution structure was compared with an NMR structure solved by the NESG using a conventional largely interactive methodology. For both proteins, the newly determined NMR structure could be superimposed with the crystal structure with r.m.s.d. values of <1.0 Å for the backbone heavy atoms, which provided a starting platform to investigate local structure variations, which may arise from either the methods used or from the different chemical environments in solution and in the crystal. Thereby, these comparative studies were further explored with the use of reference NMR and crystal structures, which were computed using the NMR software with input of upper-limit distance constraints derived from the molecular models that represent the results of structure

  18. Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of Cockayne syndrome protein A in complex with DNA damage-binding protein 1

    International Nuclear Information System (INIS)

    Meulenbroek, Elisabeth M.; Pannu, Navraj S.

    2011-01-01

    Human Cockayne syndrome protein A has been cocrystallized with human DNA damage-binding protein 1 and data have been collected to 2.9 Å resolution. Cockayne syndrome protein A is one of the main components in mammalian transcription coupled repair. Here, the overproduction, purification and crystallization of human Cockayne syndrome protein A in complex with its interacting partner DNA damage binding protein 1 are reported. The complex was coproduced in insect cells, copurified and crystallized using sitting drops with PEG 3350 and sodium citrate as crystallizing agents. The crystals had unit-cell parameters a = b = 142.03, c = 250.19 Å and diffracted to 2.9 Å resolution on beamline ID14-1 at the European Synchrotron Radiation Facility

  19. Stabilization of structure in near-infrared fluorescent proteins by binding of biliverdin chromophore

    Science.gov (United States)

    Stepanenko, Olesya V.; Stepanenko, Olga V.; Bublikov, G. S.; Kuznetsova, I. M.; Verkhusha, V. V.; Turoverov, K. K.

    2017-07-01

    Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes and their mutants with different location of Cys residues, which able to bind a biliverdin chromophore, or without these Cys residues were studied using intrinsic tryptophan fluorescence, NIR fluorescence and circular dichroism. It was shown that a covalent binding of the biliverdin chromophore to a Cys residue via thioether group substantially stabilizes the spatial structure of NIR FPs. The stability of the protein structure and the chromophore association strength strongly depends on the location of Cys residues and decreases in the following order: a protein with Cys residues in both domains, a protein with Cys in PAS domains, and a protein with Cys in GAF domains. NIR FPs without Cys residues capable to covalently attach biliverdin have the lowest stability, comparable to NIR FP apoforms.

  20. Effect of pasteurization on the protein composition and oxidative stability of beer during storage.

    Science.gov (United States)

    Lund, Marianne N; Hoff, Signe; Berner, Torben S; Lametsch, René; Andersen, Mogens L

    2012-12-19

    The impacts of pasteurization of a lager beer on protein composition and the oxidative stability were studied during storage at 22 °C for 426 days in the dark. Pasteurization clearly improved the oxidative stability of beer determined by ESR spectroscopy, whereas it had a minor negative effect on the volatile profile by increasing volatile compounds that is generally associated with heat treatment and a loss of fruity ester aroma. A faster rate of radical formation in unpasteurized beer was consistent with a faster consumption of sulfite. Beer proteins in the unpasteurized beer were more degraded, most likely due to proteolytic enzyme activity of yeast remnants and more precipitation of proteins was also observed. The differences in soluble protein content and composition are suggested to result in differences in the contents of prooxidative metals as a consequence of the proteins ability to bind metals. This also contributes to the differences in oxidative stabilities of the beers.

  1. Water-in-Oil Microemulsions for Protein Delivery: Loading Optimization and Stability.

    Science.gov (United States)

    Perinelli, Diego R; Cespi, Marco; Pucciarelli, Stefania; Vincenzetti, Silvia; Casettari, Luca; Lam, Jenny K W; Logrippo, Serena; Canala, Elisa; Soliman, Mahmoud E; Bonacucina, Giulia; Palmieri, Giovanni F

    2017-01-01

    Microemulsions are attractive delivery systems for therapeutic proteins and peptides due to their ability to enhance bioavailability. Although different proteins and peptides have been successfully delivered through such ternary systems, no information can be found about protein loading and the formulation stability when such microemulsions are prepared with pharmaceuticallyapproved oils and surfactants. The aim of this work was to optimise a ternary system consisting of water/ ethyl oleate/Span® 80-Tween® 80 and to determine its protein loading capacity and stability, using bovine serum albumin (BSA) as a model of biomolecule. The optimization was carried out using a Central Composite Design and all the prepared formulations were characterised through dynamic light scattering, rheology, optical and polarized microscopy. Subsequently, the maximum loading capacity was determined and the stability of the final microemulsion with the highest content of protein was followed over six months. To investigate the structural features of the protein, BSA was recovered from the microemulsion and analysed through fluorescence spectroscopy. After incorporation of the protein in the microemulsion, a decrease of its aqueous solubility was observed. However, the formulation remained stable over six months and the native-like state of the recovered protein was demonstrated by fluorescence spectroscopy Conclusion: This study demonstrated the feasibility of preparing microemulsions with the highest content of protein and their long-term stability. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  2. Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein

    International Nuclear Information System (INIS)

    Petit-Haertlein, Isabelle; Blakeley, Matthew P.; Howard, Eduardo; Hazemann, Isabelle; Mitschler, Andre; Haertlein, Michael; Podjarny, Alberto

    2009-01-01

    Perdeuterated type III antifreeze protein has been expressed, purified and crystallized. Preliminary neutron data collection showed diffraction to 1.85 Å resolution from a 0.13 mm 3 crystal. The highly homologous type III antifreeze protein (AFP) subfamily share the capability to inhibit ice growth at subzero temperatures. Extensive studies by X-ray crystallography have been conducted, mostly on AFPs from polar fishes. Although interactions between a defined flat ice-binding surface and a particular lattice plane of an ice crystal have now been identified, the fine structural features underlying the antifreeze mechanism still remain unclear owing to the intrinsic difficulty in identifying H atoms using X-ray diffraction data alone. Here, successful perdeuteration (i.e. complete deuteration) for neutron crystallographic studies of the North Atlantic ocean pout (Macrozoarces americanus) AFP in Escherichia coli high-density cell cultures is reported. The perdeuterated protein (AFP D) was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Well shaped perdeuterated AFP D crystals have been grown in D 2 O by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed that with a few exposures of 24 h a very low background and clear small spots up to a resolution of 1.85 Å were obtained using a ‘radically small’ perdeuterated AFP D crystal of dimensions 0.70 × 0.55 × 0.35 mm, corresponding to a volume of 0.13 mm 3

  3. Exceptional heat stability of high protein content dispersions containing whey protein particles

    NARCIS (Netherlands)

    Saglam, D.; Venema, P.; Vries, de R.J.; Linden, van der E.

    2014-01-01

    Due to aggregation and/or gelation during thermal treatment, the amount of whey proteins that can be used in the formulation of high protein foods e.g. protein drinks, is limited. The aim of this study was to replace whey proteins with whey protein particles to increase the total protein content and

  4. Expression, purification, crystallization and preliminary crystallographic studies of Rhagium inquisitor antifreeze protein

    International Nuclear Information System (INIS)

    Hakim, Aaron; Thakral, Durga; Zhu, Darren F.; Nguyen, Jennifer B.

    2012-01-01

    A novel hyperactive antifreeze protein from R. inquisitor (RiAFP) has been overexpressed, purified and crystallized. A complete native X-ray diffraction data set was recorded to 1.3 Å resolution. Antifreeze proteins (AFPs) are a specialized evolutionary adaptation of a variety of bacteria, fish, arthropods and other organisms to inhibit ice-crystal growth for survival in harsh subzero environments. The recently reported novel hyperactive AFP from Rhagium inquisitor (RiAFP) is the second distinct type of AFP in beetles and its structure could reveal important molecular insights into the evolution of AFPs. For this purpose, RiAFP was overexpressed in Escherichia coli, purified and crystallized at 293 K using a combination of 23% PEG 3350 and 0.2 M ammonium sulfate as a precipitant. X-ray diffraction data were collected to 1.3 Å resolution using a synchrotron-radiation source. The crystals belonged to the trigonal space group P3 1 21 (or P3 2 21), with unit-cell parameters a = b = 46.46, c = 193.21 Å

  5. Protein preparation, crystallization and preliminary X-ray analysis of Trypanosoma cruzi nucleoside diphosphate kinase 1

    International Nuclear Information System (INIS)

    Gómez Barroso, J. A.; Pereira, H.; Miranda, M.; Pereira, C.; Garratt, R. C.; Aguilar, C. F.

    2010-01-01

    T. cruzi TcNDPK1 was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. The flagellated protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas disease. Nucleoside diphosphate kinases (NDPKs) are enzymes that are involved in energy management and nucleoside balance in the cell. T. cruzi TcNDPK1, a canonical isoform, was overexpressed in Escherichia coli as an N-terminally poly-His-tagged fusion protein and crystallized. Crystals grew after 72 h in 0.2 M MgCl 2 , 20% PEG 3350. Data were collected to 3.5 Å resolution using synchrotron X-ray radiation at the National Synchrotron Light Laboratory (Campinas, Brazil). The crystals belonged to the trigonal space group P3, with unit-cell parameters a = b = 127.84, c = 275.49 Å. Structure determination is under way and will provide relevant information that may lead to the first step in rational drug design for the treatment of Chagas disease

  6. Interdependence of laforin and malin proteins for their stability and ...

    Indian Academy of Sciences (India)

    eisic

    2015-11-28

    Nov 28, 2015 ... Since the clinical symptoms of LD patients representing the two genetic groups are very similar and since malin is ... proteins were also implicated cellular quality control ... Discovery Series software of Bio-Rad Laboratories.

  7. Astatine-211 labelled proteins and their stability in vivo

    International Nuclear Information System (INIS)

    Yi Changhou; Jin Jannan; Zhang Shuyuan; Wang Ketai; Zhang Dayuan; Zhou Maolun

    1989-01-01

    211 At or 131 I labelled proteins, e.g. 211 At-IgG or 211 At-BSA (bovine serum albumin) were prepared by 211 At reaction with the diazo-compound of para-aminobenzoic acid, which is then conjugated with IgG or BSA via an acylation reaction. The 211 At-carbon bond was found metabolically stable under in vivo conditions. For the labelling of proteins with 211 At or 131 I, other methods of direct oxidation are also described. The results show that for the labelling of proteins with 211 At, high rate of incorporation can be obtained with hydrogen peroxide as oxidant, but the labelling of proteins with 131 I is more favourable with the strong oxidant Chloramine-T. (author) 12 refs.; 6 figs

  8. A new crystal lattice structure of Helicobacter pylori neutrophil-activating protein (HP-NAP)

    International Nuclear Information System (INIS)

    Tsuruta, Osamu; Yokoyama, Hideshi; Fujii, Satoshi

    2012-01-01

    A new crystal lattice structure of H. pylori neutrophil-activating protein has been determined. Iron loading causes a series of conformational changes at the ferroxidase centre. A new crystal lattice structure of Helicobacter pylori neutrophil-activating protein (HP-NAP) has been determined in two forms: the native state (Apo) at 2.20 Å resolution and an iron-loaded form (Fe-load) at 2.50 Å resolution. The highly solvated packing of the dodecameric shell is suitable for crystallographic study of the metal ion-uptake pathway. Like other bacterioferritins, HP-NAP forms a spherical dodecamer with 23 symmetry including two kinds of channels. Iron loading causes a series of conformational changes of amino-acid residues (Trp26, Asp52 and Glu56) at the ferroxidase centre

  9. Crystal structure of the Epithiospecifier Protein, ESP from Arabidopsis thaliana provides insights into its product specificity.

    Science.gov (United States)

    Zhang, Weiwei; Wang, Wenhe; Liu, Zihe; Xie, Yongchao; Wang, Hao; Mu, Yajuan; Huang, Yao; Feng, Yue

    2016-09-16

    Specifier proteins are important components of the glucosinolate-myrosinase system, which mediate plant defense against herbivory and pathogen attacks. Upon tissue disruption, glucosinolates are hydrolyzed to instable aglucones by myrosinases, and then aglucones will rearrange to form defensive isothiocyanates. Specifier proteins can redirect this reaction to form other products, such as simple nitriles, epithionitriles and organic thiocyanates instead of isothiocyanates based on the side chain structure of glucosinolate and the type of the specifier proteins. Nevertheless, the molecular mechanism underlying the different product spectrums of various specifier proteins was not fully understood. Here in this study, we solved the crystal structure of the Epithiospecifier Protein, ESP from Arabidopsis thaliana (AtESP) at 2.3 Å resolution. Structural comparisons with the previously solved structure of thiocyanate forming protein, TFP from Thlaspi arvense (TaTFP) reveal that AtESP shows a dimerization pattern different from TaTFP. Moreover, AtESP harbors a slightly larger active site pocket than TaTFP and several residues around the active site are different between the two proteins, which might account for the different product spectrums of the two proteins. Together, our structural study provides important insights into the molecular mechanisms of specifier proteins and shed light on the basis of their different product spectrums. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Expression, crystallization and preliminary crystallographic analysis of C-reactive protein from zebrafish

    International Nuclear Information System (INIS)

    Chen, Rong; Qi, Jianxun; Yao, Shugang; Pan, Xiaocheng; Gao, Feng; Xia, Chun

    2011-01-01

    Crystals of native and selenomethionine-substituted C-reactive protein from zebrafish diffracted to 2.3 and 1.7 Å resolution, respectively, and belonged to space group R3 with one molecule per asymmetric unit. The Matthews coefficient was calculated to be 3.28 Å 3 Da −1 . C-reactive protein (CRP) is an acute phase protein that is found in blood, the concentration of which in plasma rises rapidly in response to inflammation. It functions as a pattern-recognition molecule, recognizing dead cells and various pathogenic agents and eliminating them by utilizing the classical complement pathway and activating macrophages. CRP is phylogenetically highly conserved in invertebrates and mammals. To date, information on the CRP gene has been reported from numerous species of animals, but little is known about the structure of CRP from species other than humans. In order to solve the structure of CRP from bony fish, the CRP gene from zebrafiah (Danio rerio) was cloned and expressed in Escherichia coli. The zebrafish CRP (Dare-CRP) was then purified and crystallized. The crystal diffracted to 2.3 Å resolution and belonged to space group R3, with unit-cell parameters a = b = 114.7, c = 61.0 Å. The Matthews coefficient and solvent content were calculated to be 3.28 Å 3 Da −1 and 62.55%, respectively. Determination of the zebrafish CRP structure should be helpful in investigating the evolution of CRPs in the innate immune system

  11. Pickering emulsions stabilized by whey protein nanoparticles prepared by thermal cross-linking

    NARCIS (Netherlands)

    Wu, Jiande; Shi, Mengxuan; Li, Wei; Zhao, Luhai; Wang, Ze; Yan, Xinzhong; Norde, Willem; Li, Yuan

    2015-01-01

    A Pickering (o/w) emulsion was formed and stabilized by whey protein isolate nanoparticles (WPI NPs). Those WPI NPs were prepared by thermal cross-linking of denatured WPI proteins within w/o emulsion droplets at 80. °C for 15. min. During heating of w/o emulsions containing 10% (w/v) WPI

  12. Adsorption of plasma proteins : adsorption behaviour on apolar surfaces and effect on colloid stability

    NARCIS (Netherlands)

    van der Scheer, Albert

    1978-01-01

    In this thesis the adsorption of some plasma proteins (human albumin (HSA) and fibrinogen (HFb)) on non polar surfaces is studied, together with the influence of these proteins on the stability of polystyrene latices. The aim of these investigations is a better understanding of the processes

  13. Stability of globular proteins in H2O and D2O

    NARCIS (Netherlands)

    Efimova, Y. M.; Haemers, S.; Wierczinski, B.; Norde, W.; van Well, A. A.

    2007-01-01

    In several experimental techniques D2O rather then H2O is often used as a solvent for proteins. Concerning the influence of the solvent on the stability of the proteins, contradicting results have been reported in literature. In this paper the influence of H2O-D2O solvent substitution on the

  14. Stability of globular proteins in H2O and in D2O

    NARCIS (Netherlands)

    Efimova, Y.M.; Haemers, S.; Wierczinsky, B.; Norde, W.; Well, van A.A.

    2007-01-01

    In several experimental techniques D2O rather then H2O is often used as a solvent for proteins. Concerning the influence of the solvent on the stability of the proteins, contradicting results have been reported in literature. In this paper the influence of H2O-D2O solvent substitution on the

  15. Laboratory information management system for membrane protein structure initiative--from gene to crystal.

    Science.gov (United States)

    Troshin, Petr V; Morris, Chris; Prince, Stephen M; Papiz, Miroslav Z

    2008-12-01

    Membrane Protein Structure Initiative (MPSI) exploits laboratory competencies to work collaboratively and distribute work among the different sites. This is possible as protein structure determination requires a series of steps, starting with target selection, through cloning, expression, purification, crystallization and finally structure determination. Distributed sites create a unique set of challenges for integrating and passing on information on the progress of targets. This role is played by the Protein Information Management System (PIMS), which is a laboratory information management system (LIMS), serving as a hub for MPSI, allowing collaborative structural proteomics to be carried out in a distributed fashion. It holds key information on the progress of cloning, expression, purification and crystallization of proteins. PIMS is employed to track the status of protein targets and to manage constructs, primers, experiments, protocols, sample locations and their detailed histories: thus playing a key role in MPSI data exchange. It also serves as the centre of a federation of interoperable information resources such as local laboratory information systems and international archival resources, like PDB or NCBI. During the challenging task of PIMS integration, within the MPSI, we discovered a number of prerequisites for successful PIMS integration. In this article we share our experiences and provide invaluable insights into the process of LIMS adaptation. This information should be of interest to partners who are thinking about using LIMS as a data centre for their collaborative efforts.

  16. Structure of the ordered hydration of amino acids in proteins: analysis of crystal structures

    Energy Technology Data Exchange (ETDEWEB)

    Biedermannová, Lada, E-mail: lada.biedermannova@ibt.cas.cz; Schneider, Bohdan [Institute of Biotechnology CAS, Videnska 1083, 142 20 Prague (Czech Republic)

    2015-10-27

    The hydration of protein crystal structures was studied at the level of individual amino acids. The dependence of the number of water molecules and their preferred spatial localization on various parameters, such as solvent accessibility, secondary structure and side-chain conformation, was determined. Crystallography provides unique information about the arrangement of water molecules near protein surfaces. Using a nonredundant set of 2818 protein crystal structures with a resolution of better than 1.8 Å, the extent and structure of the hydration shell of all 20 standard amino-acid residues were analyzed as function of the residue conformation, secondary structure and solvent accessibility. The results show how hydration depends on the amino-acid conformation and the environment in which it occurs. After conformational clustering of individual residues, the density distribution of water molecules was compiled and the preferred hydration sites were determined as maxima in the pseudo-electron-density representation of water distributions. Many hydration sites interact with both main-chain and side-chain amino-acid atoms, and several occurrences of hydration sites with less canonical contacts, such as carbon–donor hydrogen bonds, OH–π interactions and off-plane interactions with aromatic heteroatoms, are also reported. Information about the location and relative importance of the empirically determined preferred hydration sites in proteins has applications in improving the current methods of hydration-site prediction in molecular replacement, ab initio protein structure prediction and the set-up of molecular-dynamics simulations.

  17. Triosephosphate isomerase is a common crystallization contaminant of soluble His-tagged proteins produced in Escherichia coli

    International Nuclear Information System (INIS)

    Kozlov, Guennadi; Vinaik, Roohi; Gehring, Kalle

    2013-01-01

    Crystals of E. coli triosephosphate isomerase were obtained as a contaminant and its structure was determined to 1.85 Å resolution. Attempts to crystallize several mammalian proteins overexpressed in Escherichia coli revealed a common contaminant, triosephosphate isomerase, a protein involved in glucose metabolism. Even with triosephosphate isomerase present in very small amounts, similarly shaped crystals appeared in the crystallization drops in a number of polyethylene glycol-containing conditions. All of the target proteins were His-tagged and their purification involved immobilized metal-affinity chromatography (IMAC), a step that was likely to lead to triosephosphate isomerase contamination. Analysis of the triosephosphate isomerase crystals led to the structure of E. coli triosephosphate isomerase at 1.85 Å resolution, which is a significant improvement over the previous structure

  18. Influence of fat crystals in the oil phase on stability of oil-in-water emulsions

    NARCIS (Netherlands)

    Boekel, van M.A.J.S.

    1980-01-01

    Coalescence at rest and during flow was studied in emulsions of paraffin oil in water with several surfactants and with crystals of solid paraffin or tristearate in the oil phase. Solid fat in the oil phase was estimated by pulsed nuclear magnetic resonance. Without crystals, oil-in-water emulsions

  19. Preparative Protein Production from Inclusion Bodies and Crystallization: A Seven-Week Biochemistry Sequence

    Science.gov (United States)

    Peterson, Megan J.; Snyder, W. Kalani; Westerman, Shelley; McFarland, Benjamin J.

    2011-01-01

    We describe how to produce and purify proteins from E. coli inclusion bodies by adapting versatile, preparative-scale techniques to the undergraduate laboratory schedule. This seven-week sequence of experiments fits into an annual cycle of research activity in biochemistry courses. Recombinant proteins are expressed as inclusion bodies, which are collected, washed, then solubilized in urea. Stepwise dialysis to dilute urea over the course of a week produces refolded protein. Column chromatography is used to purify protein into fractions, which are then analyzed with gel electrophoresis and concentration assays. Students culminate the project by designing crystallization trials in sitting-drop trays. Student evaluation of the experience has been positive, listing 5–12 new techniques learned, which are transferrable to graduate research in academia and industry. PMID:21691428

  20. Combined protein construct and synthetic gene engineering for heterologous protein expression and crystallization using Gene Composer

    Directory of Open Access Journals (Sweden)

    Walchli John

    2009-04-01

    Full Text Available Abstract Background With the goal of improving yield and success rates of heterologous protein production for structural studies we have developed the database and algorithm software package Gene Composer. This freely available electronic tool facilitates the information-rich design of protein constructs and their engineered synthetic gene sequences, as detailed in the accompanying manuscript. Results In this report, we compare heterologous protein expression levels from native sequences to that of codon engineered synthetic gene constructs designed by Gene Composer. A test set of proteins including a human kinase (P38α, viral polymerase (HCV NS5B, and bacterial structural protein (FtsZ were expressed in both E. coli and a cell-free wheat germ translation system. We also compare the protein expression levels in E. coli for a set of 11 different proteins with greatly varied G:C content and codon bias. Conclusion The results consistently demonstrate that protein yields from codon engineered Gene Composer designs are as good as or better than those achieved from the synonymous native genes. Moreover, structure guided N- and C-terminal deletion constructs designed with the aid of Gene Composer can lead to greater success in gene to structure work as exemplified by the X-ray crystallographic structure determination of FtsZ from Bacillus subtilis. These results validate the Gene Composer algorithms, and suggest that using a combination of synthetic gene and protein construct engineering tools can improve the economics of gene to structure research.

  1. Dual electrically and thermally responsive broadband reflectors based on polymer network stabilized chiral nematic liquid crystals: the role of crosslink density

    NARCIS (Netherlands)

    Khandelwal, H.; Timmermans, G.H.; Debije, M.G.; Schenning, A.P.H.J.

    2016-01-01

    A broadband reflector based on a polymer stabilized chiral nematic liquid crystal has been fabricated. The reflection bandwidth can be manually controlled by an electric field and autonomously by temperature.

  2. Polymer stabilization of the smectic C-alpha* liquid crystal phase—Over tenfold thermal stabilization by confining networks of photo-polymerized reactive mesogens

    International Nuclear Information System (INIS)

    Labeeb, A.; Gleeson, H. F.; Hegmann, T.

    2015-01-01

    The smectic C*-alpha (SmC α *) phase is one of the sub-phases of ferroelectric liquid crystals that has drawn much interest due to its electro-optical properties and ultrafast switching. Generally observed above the ferroelectric SmC* phase in temperature, the SmC α * commonly shows only very narrow phase temperature range of a few degree Celsius. To broaden the SmC α * phase, polymer stabilization was investigated for thermal phase stabilization. Two different reactive monomers were tested in three mixtures, and all three broadened the temperature range of the SmC α * phase from 3 °C to 39 °C. The current reversal method was used to determine the phase existence versus temperature. Moreover, the texture and network structure was studied by polarized optical microscopy and scanning electron microscopy, with the latter revealing the confinement of the smectic layer structure within the porous polymer network

  3. Characterization of milk proteins-lutein complexes and the impact on lutein chemical stability.

    Science.gov (United States)

    Yi, Jiang; Fan, Yuting; Yokoyama, Wallace; Zhang, Yuzhu; Zhao, Liqing

    2016-06-01

    In this study, the interaction of WPI (whey protein isolate) and SC (sodium caseinate) with hydrophobic lutein was investigated through UV-vis spectroscopy and circular dichroism (CD) as well as fluorescence. The effects on lutein's chemical stability were also examined. The decrease of turbidity of lutein suggested that lutein's aqueous solubility was improved after binding with milk proteins. CD analysis indicated lutein had little impact on the secondary structures of both proteins. Different preparation methods have significant impacts on the binding constant. Fluorescence results indicated that WPI and SC interact with lutein by hydrophobic contacts. Milk proteins have protective effects on lutein against oxidation and decomposition, and SC showed better capability in protecting lutein from oxidation than WPI during 16 days storage. The lutein's chemical stability was increased with increasing of proteins concentration. The results indicated that milk proteins may act as effective carriers for lipophilic nutraceuticals. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. New insights into structural determinants of prion protein folding and stability.

    Science.gov (United States)

    Benetti, Federico; Legname, Giuseppe

    2015-01-01

    Prions are the etiological agent of fatal neurodegenerative diseases called prion diseases or transmissible spongiform encephalopathies. These maladies can be sporadic, genetic or infectious disorders. Prions are due to post-translational modifications of the cellular prion protein leading to the formation of a β-sheet enriched conformer with altered biochemical properties. The molecular events causing prion formation in sporadic prion diseases are still elusive. Recently, we published a research elucidating the contribution of major structural determinants and environmental factors in prion protein folding and stability. Our study highlighted the crucial role of octarepeats in stabilizing prion protein; the presence of a highly enthalpically stable intermediate state in prion-susceptible species; and the role of disulfide bridge in preserving native fold thus avoiding the misfolding to a β-sheet enriched isoform. Taking advantage from these findings, in this work we present new insights into structural determinants of prion protein folding and stability.

  5. Poly(zwitterionic)protein conjugates offer increased stability without sacrificing binding affinity or bioactivity

    Science.gov (United States)

    Keefe, Andrew J.; Jiang, Shaoyi

    2012-01-01

    Treatment with therapeutic proteins is an attractive approach to targeting a number of challenging diseases. Unfortunately, the native proteins themselves are often unstable in physiological conditions, reducing bioavailability and therefore increasing the dose that is required. Conjugation with poly(ethylene glycol) (PEG) is often used to increase stability, but this has a detrimental effect on bioactivity. Here, we introduce conjugation with zwitterionic polymers such as poly(carboxybetaine). We show that poly(carboxybetaine) conjugation improves stability in a manner similar to PEGylation, but that the new conjugates retain or even improve the binding affinity as a result of enhanced protein-substrate hydrophobic interactions. This chemistry opens a new avenue for the development of protein therapeutics by avoiding the need to compromise between stability and affinity.

  6. A New Class of Amphiphiles Bearing Rigid Hydrophobic Groups for Solubilization and Stabilization of Membrane Proteins

    DEFF Research Database (Denmark)

    Chae, Pil Seok; Rasmussen, Søren G F; Rana, Rohini R

    2012-01-01

    Non-traditional amphiphiles: Conferring aqueous solubility on membrane proteins generally requires the use of a detergent or other amphiphilic agent. A new class of amphiphiles was synthesized, based on steroidal lipophilic groups, and evaluated with several membrane proteins. The results show th...... that the new amphiphile, "glyco-diosgenin" (GDN; see figure), confers enhanced stability to a variety of membrane proteins in solution relative to popular conventional detergents, such as dodecylmaltoside (DDM)....

  7. Crystallization and preliminary X-ray diffraction analysis of protein 14 from Sulfolobus islandicus filamentous virus (SIFV)

    International Nuclear Information System (INIS)

    Goulet, Adeline; Spinelli, Silvia; Campanacci, Valérie; Porciero, Sophie; Blangy, Stéphanie; Garrett, Roger A.; Tilbeurgh, Herman van; Leulliot, Nicolas; Basta, Tamara; Prangishvili, David; Cambillau, Christian

    2006-01-01

    Crystals of S. islandicus filamentous virus (SIFV) protein 14 have been grown at 293 K. Crystals belong to space group P6 2 22 or P6 4 22 and diffract to a resolution of 2.95 Å. A large-scale programme has been embarked upon aiming towards the structural determination of conserved proteins from viruses infecting hyperthermophilic archaea. Here, the crystallization of protein 14 from the archaeal virus SIFV is reported. This protein, which contains 111 residues (MW 13 465 Da), was cloned and expressed in Escherichia coli with an N-terminal His 6 tag and purified to homogeneity. The tag was subsequently cleaved and the protein was crystallized using PEG 1000 or PEG 4000 as a precipitant. Large crystals were obtained of the native and the selenomethionine-labelled protein using sitting drops of 100–300 nl. Crystals belong to space group P6 2 22 or P6 4 22, with unit-cell parameters a = b = 68.1, c = 132.4 Å. Diffraction data were collected to a maximum acceptable resolution of 2.95 and 3.20 Å for the SeMet-labelled and native protein, respectively

  8. Protein stability in pulmonary drug delivery via nebulization.

    Science.gov (United States)

    Hertel, Sebastian P; Winter, Gerhard; Friess, Wolfgang

    2015-10-01

    Protein inhalation is a delivery route which offers high potential for direct local lung application of proteins. Liquid formulations are usually available in early stages of biopharmaceutical development and nebulizers are the device of choice for atomization avoiding additional process steps like drying and enabling fast progression to clinical trials. While some proteins were proven to remain stable throughout aerosolization e.g. DNase, many biopharmaceuticals are more susceptible towards the stresses encountered during nebulization. The main reason for protein instability is unfolding and aggregation at the air-liquid interface, a problem which is of particular challenge in the case of ultrasound and jet nebulizers due to recirculation of much of the generated droplets. Surfactants are an important formulation component to protect the sensitive biomolecules. A second important challenge is warming of ultrasound and vibrating mesh devices, which can be overcome by overfilling, precooled solutions or cooling of the reservoir. Ultimately, formulation development has to go hand in hand with device evaluation. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Crystallization and preliminary crystallographic data of the PAS domain of the NifL protein from Azotobacter vinelandii.

    NARCIS (Netherlands)

    Hefti, M.H.; Hendle, J.; Enroth, C.; Vervoort, J.J.M.; Tucker, P.A.

    2001-01-01

    The Azotobacter vinelandii NifL protein is a redox-sensing flavoprotein which inhibits the activity of the nitrogen-specific transcriptional activator NifA. The N-terminal PAS domain has been overexpressed in Escherichia coli and crystallized by the hanging-drop vapour-diffusion method. The crystal

  10. Crystallization and quaternary structure analysis of an Lrp-like regulatory protein from the hyperthermophile Pyrococcus furiosus

    NARCIS (Netherlands)

    Sedelnikova, S.E.; Smits, S.H.J.; Leonard, P.M.; Brinkman, A.B.; Oost, van der J.; Rafferty, J.B.

    2001-01-01

    The LrpA transcriptional regulator from Pyrococcus furiosus, a member of the leucine-responsive regulatory protein (Lrp) family, has been crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. The crystals belong to the tetragonal system and are in

  11. NMR structure of the protein NP-247299.1: comparison with the crystal structure

    International Nuclear Information System (INIS)

    Jaudzems, Kristaps; Geralt, Michael; Serrano, Pedro; Mohanty, Biswaranjan; Horst, Reto; Pedrini, Bill; Elsliger, Marc-André; Wilson, Ian A.; Wüthrich, Kurt

    2010-01-01

    Comparison of the NMR and crystal structures of a protein determined using largely automated methods has enabled the interpretation of local differences in the highly similar structures. These differences are found in segments of higher B values in the crystal and correlate with dynamic processes on the NMR chemical shift timescale observed in solution. The NMR structure of the protein NP-247299.1 in solution at 313 K has been determined and is compared with the X-ray crystal structure, which was also solved in the Joint Center for Structural Genomics (JCSG) at 100 K and at 1.7 Å resolution. Both structures were obtained using the current largely automated crystallographic and solution NMR methods used by the JCSG. This paper assesses the accuracy and precision of the results from these recently established automated approaches, aiming for quantitative statements about the location of structure variations that may arise from either one of the methods used or from the different environments in solution and in the crystal. To evaluate the possible impact of the different software used for the crystallographic and the NMR structure determinations and analysis, the concept is introduced of reference structures, which are computed using the NMR software with input of upper-limit distance constraints derived from the molecular models representing the results of the two structure determinations. The use of this new approach is explored to quantify global differences that arise from the different methods of structure determination and analysis versus those that represent interesting local variations or dynamics. The near-identity of the protein core in the NMR and crystal structures thus provided a basis for the identification of complementary information from the two different methods. It was thus observed that locally increased crystallographic B values correlate with dynamic structural polymorphisms in solution, including that the solution state of the protein involves

  12. Biophysical characterization and crystal structure of the Feline Immunodeficiency Virus p15 matrix protein.

    Science.gov (United States)

    Serrière, Jennifer; Robert, Xavier; Perez, Magali; Gouet, Patrice; Guillon, Christophe

    2013-06-24

    Feline Immunodeficiency Virus (FIV) is a viral pathogen that infects domestic cats and wild felids. During the viral replication cycle, the FIV p15 matrix protein oligomerizes to form a closed matrix that underlies the lipidic envelope of the virion. Because of its crucial role in the early and late stages of viral morphogenesis, especially in viral assembly, FIV p15 is an interesting target in the development of potential new therapeutic strategies. Our biochemical study of FIV p15 revealed that it forms a stable dimer in solution under acidic conditions and at high concentration, unlike other retroviral matrix proteins. We determined the crystal structure of full-length FIV p15 to 2 Å resolution and observed a helical organization of the protein, typical for retroviral matrix proteins. A hydrophobic pocket that could accommodate a myristoyl group was identified, and the C-terminal end of FIV p15, which is mainly unstructured, was visible in electron density maps. As FIV p15 crystallizes in acidic conditions but with one monomer in the asymmetric unit, we searched for the presence of a biological dimer in the crystal. No biological assembly was detected by the PISA server, but the three most buried crystallographic interfaces have interesting features: the first one displays a highly conserved tryptophan acting as a binding platform, the second one is located along a 2-fold symmetry axis and the third one resembles the dimeric interface of EIAV p15. Because the C-terminal end of p15 is involved in two of these three interfaces, we investigated the structure and assembly of a C-terminal-truncated form of p15 lacking 14 residues. The truncated FIV p15 dimerizes in solution at a lower concentration and crystallizes with two molecules in the asymmetric unit. The EIAV-like dimeric interface is the only one to be retained in the new crystal form. The dimeric form of FIV p15 in solution and its extended C-terminal end are characteristic among lentiviral matrix proteins

  13. Physicochemical properties and storage stability of soybean protein nanoemulsions prepared by ultra-high pressure homogenization.

    Science.gov (United States)

    Xu, Jing; Mukherjee, Dipaloke; Chang, Sam K C

    2018-02-01

    This study investigated the effects of the ultrahigh pressure homogenization (pressure, protein concentration, oil phase fraction, pH, temperature, and ionic strength) and storage on the properties of nanoemulsions (100-500nm range), which were stabilized by laboratory-prepared soybean protein isolate (SPI), β-conglycinin (7S) and glycinin (11S). The nanoemulsions made with SPI, 7S and 11S proteins exhibited considerable stability over various ionic strengths (0-500mM NaCl), pH (7), thermal treatments (30-60°C) and storage (0-45days). The far-UV spectra of SPI, 7S, 11S dispersions, and SPI-, 7S-, 11S protein-stabilized nanoemulsions were analyzed for the protein structural changes following lipid removal. The ultra-high pressure homogenization changed the secondary structure of SPI, 7S, 11S proteins in the nanoemulsions, and enhanced their stability. This study demonstrated that SPI, 7S, and 11S proteins can be used as effective emulsifiers in nanoemulsions prepared by ultra-high pressure homogenization. Copyright © 2017. Published by Elsevier Ltd.

  14. Preparation, crystallization and preliminary X-ray analysis of YjcG protein from Bacillus subtilis

    International Nuclear Information System (INIS)

    Li, Dan; Chan, Chiomui; Liang, Yu-He; Zheng, Xiaofeng; Li, Lanfen; Su, Xiao-Dong

    2005-01-01

    B. subtilis YjcG protein was expressed, purified and crystallized. A complete diffraction data set was collected at BSRF beamline 3W1A and processed to 2.3 Å resolution. Bacillus subtilis YjcG is a functionally uncharacterized protein with 171 residues that has no structural homologue in the Protein Data Bank. However, it shows sequence homology to bacterial and archaeal 2′–5′ RNA ligases. In order to identify its exact function via structural studies, the yjcG gene was amplified from B. subtilis genomic DNA and cloned into the expression vector pET21-DEST. The protein was expressed in a soluble form in Escherichia coli and was purified to homogeneity. Crystals suitable for X-ray analysis were obtained that diffracted to 2.3 Å and belonged to space group C2, with unit-cell parameters a = 99.66, b = 73.93, c = 61.77 Å, β = 113.56°

  15. Crystal structure of the β2 adrenergic receptor-Gs protein complex

    Energy Technology Data Exchange (ETDEWEB)

    Rasmussen, Søren G.F.; DeVree, Brian T; Zou, Yaozhong; Kruse, Andrew C; Chung, Ka Young; Kobilka, Tong Sun; Thian, Foon Sun; Chae, Pil Seok; Pardon, Els; Calinski, Diane; Mathiesen, Jesper M; Shah, Syed T.A.; Lyons, Joseph A; Caffrey, Martin; Gellman, Samuel H; Steyaert, Jan; Skiniotis, Georgios; Weis, William I; Sunahara, Roger K; Kobilka, Brian K [Brussels; (Trinity); (Michigan); (Stanford-MED); (Michigan-Med); (UW)

    2011-12-07

    G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β2 adrenergic receptor (β2AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β2AR and nucleotide-free Gs heterotrimer. The principal interactions between the β2AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β2AR include a 14Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.

  16. Novel Gyroscopic Mounting for Crystal Oscillators to Increase Short and Medium Term Stability under Highly Dynamic Conditions.

    Science.gov (United States)

    Abedi, Maryam; Jin, Tian; Sun, Kewen

    2015-06-17

    In this paper, a gyroscopic mounting method for crystal oscillators to reduce the impact of dynamic loads on their output stability has been proposed. In order to prove the efficiency of this mounting approach, each dynamic load-induced instability has been analyzed in detail. A statistical study has been performed on the elevation angle of the g-sensitivity vector of Stress Compensated-cut (SC-cut) crystals. The analysis results show that the proposed gyroscopic mounting method gives good performance for host vehicle attitude changes. A phase noise improvement of 27 dB maximum and 5.7 dB on average can be achieved in the case of steady state loads, while under sinusoidal vibration conditions, the maximum and average phase noise improvement are as high as 24 dB and 7.5 dB respectively. With this gyroscopic mounting method, random vibration-induced phase noise instability is reduced 30 dB maximum and 8.7 dB on average. Good effects are apparent for crystal g-sensitivity vectors with low elevation angle φ and azimuthal angle β. under highly dynamic conditions, indicating the probability that crystal oscillator instability will be significantly reduced by using the proposed mounting approach.

  17. Structure and Stability of Molecular Crystals with Many-Body Dispersion-Inclusive Density Functional Tight Binding.

    Science.gov (United States)

    Mortazavi, Majid; Brandenburg, Jan Gerit; Maurer, Reinhard J; Tkatchenko, Alexandre

    2018-01-18

    Accurate prediction of structure and stability of molecular crystals is crucial in materials science and requires reliable modeling of long-range dispersion interactions. Semiempirical electronic structure methods are computationally more efficient than their ab initio counterparts, allowing structure sampling with significant speedups. We combine the Tkatchenko-Scheffler van der Waals method (TS) and the many-body dispersion method (MBD) with third-order density functional tight-binding (DFTB3) via a charge population-based method. We find an overall good performance for the X23 benchmark database of molecular crystals, despite an underestimation of crystal volume that can be traced to the DFTB parametrization. We achieve accurate lattice energy predictions with DFT+MBD energetics on top of vdW-inclusive DFTB3 structures, resulting in a speedup of up to 3000 times compared with a full DFT treatment. This suggests that vdW-inclusive DFTB3 can serve as a viable structural prescreening tool in crystal structure prediction.

  18. Crystal Structure of the Carboxy-Terminal Region of the Bacteriophage T4 Proximal Long Tail Fiber Protein Gp34

    Directory of Open Access Journals (Sweden)

    Meritxell Granell

    2017-06-01

    Full Text Available Long tail fibers of bacteriophage T4 are formed by proteins gp34, gp35, gp36, and gp37, with gp34 located at the phage-proximal end and gp37 at the phage-distal, receptor-binding end. We have solved the structure of the carboxy-terminal region of gp34, consisting of amino acids 894–1289, by single-wavelength anomalous diffraction and extended the structure to amino acids 744–1289 using data collected from crystals containing longer gp34-fragments. The structure reveals three repeats of a mixed α-β fibrous domain in residues 744 to 877. A triple-helical neck connects to an extended triple β-helix domain (amino acids 900–1127 punctuated by two β-prism domains. Next, a β-prism domain decorated with short helices and extended β-helices is present (residues 1146–1238, while the C-terminal end is capped with another short β-helical region and three β-hairpins. The structure provides insight into the stability of the fibrous gp34 protein.

  19. Crystallization and preliminary X-ray diffraction analysis of central structure domains from mumps virus F protein

    International Nuclear Information System (INIS)

    Liu, Yueyong; Xu, Yanhui; Zhu, Jieqing; Qiu, Bingsheng; Rao, Zihe; Gao, George F.; Tien, Po

    2005-01-01

    Single crystals of the central structure domains from mumps virus F protein have been obtained by the hanging-drop vapour-diffusion method. A diffraction data set has been collected to 2.2 Å resolution. Fusion of members of the Paramyxoviridae family involves two glycoproteins: the attachment protein and the fusion protein. Changes in the fusion-protein conformation were caused by binding of the attachment protein to the cellular receptor. In the membrane-fusion process, two highly conserved heptad-repeat (HR) regions, HR1 and HR2, are believed to form a stable six-helix coiled-coil bundle. However, no crystal structure has yet been determined for this state in the mumps virus (MuV, a member of the Paramyxoviridae family). In this study, a single-chain protein consisting of two HR regions connected by a flexible amino-acid linker (named 2-Helix) was expressed, purified and crystallized by the hanging-drop vapour-diffusion method. A complete X-ray data set was obtained in-house to 2.2 Å resolution from a single crystal. The crystal belongs to space group C2, with unit-cell parameters a = 161.2, b = 60.8, c = 40.1 Å, β = 98.4°. The crystal structure will help in understanding the molecular mechanism of Paramyxoviridae family membrane fusion

  20. ContaMiner and ContaBase: a webserver and database for early identification of unwantedly crystallized protein contaminants

    Science.gov (United States)

    Hungler, Arnaud; Momin, Afaque; Diederichs, Kay; Arold, Stefan, T.

    2016-01-01

    Solving the phase problem in protein X-ray crystallography relies heavily on the identity of the crystallized protein, especially when molecular replacement (MR) methods are used. Yet, it is not uncommon that a contaminant crystallizes instead of the protein of interest. Such contaminants may be proteins from the expression host organism, protein fusion tags or proteins added during the purification steps. Many contaminants co-purify easily, crystallize and give good diffraction data. Identification of contaminant crystals may take time, since the presence of the contaminant is unexpected and its identity unknown. A webserver (ContaMiner) and a contaminant database (ContaBase) have been established, to allow fast MR-based screening of crystallographic data against currently 62 known contaminants. The web-based ContaMiner (available at http://strube.cbrc.kaust.edu.sa/contaminer/) currently produces results in 5 min to 4 h. The program is also available in a github repository and can be installed locally. ContaMiner enables screening of novel crystals at synchrotron beamlines, and it would be valuable as a routine safety check for ‘crystallization and preliminary X-ray analysis’ publications. Thus, in addition to potentially saving X-ray crystallographers much time and effort, ContaMiner might considerably lower the risk of publishing erroneous data. PMID:27980519

  1. Development and Stability Evaluation of Liquid Crystal-Based Formulations Containing Glycolic Plant Extracts and Nano-Actives

    Directory of Open Access Journals (Sweden)

    Andreza Rodrigues Ueoka

    2018-03-01

    Full Text Available Emulsions are of great use in cosmetic formulations due to their stability. The aim of this work was to develop and assess organoleptic, physicochemical, and microscopic properties of four auto-emulsifiable oil-in-water formulations. Such formulations were developed containing 4.0% cetearyl alcohol, dicetyl phosphate, and ceteth-10 phosphate (Formulation A, nano-actives obtained from safflower, coconut, and clove oils (Formulation B; a mixture of glycolic extracts from Centella asiatica leaves, Aesculus hippocastanum seeds, and Hamamelis virginiana leaves (Formulation C; association between the nano-actives and glycolic extracts described above (Formulation D. The formulations were trialed for 90 days under the normal stability test. The developed formulations were considered all stable and homogeneous, with liquid crystals possibly being formed. Organoleptic parameters and pH of Formulations A and B remained unchanged, but the color of Formulations C and D changed due to the natural color of the glycolic extracts used. It can be concluded that the formation of liquid crystals increased the stability of the formulations, and future tests should be carried out in order to assess the rheological properties and hydration potential of the developed formulations.

  2. Preparation of shape-stabilized co-crystallized poly (ethylene glycol) composites as thermal energy storage materials

    International Nuclear Information System (INIS)

    Qian, Yong; Wei, Ping; Jiang, Pingkai; Li, Zhi; Yan, Yonggang; Ji, Kejian; Deng, Weihua

    2013-01-01

    Highlights: • Shape-stabilized PEG composites were prepared by sol–gel process. • The increased energy storage ability of composite was from cocrystallization effect. • Diammonium phosphate improved flame retardancy properties of PEG composite. • PEG composites had potential to be used as thermal energy storage materials. - Abstract: Shape-stabilized co-crystallized poly (ethylene glycol) (PEG) composites were prepared by sol–gel process. Tetraethoxysilane was utilized as supporting matrix precursor. The crystallization property as well as thermal energy storage properties of PEG was influenced by silica network. The combination of PEG 2k and PEG 10k with suitable ratio (3:1 by weight) led to synergistically increased fusion enthalpy attributed to cocrystallization effect. Furthermore, halogen-free flame retarded PEG composites were obtained using diammonium phosphate as flame retardant. With suitable composition, the latent heat value of flame retarded PEG composite was 96.7 kJ/kg accompanied with good thermal stability and improved flame retardancy properties. Fourier transform infrared spectrum (FT-IR), X-ray diffraction (XRD), polarized optical microscope (POM) and scanning electron microscope (SEM) were used to characterize the structure of PEG composites. Thermal stability properties of PEG composites were investigated by thermogravimetric analyzer (TGA). Char residue obtained from muffle furnace of PEG composites was analyzed by SEM and FT-IR. Flame retardancy properties of PEG composites were estimated by pyrolysis combustion flow calorimeter. Results showed that it was potential for shape-stabilized halogen-free flame retarded PEG composite to be applied in thermal energy storage field

  3. TOPICAL REVIEW: Protein stability and enzyme activity at extreme biological temperatures

    Science.gov (United States)

    Feller, Georges

    2010-08-01

    Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 °C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins.

  4. Stability of (Fe-Tm-B) amorphous alloys: relaxation and crystallization phenomena

    International Nuclear Information System (INIS)

    Zemcik, T.

    1994-01-01

    Fe-Tm-B base (TM = transition metal) amorphous alloys (metallic glasses) are thermodynamically metastable. This limits their use as otherwise favourable materials, e.g. magnetically soft, corrosion resistant and mechanically firm. By analogy of the mechanical strain-stress dependence, at a certain degree of thermal activation the amorphous structure reaches its limiting state where it changes its character and physical properties. Relaxation and early crystallization processes in amorphous alloys, starting already around 100 C, are reviewed involving subsequently stress relief, free volume shrinking, topological and chemical ordering, pre-crystallization phenomena up to partial (primary) crystallization. Two diametrically different examples are demonstrated from among the soft magnetic materials: relaxation and early crystallization processes in the Fe-Co-B metallic glasses and controlled crystallization of amorphous ribbons yielding rather modern nanocrystalline ''Finemet'' alloys where late relaxation and pre-crystallization phenomena overlap when forming extremely dispersive and fine-grained nanocrystals-in-amorphous-sauce structure. Moessbauer spectroscopy seems to be unique for magnetic and phase analysis of such complicated systems. (orig.)

  5. Polarization switching detection method using a ferroelectric liquid crystal for dichroic atomic vapor laser lock frequency stabilization techniques.

    Science.gov (United States)

    Dudzik, Grzegorz; Rzepka, Janusz; Abramski, Krzysztof M

    2015-04-01

    We present a concept of the polarization switching detection method implemented for frequency-stabilized lasers, called the polarization switching dichroic atomic vapor laser lock (PSDAVLL) technique. It is a combination of the well-known dichroic atomic vapor laser lock method for laser frequency stabilization with a synchronous detection system based on the surface-stabilized ferroelectric liquid crystal (SSFLC).The SSFLC is a polarization switch and quarter wave-plate component. This technique provides a 9.6 dB better dynamic range ratio (DNR) than the well-known two-photodiode detection configuration known as the balanced polarimeter. This paper describes the proposed method used practically in the VCSEL laser frequency stabilization system. The applied PSDAVLL method has allowed us to obtain a frequency stability of 2.7×10⁻⁹ and a reproducibility of 1.2×10⁻⁸, with a DNR of detected signals of around 81 dB. It has been shown that PSDAVLL might be successfully used as a method for spectra-stable laser sources.

  6. Monoclonal Antibody Analysis and Insecticidal Spectrum of Three Types of Lepidopteran-Specific Insecticidal Crystal Proteins of Bacillus thuringiensis

    Science.gov (United States)

    Höfte, Herman; Van Rie, Jeroen; Jansens, Stefan; Van Houtven, Annemie; Vanderbruggen, Hilde; Vaeck, Mark

    1988-01-01

    We have investigated the protein composition and the insecticidal spectrum of crystals of 29 Bacillus thuringiensis strains active against lepidopteran larvae. All crystals contained proteins of 130 to 140 kilodaltons (kDa) which could be grouped into three types by the molecular weight of the protoxin and the trypsin-activated core fragment. Proteins of the three types showed a characteristic insecticidal spectrum when tested against five lepidopteran species. Type A crystal proteins were protoxins of 130 or 133 kDa, which were processed into 60-kDa toxins by trypsin. Several genes encoding crystal proteins of this type have been cloned and sequenced earlier. They are highly conserved in the N-terminal half of the toxic fragment and were previously classified in three subtypes (the 4.5-, 5.3-, and 6.6-kilobase subtypes) based on the restriction map of their genes. The present study shows that different proteins of these three subtypes were equally toxic against Manduca sexta and Pieris brassicae and had no detectable activity against Spodoptera littoralis. However, the 4.5-, 5.3-, and 6.6-kilobase subtypes differed in their toxicity against Heliothis virescens and Mamestra brassicae. Type B crystal proteins consisted of 140-kDa protoxins with a 55-kDa tryptic core fragment. These were only active against one of the five insect species tested (P. brassicae). The protoxin and the trypsin-activated toxin of type C were 135- and 63-kDa proteins, respectively. Proteins of this type were associated with high toxicity against S. littoralis and M. brassicae. A panel of 35 monoclonal antibodies was used to compare the structural characteristics of crystal proteins of the three different types and subtypes. Each type of protein could be associated with a typical epitope structure, indicating an unambiguous correlation between antigenic structure and insect specificity. Images PMID:16347711

  7. Crystal structure of an eIF4G-like protein from Danio rerio

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Euiyoung; Bitto, Eduard; Bingman, Craig A.; McCoy, Jason G.; Wesenberg, Gary E.; Phillips, Jr., George N. (UW)

    2012-04-18

    The gene LOC 91917 Danio rerio (zebrafish) encodes a protein annotated in the UniProt knowledgebase as the middle domain of eukaryotic initiation factor 4G domain containing protein b (MIF4Gdb). Its molecular weight is 25.8 kDa, and it comprises 222 amino acid residues. BLAST searches revealed homologues of D. rerio MIF4Gdb in many eukaryotes including humans. The homologue sand MIF4Gdb were identified as members of the Pfam family, MIF4G (PF2854), which is named after the middle domain of eukaryotic initiation factor 4G (eIF4G). eIF4G is a component of eukaryotic translational initiation complex, and contains binding sites for other initiation factors, suggesting its critical role in translational initiation. The MIF4G domain also occurs in several other proteins involved in RNA metabolism, including the Nonsense-mediated mRNA decay 2 protein (NMD2/UPF2), and the nuclear cap-binding protein 80-kDa subunit (CBP80). Sequence and structure analysis of the MIF4G domains in many proteins indicate that the domain assumes an all helical fold and has tandem repeated motifs. The zebrafish protein described here has homology to domains of other proteins variously referred to as NIC-containing proteins (NMD2, eIF4G, CBP80). The biological function of D. rerio MIF4Gdb has not yet been experimentally characterized, and the annotation is based on amino acid sequence comparison. D. rerio MIF4Gdb did not share more than 25% sequence identity with any protein for which the three-dimensional structure is known and was selected as a target for structure determination by the Center for Eukaryotic Structural Genomics (CESG). Here, they report the crystal structure of D. rerio MIF4Gdb (UniGene code Dr.79360, UniProt code Q5EAQ1, CESG target number GO.79294).

  8. Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

    Science.gov (United States)

    Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

    2003-01-01

    Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

  9. On the behavior of water at subfreezing temperatures in a protein crystal: evidence of higher mobility than in bulk water.

    Science.gov (United States)

    Wang, Dongqi; Böckmann, Anja; Dolenc, Jožica; Meier, Beat H; van Gunsteren, Wilfred F

    2013-10-03

    NMR experiments have shown that water molecules in the crystal of the protein Crh are still mobile at temperatures well below 273 K. In order to investigate this water anomaly, a molecular dynamics (MD) simulation study of crystalline Crh was carried out to determine the mobility of water in this crystal. The simulations were carried out at three temperatures, 150, 200, and 291 K. Simulations of bulk water at these temperatures were also done to obtain the properties of the simple point charge (SPC) water model used at these temperatures and to allow a comparison of the properties of water in the Crh crystal with those of bulk water at the same temperatures. According to the simulations, water is immobilized at 150 K both in crystal and in bulk water. As expected, at 291 K it diffuses and rotates more slowly in the protein crystal than in bulk water. However, at 200 K, the translational and rotational mobility of the water molecules is larger in the crystal than in bulk water. The enhancement of water mobility in the crystal at 200 K was further investigated by MD simulations in which the backbone or all protein atoms were positionally restrained, and in which additionally the electrostatic protein-water interactions were removed. Of these changes in the environment of the water molecules, rigidifying the protein backbones slightly enhanced water diffusion, while it slowed down rotation. In contrast, removal of electrostatic protein-water interactions did not change water diffusion but enhanced rotational motion significantly. Further investigations are required to delineate particular features of the protein crystal that induce the anomalous behavior of water at 200 K.

  10. Crystallization and preliminary X-ray analysis of Ebola VP35 interferon inhibitory domain mutant proteins

    International Nuclear Information System (INIS)

    Leung, Daisy W.; Borek, Dominika; Farahbakhsh, Mina; Ramanan, Parameshwaran; Nix, Jay C.; Wang, Tianjiao; Prins, Kathleen C.; Otwinowski, Zbyszek; Honzatko, Richard B.; Helgeson, Luke A.; Basler, Christopher F.; Amarasinghe, Gaya K.

    2010-01-01

    Three mutant forms of Ebola VP35 interferon inhibitory domain were crystallized in three different space groups. VP35 is one of seven structural proteins encoded by the Ebola viral genome and mediates viral replication, nucleocapsid formation and host immune suppression. The C-terminal interferon inhibitory domain (IID) of VP35 is critical for dsRNA binding and interferon inhibition. The wild-type VP35 IID structure revealed several conserved residues that are important for dsRNA binding and interferon antagonism. Here, the expression, purification and crystallization of recombinant Zaire Ebola VP35 IID mutants R312A, K319A/R322A and K339A in space groups P6 1 22, P2 1 2 1 2 1 and P2 1 , respectively, are described. Diffraction data were collected using synchrotron sources at the Advanced Light Source and the Advanced Photon Source

  11. A new crystal structure fragment-based pharmacophore method for G protein-coupled receptors

    DEFF Research Database (Denmark)

    Fidom, Kimberley; Isberg, Vignir; Hauser, Alexander Sebastian

    2015-01-01

    and receptor residue pairs, from crystal structure complexes. We describe the procedure to collect a library with more than 250 fragments covering 29 residue positions within the generic transmembrane binding pocket. We describe how the library fragments are recombined and inferred to build pharmacophores...... for new targets. A validating retrospective virtual screening of histamine H1 and H3 receptor pharmacophores yielded area-under-the-curves of 0.88 and 0.82, respectively. The fragment-based method has the unique advantage that it can be applied to targets for which no (homologous) crystal structures...... or ligands are known. 47% of the class A G protein-coupled receptors can be targeted with at least four-element pharmacophores. The fragment libraries can also be used to grow known ligands or for rotamer refinement of homology models. Researchers can download the complete fragment library or a subset...

  12. Synergistic and antagonistic effects of plant and dairy protein blends on the physicochemical stability of lycopene-loaded emulsions

    NARCIS (Netherlands)

    Ho, Kacie K.H.Y.; Schroën, Karin; San Martín-González, M.F.; Berton-Carabin, Claire C.

    2018-01-01

    Whey-plant protein-based emulsions had high physicochemical stability. Whey and plant protein blend-based interfaces were viscoelastic while casein-based interfaces were relatively viscous. Whey-plant and plant-plant protein blends behaved synergistically leading to enhanced emulsion stability.

  13. The Effect of Protein PEGylation on Physical Stability in Liquid Formulation

    DEFF Research Database (Denmark)

    Holm, Louise Stenstrup; Mcumber, Aaron; Rasmussen, Jakob Ewald

    2014-01-01

    The presence of micron aggregates in protein formulations has recently attracted increased interest from regulatory authorities, industry, and academia because of the potential undesired side effects of their presence. In this study, we characterized the micron aggregate formation of hen egg...... approximately half as many particles as Lyz, despite its lower apparent thermodynamic stability and more loose protein fold. Further characterization showed that the PEGylation led to a change from attractive to repulsive protein-protein interactions, which may partly explain the reduced particle formation...

  14. Insights into the role of hydration in protein structure and stability obtained through hydrostatic pressure studies

    Directory of Open Access Journals (Sweden)

    C.A. Royer

    2005-08-01

    Full Text Available A thorough understanding of protein structure and stability requires that we elucidate the molecular basis for the effects of both temperature and pressure on protein conformational transitions. While temperature effects are relatively well understood and the change in heat capacity upon unfolding has been reasonably well parameterized, the state of understanding of pressure effects is much less advanced. Ultimately, a quantitative parameterization of the volume changes (at the basis of pressure effects accompanying protein conformational transitions will be required. The present report introduces a qualitative hypothesis based on available model compound data for the molecular basis of volume change upon protein unfolding and its dependence on temperature.

  15. Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases

    OpenAIRE

    Felsovalyi, Flora; Patel, Tushar; Mangiagalli, Paolo; Kumar, Sanat K; Banta, Scott

    2012-01-01

    Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only...

  16. Isolation, purification, crystallization and preliminary crystallographic studies of amaryllin, a plant pathogenesis-related protein from Amaryllis belladonna

    International Nuclear Information System (INIS)

    Kumar, Sanjit; Singh, Nagendra; Sinha, Mau; Kaur, Punit; Srinivasan, A.; Sharma, Sujata; Singh, T. P.

    2009-01-01

    A novel 15 kDa antifungal protein amaryllin has been crystallized using 30% PEG 8000 as the precipitating agent. The crystals belong to orthorhombic space group I or I2 1 2 1 2 1 with cell dimensions, a = 48.6, b = 61.9 and c = 79.6–Å. A novel antifungal protein, amaryllin, has been isolated from the underground bulbs of Amaryllis belladonna, purified to homogeneity and crystallized. The protein was extracted using ammonium sulfate fractionation. The purified protein samples indicated a molecular weight of 15 kDa on SDS–PAGE. The protein showed antifungal activity against Aspergillus flavus and Fusarium oxysporum. The N-terminal sequence of the first 15 amino-acid residues was determined using Edman degradation and did not show significant sequence identity to any known protein. The protein was crystallized using the hanging-drop vapour-diffusion method with 30% PEG 8000 as precipitating agent. The crystals diffracted to 2.7 Å resolution and belonged to the orthorhombic space group I222 or I2 1 2 1 2 1 , with unit-cell parameters a = 48.6, b = 61.9, c = 79.6 Å. The complete sequence and structure determination of amaryllin are in progress

  17. TargetCrys: protein crystallization prediction by fusing multi-view features with two-layered SVM.

    Science.gov (United States)

    Hu, Jun; Han, Ke; Li, Yang; Yang, Jing-Yu; Shen, Hong-Bin; Yu, Dong-Jun

    2016-11-01

    The accurate prediction of whether a protein will crystallize plays a crucial role in improving the success rate of protein crystallization projects. A common critical problem in the development of machine-learning-based protein crystallization predictors is how to effectively utilize protein features extracted from different views. In this study, we aimed to improve the efficiency of fusing multi-view protein features by proposing a new two-layered SVM (2L-SVM) which switches the feature-level fusion problem to a decision-level fusion problem: the SVMs in the 1st layer of the 2L-SVM are trained on each of the multi-view feature sets; then, the outputs of the 1st layer SVMs, which are the "intermediate" decisions made based on the respective feature sets, are further ensembled by a 2nd layer SVM. Based on the proposed 2L-SVM, we implemented a sequence-based protein crystallization predictor called TargetCrys. Experimental results on several benchmark datasets demonstrated the efficacy of the proposed 2L-SVM for fusing multi-view features. We also compared TargetCrys with existing sequence-based protein crystallization predictors and demonstrated that the proposed TargetCrys outperformed most of the existing predictors and is competitive with the state-of-the-art predictors. The TargetCrys webserver and datasets used in this study are freely available for academic use at: http://csbio.njust.edu.cn/bioinf/TargetCrys .

  18. X-ray-excited optical luminescence of protein crystals: a new tool for studying radiation damage during diffraction data collection.

    Science.gov (United States)

    Owen, Robin L; Yorke, Briony A; Pearson, Arwen R

    2012-05-01

    During X-ray irradiation protein crystals radiate energy in the form of small amounts of visible light. This is known as X-ray-excited optical luminescence (XEOL). The XEOL of several proteins and their constituent amino acids has been characterized using the microspectrophotometers at the Swiss Light Source and Diamond Light Source. XEOL arises primarily from aromatic amino acids, but the effects of local environment and quenching within a crystal mean that the XEOL spectrum of a crystal is not the simple sum of the spectra of its constituent parts. Upon repeated exposure to X-rays XEOL spectra decay non-uniformly, suggesting that XEOL is sensitive to site-specific radiation damage. However, rates of XEOL decay were found not to correlate to decays in diffracting power, making XEOL of limited use as a metric for radiation damage to protein crystals. © 2012 International Union of Crystallography

  19. Purification, crystallization and preliminary X-ray diffraction study of human ribosomal protein L10 core domain

    International Nuclear Information System (INIS)

    Nishimura, Mitsuhiro; Kaminishi, Tatsuya; Kawazoe, Masahito; Shirouzu, Mikako; Takemoto, Chie; Yokoyama, Shigeyuki; Tanaka, Akiko; Sugano, Sumio; Yoshida, Takuya; Ohkubo, Tadayasu; Kobayashi, Yuji

    2007-01-01

    A truncated variant of human ribosomal protien L10 was prepared and crystallized. Diffraction data were collected to 2.5 Å resolution. Eukaryotic ribosomal protein L10 is an essential component of the large ribosomal subunit, which organizes the architecture of the aminoacyl-tRNA binding site. The human L10 protein is also called the QM protein and consists of 214 amino-acid residues. For crystallization, the L10 core domain (L10CD, Phe34–Glu182) was recombinantly expressed in Escherichia coli and purified to homogeneity. A hexagonal crystal of L10CD was obtained by the sitting-drop vapour-diffusion method. The L10CD crystal diffracted to 2.5 Å resolution and belongs to space group P3 1 21 or P3 2 21

  20. Principles and equations for measuring and interpreting protein stability: From monomer to tetramer.

    Science.gov (United States)

    Bedouelle, Hugues

    2016-02-01

    The ability to measure the thermodynamic stability of proteins with precision is important for both academic and applied research. Such measurements rely on mathematical models of the protein denaturation profile, i.e. the relation between a global protein signal, corresponding to the folding states in equilibrium, and the variable value of a denaturing agent, either heat or a chemical molecule, e.g. urea or guanidinium hydrochloride. In turn, such models rely on a handful of physical laws: the laws of mass action and conservation, the law that relates the protein signal and concentration, and the one that relates stability and denaturant value. So far, equations have been derived mainly for the denaturation profiles of homomeric proteins. Here, we review the underlying basic physical laws and show in detail how to derive model equations for the unfolding equilibria of homomeric or heteromeric proteins up to trimers and potentially tetramers, with or without folding intermediates, and give full demonstrations. We show that such equations cannot be derived for pentamers or higher oligomers except in special degenerate cases. We expand the method to signals that do not correspond to extensive protein properties. We review and expand methods for uncovering hidden intermediates of unfolding. Finally, we review methods for comparing and interpreting the thermodynamic parameters that derive from stability measurements for cognate wild-type and mutant proteins. This work should provide a robust theoretical basis for measuring the stability of complex proteins. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  1. Effect of thermal stability on protein adsorption to silica using homologous aldo-keto reductases.

    Science.gov (United States)

    Felsovalyi, Flora; Patel, Tushar; Mangiagalli, Paolo; Kumar, Sanat K; Banta, Scott

    2012-08-01

    Gaining more insight into the mechanisms governing the behavior of proteins at solid/liquid interfaces is particularly relevant in the interaction of high-value biologics with storage and delivery device surfaces, where adsorption-induced conformational changes may dramatically affect biocompatibility. The impact of structural stability on interfacial behavior has been previously investigated by engineering nonwild-type stability mutants. Potential shortcomings of such approaches include only modest changes in thermostability, and the introduction of changes in the topology of the proteins when disulfide bonds are incorporated. Here we employ two members of the aldo-keto reductase superfamily (alcohol dehydrogenase, AdhD and human aldose reductase, hAR) to gain a new perspective on the role of naturally occurring thermostability on adsorbed protein arrangement and its subsequent impact on desorption. Unexpectedly, we find that during initial adsorption events, both proteins have similar affinity to the substrate and undergo nearly identical levels of structural perturbation. Interesting differences between AdhD and hAR occur during desorption and both proteins exhibit some level of activity loss and irreversible conformational change upon desorption. Although such surface-induced denaturation is expected for the less stable hAR, it is remarkable that the extremely thermostable AdhD is similarly affected by adsorption-induced events. These results question the role of thermal stability as a predictor of protein adsorption/desorption behavior. Copyright © 2012 The Protein Society.

  2. High-quality Thermodynamic Data on the Stability Changes of Proteins Upon Single-site Mutations

    Energy Technology Data Exchange (ETDEWEB)

    Pucci, Fabrizio, E-mail: fapucci@ulb.ac.be; Bourgeas, Raphaël, E-mail: rbourgeas@ulb.ac.be; Rooman, Marianne, E-mail: mrooman@ulb.ac.be [Department of BioModeling, BioInformatics and BioProcesses, Université Libre de Bruxelles, CP 165/61, Roosevelt Avenue 50, 1050 Brussels, Belgium and Interuniversity Institute of Bioinformatics in Brussels, CP 263, Triumph Bld, 1050 Brussels (Belgium)

    2016-06-15

    We have set up and manually curated a dataset containing experimental information on the impact of amino acid substitutions in a protein on its thermal stability. It consists of a repository of experimentally measured melting temperatures (T{sub m}) and their changes upon point mutations (ΔT{sub m}) for proteins having a well-resolved x-ray structure. This high-quality dataset is designed for being used for the training or benchmarking of in silico thermal stability prediction methods. It also reports other experimentally measured thermodynamic quantities when available, i.e., the folding enthalpy (ΔH) and heat capacity (ΔC{sub P}) of the wild type proteins and their changes upon mutations (ΔΔH and ΔΔC{sub P}), as well as the change in folding free energy (ΔΔG) at a reference temperature. These data are analyzed in view of improving our insights into the correlation between thermal and thermodynamic stabilities, the asymmetry between the number of stabilizing and destabilizing mutations, and the difference in stabilization potential of thermostable versus mesostable proteins.

  3. Structure determination of an integral membrane protein at room temperature from crystals in situ

    International Nuclear Information System (INIS)

    Axford, Danny; Foadi, James; Hu, Nien-Jen; Choudhury, Hassanul Ghani; Iwata, So; Beis, Konstantinos; Evans, Gwyndaf; Alguel, Yilmaz

    2015-01-01

    The X-ray structure determination of an integral membrane protein using synchrotron diffraction data measured in situ at room temperature is demonstrated. The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines

  4. Structure determination of an integral membrane protein at room temperature from crystals in situ

    Energy Technology Data Exchange (ETDEWEB)

    Axford, Danny [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Foadi, James [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Hu, Nien-Jen; Choudhury, Hassanul Ghani [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Rutherford Appleton Laboratory, Oxfordshire OX11 0FA (United Kingdom); Iwata, So [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Rutherford Appleton Laboratory, Oxfordshire OX11 0FA (United Kingdom); Kyoto University, Kyoto 606-8501 (Japan); Beis, Konstantinos [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Rutherford Appleton Laboratory, Oxfordshire OX11 0FA (United Kingdom); Evans, Gwyndaf, E-mail: gwyndaf.evans@diamond.ac.uk [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Alguel, Yilmaz, E-mail: gwyndaf.evans@diamond.ac.uk [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Rutherford Appleton Laboratory, Oxfordshire OX11 0FA (United Kingdom)

    2015-05-14

    The X-ray structure determination of an integral membrane protein using synchrotron diffraction data measured in situ at room temperature is demonstrated. The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines.

  5. Crystallization and preliminary crystallographic characterization of the extrinsic PsbP protein of photosystem II from Spinacia oleracea

    International Nuclear Information System (INIS)

    Kohoutová, J.; Kutá Smatanová, I.; Brynda, J.; Lapkouski, M.; Revuelta, J. L.; Arellano, J. B.; Ettrich, R.

    2009-01-01

    Degradation-free crystalization of thrombin-digested recombinant His-tagged PsbP protein of photosystem II from Spinacia oleracea resulting in crystals diffracting to 2.06 Å. Preliminary X-ray diffraction analysis of the extrinsic PsbP protein of photosystem II from spinach (Spinacia oleracea) was performed using N-terminally His-tagged recombinant PsbP protein overexpressed in Escherichia coli. Recombinant PsbP protein (thrombin-digested recombinant His-tagged PsbP) stored in bis-Tris buffer pH 6.00 was crystallized using the sitting-drop vapour-diffusion technique with PEG 550 MME as a precipitant and zinc sulfate as an additive. SDS–PAGE analysis of a dissolved crystal showed that the crystals did not contain the degradation products of recombinant PsbP protein. PsbP crystals diffracted to 2.06 Å resolution in space group P2 1 2 1 2 1 , with unit-cell parameters a = 38.68, b = 46.73, c = 88.9 Å

  6. Dissecting the critical factors for thermodynamic stability of modular proteins using molecular modeling approach.

    Directory of Open Access Journals (Sweden)

    Yuno Lee

    Full Text Available Repeat proteins have recently attracted much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural and biophysical features. In particular, repeat proteins show high stability against temperature and chaotic agents. Despite many studies, structural features for the stability of repeat proteins remain poorly understood. Here we present an interesting result from in silico analyses pursuing the factors which affect the stability of repeat proteins. Previously developed repebody structure based on variable lymphocytes receptors (VLRs which consists of leucine-rich repeat (LRR modules was used as initial structure for the present study. We constructed extra six repebody structures with varying numbers of repeat modules and those structures were used for molecular dynamics simulations. For the structures, the intramolecular interactions including backbone H-bonds, van der Waals energy, and hydrophobicity were investigated and then the radius of gyration, solvent-accessible surface area, ratio of secondary structure, and hydration free energy were also calculated to find out the relationship between the number of LRR modules and stability of the protein. Our results show that the intramolecular interactions lead to more compact structure and smaller surface area of the repebodies, which are critical for the stability of repeat proteins. The other features were also well compatible with the experimental results. Based on our observations, the repebody-5 was proposed as the best structure from the all repebodies in structure optimization process. The present study successfully demonstrated that our computer-based molecular modeling approach can significantly contribute to the experiment-based protein engineering challenge.

  7. Crystal Structure of VC0702 at 2.0 Angstrom: Conserved Hypothetical Protein from Vibrio Cholerae

    International Nuclear Information System (INIS)

    Ni, S.; Forouhar, F.; Bussiere, D.; Robinson, H.; Kennedy, M.

    2006-01-01

    VC0702, a conserved hypothetical protein of unknown function from Vibrio cholerae, resides in a three-gene operon containing the MbaA gene that encodes for a GGDEF and EAL domain-containing protein which is involved in regulating formation of the extracellular matrix of biofilms in Vibrio cholerae. The VC0702 crystal structure has been determined at 2.0 Angstroms and refined to R work = 22.8% and R free = 26.3%. VC0702 crystallized in an orthorhombic crystal lattice in the C2221 space group with dimensions of a = 66.61 Angstroms, b = 88.118 Angstroms, and c = 118.35 Angstroms with a homodimer in the asymmetric unit. VC0702, which forms a mixed α + β three-layered αβα sandwich, belongs to the Pfam DUF84 and COG1986 families of proteins. Sequence conservation within the DUF84 and COG1986 families was used to identify a conserved patch of surface residues that define a cleft and potential substrate-binding site in VC0702. The three-dimensional structure of VC0702 is similar to that of Mj0226 from Methanococcus janeschii, which has been identified as a novel NTPase that binds NTP in a deep cleft similarly located to the conserved patch of surface residues that define an analogous cleft in VC0702. Collectively, the data suggest that VC0702 may have a biochemical function that involves NTP binding and phosphatase activity of some kind, and is likely involved in regulation of the signaling pathway that controls biofilm formation and maintenance in Vibrio cholerae

  8. Sample Stability and Protein Composition of Saliva: Implications for Its Use as a Diagnostic Fluid

    Directory of Open Access Journals (Sweden)

    Han Roelofsen

    2008-01-01

    Full Text Available Saliva is an easy accessible plasma ultra-filtrate. Therefore, saliva can be an attractive alternative to blood for measurement of diagnostic protein markers. Our aim was to determine stability and protein composition of saliva. Protein stability at room temperature was examined by incubating fresh whole saliva with and without inhibitors of proteases and bacterial metabolism followed by Surface Enhanced Laser Desorption/Ionization (SELDI analyses. Protein composition was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE fractionation of saliva proteins followed by digestion of excised bands and identification by liquid chromatography tandem mass spectrometry (LC-MS/MS. Results show that rapid protein degradation occurs within 30 minutes after sample collection. Degradation starts already during collection. Protease inhibitors partly prevented degradation while inhibition of bacterial metabolism did not affect degradation. Three stable degradation products of 2937 Da, 3370 Da and 4132 Da were discovered which can be used as markers to monitor sample quality. Saliva proteome analyses revealed 218 proteins of which 84 can also be found in blood plasma. Based on a comparison with seven other proteomics studies on whole saliva we identified 83 new saliva proteins. We conclude that saliva is a promising diagnostic fl uid when precautions are taken towards protein breakdown.

  9. StAR Protein Stability in Y1 and Kin-8 Mouse Adrenocortical Cells.

    Science.gov (United States)

    Clark, Barbara J; Hudson, Elizabeth A

    2015-03-04

    The steroidogenic acute regulatory protein (STAR) protein expression is required for cholesterol transport into mitochondria to initiate steroidogenesis in the adrenal and gonads. STAR is synthesized as a 37 kDa precursor protein which is targeted to the mitochondria and imported and processed to an intra-mitochondrial 30 kDa protein. Tropic hormone stimulation of the cAMP-dependent protein kinase A (PKA) signaling pathway is the major contributor to the transcriptional and post-transcriptional regulation of STAR synthesis. Many studies have focused on the mechanisms of cAMP-PKA mediated control of STAR synthesis while there are few reports on STAR degradation pathways. The objective of this study was to determine the effect of cAMP-PKA-dependent signaling on STAR protein stability. We have used the cAMP-PKA responsive Y1 mouse adrenocortical cells and the PKA-deficient Kin-8 cells to measure STAR phosphorylation and protein half-life. Western blot analysis and standard radiolabeled pulse-chase experiments were used to determine STAR phosphorylation status and protein half-life, respectively. Our data demonstrate that PKA-dependent STAR phosphorylation does not contribute to 30 kDa STAR protein stability in the mitochondria. We further show that inhibition of the 26S proteasome does not block precursor STAR phosphorylation or steroid production in Y1 cells. These data suggest STAR can maintain function and promote steroidogenesis under conditions of proteasome inhibition.

  10. ContaMiner and ContaBase: a webserver and database for early identification of unwantedly crystallized protein contaminants

    KAUST Repository

    Hungler, Arnaud; Momin, Afaque Ahmad Imtiyaz; Diederichs, Kay; Arold, Stefan T.

    2016-01-01

    Solving the phase problem in protein X-ray crystallography relies heavily on the identity of the crystallized protein, especially when molecular replacement (MR) methods are used. Yet, it is not uncommon that a contaminant crystallizes instead of the protein of interest. Such contaminants may be proteins from the expression host organism, protein fusion tags or proteins added during the purification steps. Many contaminants co-purify easily, crystallize and give good diffraction data. Identification of contaminant crystals may take time, since the presence of the contaminant is unexpected and its identity unknown. A webserver (ContaMiner) and a contaminant database (ContaBase) have been established, to allow fast MR-based screening of crystallographic data against currently 62 known contaminants. The web-based ContaMiner (available at http://strube.cbrc.kaust.edu.sa/contaminer/) currently produces results in 5 min to 4 h. The program is also available in a github repository and can be installed locally. ContaMiner enables screening of novel crystals at synchrotron beamlines, and it would be valuable as a routine safety check for 'crystallization and preliminary X-ray analysis' publications. Thus, in addition to potentially saving X-ray crystallographers much time and effort, ContaMiner might considerably lower the risk of publishing erroneous data. A web server, titled ContaMiner, has been established, which allows fast molecular-replacement-based screening of crystallographic data against a database (ContaBase) of currently 62 potential contaminants. ContaMiner enables systematic screening of novel crystals at synchrotron beamlines, and it would be valuable as a routine safety check for 'crystallization and preliminary X-ray analysis' publications. © Arnaud Hungler et al. 2016.

  11. ContaMiner and ContaBase: a webserver and database for early identification of unwantedly crystallized protein contaminants

    KAUST Repository

    Hungler, Arnaud

    2016-11-02

    Solving the phase problem in protein X-ray crystallography relies heavily on the identity of the crystallized protein, especially when molecular replacement (MR) methods are used. Yet, it is not uncommon that a contaminant crystallizes instead of the protein of interest. Such contaminants may be proteins from the expression host organism, protein fusion tags or proteins added during the purification steps. Many contaminants co-purify easily, crystallize and give good diffraction data. Identification of contaminant crystals may take time, since the presence of the contaminant is unexpected and its identity unknown. A webserver (ContaMiner) and a contaminant database (ContaBase) have been established, to allow fast MR-based screening of crystallographic data against currently 62 known contaminants. The web-based ContaMiner (available at http://strube.cbrc.kaust.edu.sa/contaminer/) currently produces results in 5 min to 4 h. The program is also available in a github repository and can be installed locally. ContaMiner enables screening of novel crystals at synchrotron beamlines, and it would be valuable as a routine safety check for \\'crystallization and preliminary X-ray analysis\\' publications. Thus, in addition to potentially saving X-ray crystallographers much time and effort, ContaMiner might considerably lower the risk of publishing erroneous data. A web server, titled ContaMiner, has been established, which allows fast molecular-replacement-based screening of crystallographic data against a database (ContaBase) of currently 62 potential contaminants. ContaMiner enables systematic screening of novel crystals at synchrotron beamlines, and it would be valuable as a routine safety check for \\'crystallization and preliminary X-ray analysis\\' publications. © Arnaud Hungler et al. 2016.

  12. Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin.

    Science.gov (United States)

    Oulhen, Nathalie; Wessel, Gary M

    2016-10-01

    Nanos is a translational regulator required for the survival and maintenance of primordial germ cells. In the sea urchin, Strongylocentrotus purpuratus (Sp), Nanos 2 mRNA is broadly transcribed but accumulates specifically in the small micromere (sMic) lineage, in part because of the 3'UTR element GNARLE leads to turnover in somatic cells but retention in the sMics. Here we found that the Nanos 2 protein is also selectively stabilized; it is initially translated throughout the embryo but turned over in the future somatic cells and retained only in the sMics, the future germ line in this animal. This differential stability of Nanos protein is dependent on the open reading frame (ORF), and is independent of the sumoylation and ubiquitylation pathways. Manipulation of the ORF indicates that 68 amino acids in the N terminus of the Nanos protein are essential for its stability in the sMics whereas a 45 amino acid element adjacent to the zinc fingers targets its degradation. Further, this regulation of Nanos protein is cell autonomous, following formation of the germ line. These results are paradigmatic for the unique presence of Nanos in the germ line by a combination of selective RNA retention, distinctive translational control mechanisms (Oulhen et al., 2013), and now also by defined Nanos protein stability. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Crystal structure of AFV3-109, a highly conserved protein from crenarchaeal viruses

    Directory of Open Access Journals (Sweden)

    Quevillon-Cheruel Sophie

    2007-01-01

    Full Text Available Abstract The extraordinary morphologies of viruses infecting hyperthermophilic archaea clearly distinguish them from bacterial and eukaryotic viruses. Moreover, their genomes code for proteins that to a large extend have no related sequences in the extent databases. However, a small pool of genes is shared by overlapping subsets of these viruses, and the most conserved gene, exemplified by the ORF109 of the Acidianus Filamentous Virus 3, AFV3, is present on genomes of members of three viral familes, the Lipothrixviridae, Rudiviridae, and "Bicaudaviridae", as well as of the unclassified Sulfolobus Turreted Icosahedral Virus, STIV. We present here the crystal structure of the protein (Mr = 13.1 kD, 109 residues encoded by the AFV3 ORF 109 in two different crystal forms at 1.5 and 1.3 Å resolution. The structure of AFV3-109 is a five stranded β-sheet with loops on one side and three helices on the other. It forms a dimer adopting the shape of a cradle that encompasses the best conserved regions of the sequence. No protein with a related fold could be identified except for the ortholog from STIV1, whose structure was deposited at the Protein Data Bank. We could clearly identify a well bound glycerol inside the cradle, contacting exclusively totally conserved residues. This interaction was confirmed in solution by fluorescence titration. Although the function of AFV3-109 cannot be deduced directly from its structure, structural homology with the STIV1 protein, and the size and charge distribution of the cavity suggested it could interact with nucleic acids. Fluorescence quenching titrations also showed that AFV3-109 interacts with dsDNA. Genomic sequence analysis revealed bacterial homologs of AFV3-109 as a part of a putative previously unidentified prophage sequences in some Firmicutes.

  14. Solution structure of detergent micelles at conditions relevant to membrane protein crystallization.

    Energy Technology Data Exchange (ETDEWEB)

    Littrell, K.; Thiyagarajan, P.; Tiede, D.; Urban, V.

    1999-07-02

    In this study small angle neutron scattering was used to characterize the formation of micelles in aqueous solutions of the detergents DMG and SPC as a function of detergent concentration and ionic strength of the solvent. The effects on the micelle structure of the additives glycerol and PEG, alone as well as in combination typical for actual membrane protein crystallization, were also explored. This research suggests that the micelles are cigar-like in form at the concentrations studied. The size of the micelles was observed to increase with increasing ionic strength but decrease with the addition of glycerol or PEG.

  15. Model for determining vapor equilibrium rates in the hanging drop method for protein crystal growth

    Science.gov (United States)

    Baird, James K.; Frieden, Richard W.; Meehan, E. J., Jr.; Twigg, Pamela J.; Howard, Sandra B.; Fowlis, William A.

    1987-01-01

    An engineering analysis of the rate of evaporation of solvent in the hanging drop method of protein crystal growth is presented. Results are applied to 18 drop and well arrangements commonly encountered in the laboratory. The chemical nature of the salt, drop size and shape, drop concentration, well size, well concentration, and temperature are taken into account. The rate of evaporation increases with temperature, drop size, and the salt concentration difference between the drop and the well. The evaporation in this model possesses no unique half-life. Once the salt in the drop achieves 80 percent of its final concentration, further evaporation suffers from the law of diminishing returns.

  16. Expression, crystallization and preliminary X-ray crystallographic studies of the outer membrane protein OmpW from Escherichia coli

    International Nuclear Information System (INIS)

    Albrecht, Reinhard; Zeth, Kornelius; Söding, Johannes; Lupas, Andrei; Linke, Dirk

    2006-01-01

    The outer membrane protein OmpW from E. coli was overexpressed in inclusion bodies and refolded with the help of detergent. The protein has been crystallized and the crystals diffract to 3.5 Å resolution. OmpW is an eight-stranded 21 kDa molecular-weight β-barrel protein from the outer membrane of Gram-negative bacteria. It is a major antigen in bacterial infections and has implications in antibiotic resistance and in the oxidative degradation of organic compounds. OmpW from Escherichia coli was cloned and the protein was expressed in inclusion bodies. A method for refolding and purification was developed which yields properly folded protein according to circular-dichroism measurements. The protein has been crystallized and crystals were obtained that diffracted to a resolution limit of 3.5 Å. The crystals belong to space group P422, with unit-cell parameters a = 122.5, c = 105.7 Å. A homology model of OmpW is presented based on known structures of eight-stranded β-barrels, intended for use in molecular-replacement trials

  17. Stabilizing Protein Effects on the Pressure Sensitivity of Fluorescent Gold Nanoclusters

    Science.gov (United States)

    2016-01-13

    affected by the environment of the stabilizing protein, allowing these hybrid systems to act as sensors in many applications.2,9,14–19 This has led...Biosens Bioelectron. 2012;32:297–299. 8. Joseph D, Geckeler KE. Synthesis of highly fluorescent gold nanoclusters using egg white proteins. Colloids Surf...Chang HW, Chien YC, Hsiao JK, Cheng JT, Chou PT. Insulin -directed synthesis of fluorescent gold nanoclusters: preservation of insulin bioactivity and

  18. Stability and structure of the membrane protein transporter Ffh is modulated by substrates and lipids

    DEFF Research Database (Denmark)

    Reinau, Marika Ejby; Otzen, Daniel

    2009-01-01

    the apoprotein. Escherichia coli lipid and DOPG (and to a smaller extent DOPC) increase Ffh's α-helical content, possibly related to Ffh's role in guiding membrane proteins to the membrane. Binding is largely mediated by electrostatic interactions but does not protect Ffh against trypsinolysis. We conclude...... that Ffh is a structurally flexible and dynamic protein whose stability is significantly modulated by the environment. © 2009 Elsevier Inc. All rights reserved....

  19. Purification, crystallization and preliminary crystallographic analysis of GTP-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Wu Hao,; Sun, L.; Brouns, S.J.J.; Fu, S.; Akerboom, A.P.; Li, X.; Oost, van der J.

    2007-01-01

    A predicted GTP-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus, termed SsGBP, has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized using the hanging-drop vapour-diffusion technique in the presence of 0.05 M cadmium sulfate and 0.8

  20. Purification, crystallization and preliminary X-ray diffraction analysis of an oomycete-derived Nep1-like protein

    NARCIS (Netherlands)

    Luberacki, B.; Weyand, M.; Seitz, H.U.; Koch, W.; Oecking, C.; Ottmann, C.

    2008-01-01

    The elicitor protein Nep1-like protein from the plant pathogen Pythium aphanidermatum was purified and crystallized using the hanging-drop vapour-diffusion method. A native data set was collected to 1.35 angstrom resolution at 100 K using synchrotron radiation. Since selenomethionine-labelled

  1. Co-ordinated functions of Mms proteins define the surface structure of cubo-octahedral magnetite crystals in magnetotactic bacteria.

    Science.gov (United States)

    Arakaki, Atsushi; Yamagishi, Ayana; Fukuyo, Ayumi; Tanaka, Masayoshi; Matsunaga, Tadashi

    2014-08-01

    Magnetotactic bacteria synthesize magnetosomes comprised of membrane-enveloped single crystalline magnetite (Fe3 O4 ). The size and morphology of the nano-sized magnetite crystals (Mms (Mms5, Mms6, Mms7, and Mms13), was previously isolated from the surface of cubo-octahedral magnetite crystals in Magnetospirillum magneticum strain AMB-1. Analysis of an mms6 gene deletion mutant suggested that the Mms6 protein plays a major role in the regulation of magnetite crystal size and morphology. In this study, we constructed various mms gene deletion mutants and characterized the magnetite crystals formed by the mutant strains. Comparative analysis showed that all mms genes were involved in the promotion of crystal growth in different manners. The phenotypic characterization of magnetites also suggested that these proteins are involved in controlling the geometries of the crystal surface structures. Thus, the co-ordinated functions of Mms proteins regulate the morphology of the cubo-octahedral magnetite crystals in magnetotactic bacteria. © 2014 John Wiley & Sons Ltd.

  2. Crystallization and preliminary X-ray analysis of the chemokine-binding protein from orf virus (Poxviridae)

    International Nuclear Information System (INIS)

    Couñago, Rafael Miguez; Fleming, Stephen B.; Mercer, Andrew A.; Krause, Kurt L.

    2010-01-01

    The chemokine-binding protein from orf virus was purified and crystallized. The morphology and diffraction behaviour of these crystals was significantly improved through the use of additives known as Silver Bullets. The parapoxvirus orf virus (ORFV) encodes a chemokine-binding protein (CBP) that functions to downregulate the host’s immune response at the site of infection by blocking the chemokine-induced recruitment of immune cells. In order to shed light on the structural determinants of CBP–chemokine binding, ORFV CBP was crystallized as part of an ongoing structure–function study on this protein. ORFV CBP crystals were obtained by the sitting-drop vapour-diffusion technique using ammonium citrate as a precipitant. The crystal quality was greatly improved through the addition of small-molecule additives to the crystallization mother liquor. ORFV CBP crystals diffracted X-rays to 2.50 Å resolution and belonged to the hexagonal space group P6 1 22 or its enantiomorph P6 5 22, with unit-cell parameters a = b = 75.62, c = 282.49 Å, α = 90, β = 90, γ = 120°

  3. Crystal Structure Analysis and the Identification of Distinctive Functional Regions of the Protein Elicitor Mohrip2.

    Science.gov (United States)

    Liu, Mengjie; Duan, Liangwei; Wang, Meifang; Zeng, Hongmei; Liu, Xinqi; Qiu, Dewen

    2016-01-01

    The protein elicitor MoHrip2, which was extracted from Magnaporthe oryzae as an exocrine protein, triggers the tobacco immune system and enhances blast resistance in rice. However, the detailed mechanisms by which MoHrip2 acts as an elicitor remain unclear. Here, we investigated the structure of MoHrip2 to elucidate its functions based on molecular structure. The three-dimensional structure of MoHrip2 was obtained. Overall, the crystal structure formed a β-barrel structure and showed high similarity to the pathogenesis-related (PR) thaumatin superfamily protein thaumatin-like xylanase inhibitor (TL-XI). To investigate the functional regions responsible for MoHrip2 elicitor activities, the full length and eight truncated proteins were expressed in Escherichia coli and were evaluated for elicitor activity in tobacco. Biological function analysis showed that MoHrip2 triggered the defense system against Botrytis cinerea in tobacco. Moreover, only MoHrip2M14 and other fragments containing the 14 amino acids residues in the middle region of the protein showed the elicitor activity of inducing a hypersensitive response and resistance related pathways, which were similar to that of full-length MoHrip2. These results revealed that the central 14 amino acid residues were essential for anti-pathogenic activity.

  4. Crystal Structure Analysis and the Identification of Distinctive Functional Regions of the Protein Elicitor Mohrip2

    Directory of Open Access Journals (Sweden)

    Mengjie Liu

    2016-07-01

    Full Text Available The protein elicitor MoHrip2, which was extracted from Magnaporthe oryzae as an exocrine protein, triggers the tobacco immune system and enhances blast resistance in rice. However, the detailed mechanisms by which MoHrip2 acts as an elicitor remain unclear. Here, we investigated the structure of MoHrip2 to elucidate its functions based on molecular structure. The 3-dimensional structure of MoHrip2 was obtained. Overall, the crystal structure formed a β-barrel structure and showed high similarity to the pathogenesis-related (PR thaumatin superfamily protein thaumatin-like xylanase inhibitor (TL-XI. To investigate the functional regions responsible for MoHrip2 elicitor activities, the full length and 8 truncated proteins were expressed in Escherichia coli and were evaluated for elicitor activity in tobacco. Biological function analysis showed that MoHrip2 triggered the defense system against Botrytis cinerea in tobacco. Moreover, only MoHrip2M14 and other fragments containing the 14 amino acids residues in the middle region of the protein showed the elicitor activity of inducing a hypersensitive response and resistance related pathways, which were similar to that of full-length MoHrip2. These results revealed that the central 14 amino acid residues were essential for anti-pathogenic activity.

  5. Laser scattering in a hanging drop vapor diffusion apparatus for protein crystal growth in a microgravity environment

    Science.gov (United States)

    Casay, G. A.; Wilson, W. W.

    1992-01-01

    One type of hardware used to grow protein crystals in the microgravity environment aboard the U.S. Space Shuttle is a hanging drop vapor diffusion apparatus (HDVDA). In order to optimize crystal growth conditions, dynamic control of the HDVDA is desirable. A critical component in the dynamically controlled system is a detector for protein nucleation. We have constructed a laser scattering detector for the HDVDA capable of detecting the nucleation stage. The detector was successfully tested for several scatterers differing in size using dynamic light scattering techniques. In addition, the ability to detect protein nucleation using the HDVDA was demonstrated for lysozyme.

  6. Magnetically induced crystal structure and phase stability in Fe1-cCoc

    DEFF Research Database (Denmark)

    Abrikosov, I.A.; James, P.; Eriksson, O.

    1996-01-01

    We present an ab initio determination of the crystallographic phase stability of Fe-Co alloys as a function of concentration, using the coherent potential approximation. A bcc --> hcp phase transition is found at a concentration of 85 at.% of Co, in good agreement with the experimental phase...... diagram. We demonstrate that for the Pe-rich random alloys magnetism-stabilizes the bce phase relative to the close-packed fee and hcp phases. Magnetism also favors the partially ordered alpha' phase relative to the random bce alloy. This unique relation between magnetism and phase stability for the Fe...

  7. Broadband reflection of polymer-stabilized chiral nematic liquid crystals induced by a chiral azobenzene compound.

    Science.gov (United States)

    Chen, Xingwu; Wang, Ling; Chen, Yinjie; Li, Chenyue; Hou, Guoyan; Liu, Xin; Zhang, Xiaoguang; He, Wanli; Yang, Huai

    2014-01-21

    A chiral nematic liquid crystal-photopolymerizable monomer-chiral azobenzene compound composite was prepared and then polymerized under UV irradiation. The reflection wavelength of the composite can be extended to cover the 1000-2400 nm range and also be adjusted to the visible light region by controlling the concentration of chiral compounds.

  8. Na2TiGeO5: Crystal structure stability at low temperature and high pressure

    DEFF Research Database (Denmark)

    Waskowska, A.; Gerward, Leif; Olsen, J.S.

    2008-01-01

    The temperature evolution of the lattice parameters measured from 295 to 125 K exhibits a small instability below T-c approximate to 278 K, indicating ferroelastic properties of Na2TiGeO5. The behavior is related to the specific crystal structure built of polyhedral layers with shared TiO5 pyrami...

  9. Crystallization of a 79 kDa fragment of the hook protein FlgE from Campylobacter jejuni

    International Nuclear Information System (INIS)

    Kido, Yasuji; Yoon, Young-Ho; Samatey, Fadel A.

    2011-01-01

    A 79 kDa fragment of FlgE from C. jejuni has been crystallized. A 79 kDa fragment of the bacterial flagellar hook protein FlgE from Campylobacter jejuni was cloned, overexpressed, purified and crystallized. Two different crystal forms were obtained. Synchrotron X-ray diffraction data showed that the first crystal form, which diffracted to 4.9 Å resolution, belonged to the tetragonal crystal system, with space group I4 1 22 and unit-cell parameters a = b = 186.2, c = 386.6 Å, α = β = γ = 90°. The second crystal form diffracted to 2.5 Å resolution and belonged to the monoclinic crystal system, with space group P2 1 and unit-cell parameters a = 75.7, b = 173.8, c = 150.8 Å, α = γ = 90, β = 106.5°. SeMet protein was also overexpressed, purified and crystallized, and a 2.6 Å resolution MAD data set was collected

  10. Crystallization and preliminary X-ray crystallographic analysis of human PACSIN 1 protein

    International Nuclear Information System (INIS)

    Bai, Xiaoyun; Meng, Geng; Li, Guoming; Luo, Ming; Zheng, Xiaofeng

    2009-01-01

    A C-terminal truncation construct of human PACSIN 1 (1–344) has been purified and crystallized. Diffraction data were collected to 3.0 Å resolution. PACSIN 1, which is mainly detected in brain tissue, is one of the PACSIN-family proteins involved in endocytosis and recruitment of synaptic vesicles. It binds to dynamin, synaptojanin 1 and N-WASP, and functions in vesicle formation and transport. However, the mechanisms of action of PACSIN 1 in these processes are largely unknown. Here, full-length and five C-terminal truncation constructs of human PACSIN 1 have been successfully expressed and purified in Escherichia coli. PACSIN 1 (1–344) was crystallized and diffracted to a resolution of 3.0 Å. The crystal belonged to space group C2, with unit-cell parameters a = 158.65, b = 87.38, c = 91.76 Å, α = 90.00, β = 113.61, γ = 90.00°. There were two molecules in the asymmetric unit and the solvent content was estimated to be about 70.47%

  11. High-throughput protein crystallization on the World Community Grid and the GPU

    International Nuclear Information System (INIS)

    Kotseruba, Yulia; Cumbaa, Christian A; Jurisica, Igor

    2012-01-01

    We have developed CPU and GPU versions of an automated image analysis and classification system for protein crystallization trial images from the Hauptman Woodward Institute's High-Throughput Screening lab. The analysis step computes 12,375 numerical features per image. Using these features, we have trained a classifier that distinguishes 11 different crystallization outcomes, recognizing 80% of all crystals, 94% of clear drops, 94% of precipitates. The computing requirements for this analysis system are large. The complete HWI archive of 120 million images is being processed by the donated CPU cycles on World Community Grid, with a GPU phase launching in early 2012. The main computational burden of the analysis is the measure of textural (GLCM) features within the image at multiple neighbourhoods, distances, and at multiple greyscale intensity resolutions. CPU runtime averages 4,092 seconds (single threaded) on an Intel Xeon, but only 65 seconds on an NVIDIA Tesla C2050. We report on the process of adapting the C++ code to OpenCL, optimized for multiple platforms.

  12. Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging.

    Science.gov (United States)

    Warren, Anna J; Armour, Wes; Axford, Danny; Basham, Mark; Connolley, Thomas; Hall, David R; Horrell, Sam; McAuley, Katherine E; Mykhaylyk, Vitaliy; Wagner, Armin; Evans, Gwyndaf

    2013-07-01

    The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required.

  13. Crystal structure of the extracellular domain of human myelin protein zero

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhigang; Wang, Yong; Yedidi, Ravikiran S.; Brunzelle, Joseph S.; Kovari, Iulia A.; Sohi, Jasloveleen; Kamholz, John; Kovari, Ladislau C. (WSU-MED); (NWU)

    2012-03-27

    different mutations in the MPZ gene leading to peripheral neuropathy in patients have been reported worldwide (http://www.molgen. ua.ac.be/CMTMutations). All identified mutations resulting in a change or deletion of amino acid residues in MPZ give rise to neuropathy with the exception of R215L, which instead causes a benign polymorphism. Furthermore, more detailed analysis has classified the MPZ mutations into two major groups. In the first group, the mutations disrupt the intracellular processing of MPZ and are primarily associated with early onset neuropathy. It has been proposed that the mutated MPZ is trapped inside the cell rather than being transported to the plasma membrane. However, other evidence suggests that the mutated MPZ protein is expressed on the plasma membrane, but dominant-negatively disrupts the structure of myelin. In the second group, the MPZ mutations are associated with late onset neuropathy as these mutations cause only mild demyelination. The underlying mechanism is elusive with the hypothesis being that the second group of mutations cause minor abnormalities in the myelin sheath that over time may lead to aberrant Schwann cell-axon interactions and subsequently to axonal degeneration. The crystal structure of the extracellular domain of human MPZ (hP0ex) fused with maltose binding protein (MBP) is reported at 2.1 {angstrom} resolution. While the crystal structure of rat MPZ extracellular domain (rP0ex) is available, the crystal structure of the human counterpart is useful for the analysis of the two homologs as well as a comparison between the two species. The hP0ex molecule reveals subtle structural variations between two homologs allowing comparison of the human myelin protein zero to that of the rat protein. The alignment of these homologs is shown in Figure 1(a).

  14. [Genes of insecticidal crystal proteins with dual specificity in Bacillus thuringiensis strains, isolated in the Crimea territory].

    Science.gov (United States)

    Rymar, S Iu; Isakova, I A; Kuznietsova, L M; Kordium, V A

    2006-01-01

    The insecticidal crystal proteins of 15 B. thuringiensis strains, isolated in the Crimea territory that are toxical for some Lepidoptera and Colorado potato beetle larvae were identified by PAGE electrophoresis. Ten strains produced the crystal proteins with high molecular weight (> 120 kD). PCR with use of broad specificity primers and DNA of these B. thuringiensis strains as template demonstrated the specific PCR products (1000 bp). Amplified DNA fragments were cloned and sequenced. The nucleotide sequence analysis revealed that the genomes of ten strains of B. thuringiensis carried Cry1B genes, which are responsible for production of the insecticidal crystal proteins with dual specificity. The influence of the solubilization conditions on the structure and toxicity of Cry1B protein for Colorado potato beetle larvae was shown. The dual toxicity of studied B. thuringiensis strains is explained by the Cry1B genes presence in their genomes. These strains may be used to develop the broad specificity bioinsecticides.

  15. Stabilization and delivery approaches for protein and peptide pharmaceuticals: an extensive review of patents.

    Science.gov (United States)

    Swain, Suryakanta; Mondal, Debanik; Beg, Sarwar; Patra, Chinam Niranjan; Dinda, Subas Chandra; Sruti, Jammula; Rao, Muddana Eswara Bhanoji

    2013-04-01

    Proteins and peptides are the building blocks of human body and act as the arsenal to combat against the invading pathogenic organisms for treatment and management of diseases. Majority of such biomacromolecules are synthesized by the human body itself. However, entry of disease causing pathogens causes misleading in the synthesis of desired proteins for antibody formation. In such alarming situations, the delivery of requisite protein and peptide from external source helps in augmenting the body's immunity. The major drawbacks underlying poor biopharmaceutical performance of high molecular weight protein and peptide drugs are due to poor oral absorption, formulation stability, degradation in the gastric milieu, susceptible to presystemic metabolism. Numerous literature recounts the application of myriad drug delivery strategies for the effective delivery of protein and peptides viz. parentral, oral, transdermal, nasal, pulmonary, rectal, buccal and ocular drug delivery systems. There are many reviews on various delivery strategies for protein and peptide pharmaceuticals, but the present review article provides a bird's eye view on various novel drug delivery systems used for enhanced delivery of protein and peptide pharmaceuticals in the light of patent literature. Apart from this, the present manuscript endeavor provides idea on possible causes and major degradation pathways responsible for poor stability of protein and peptide drugs along with recent market instances on them utilizing novel drug delivery systems.

  16. In vivo stability and inertness of various direct labelled and chelate-tagged protein

    International Nuclear Information System (INIS)

    Janoki, A.; Korosi, L.; Klivenyi, G.; Spett, B.

    1987-01-01

    There were looking for methods giving precise information about composition and activity distribution of protein components, both in the initial samples and serum samples after intravenous administration. It was tested the applicability of electroimmunoassay, polyacrilamide gel electrophoresis and high performance liquid chromatography for the assessment of in vivo stability and labelled proteins. The model compound was human serum albumin (HSA) labelled with 99m Tc and 125 I, respectively. Bifunctional chelate labelling was done with desferrioxamine, in this case protein was labelled with 67 Ga. Biodistribution of the labelled compounds and their elimination from the blood were studied in rabbits. Experience with various labelling proteins, especially with Tc-Sn-HSA system indicate that in vivo stability of this compounds are generally low. Following intravenous injection of proteins labelled with metal isotopes, due to dilution and to the presence of considerable amount of compatitive protein in the serum, part of the label is being detached from the carrier protein. Distribution of the detached metal is different from the original distribution of the protein. This problem arises also with radiopharmaceuticals based on monoclonal antibodies. (M.E.L.) [es

  17. Nanoporous microbead supported bilayers: stability, physical characterization, and incorporation of functional transmembrane proteins.

    Energy Technology Data Exchange (ETDEWEB)

    Davis, Ryan W. (University of New Mexico, Albuquerque, NM); Brozik, James A. (University of New Mexico, Albuquerque, NM); Brozik, Susan Marie; Cox, Jason M. (University of New Mexico, Albuquerque, NM); Lopez, Gabriel P. (University of New Mexico, Albuquerque, NM); Barrick, Todd A. (University of New Mexico, Albuquerque, NM); Flores, Adrean (University of New Mexico, Albuquerque, NM)

    2007-03-01

    The introduction of functional transmembrane proteins into supported bilayer-based biomimetic systems presents a significant challenge for biophysics. Among the various methods for producing supported bilayers, liposomal fusion offers a versatile method for the introduction of membrane proteins into supported bilayers on a variety of substrates. In this study, the properties of protein containing unilamellar phosphocholine lipid bilayers on nanoporous silica microspheres are investigated. The effects of the silica substrate, pore structure, and the substrate curvature on the stability of the membrane and the functionality of the membrane protein are determined. Supported bilayers on porous silica microspheres show a significant increase in surface area on surfaces with structures in excess of 10 nm as well as an overall decrease in stability resulting from increasing pore size and curvature. Comparison of the liposomal and detergent-mediated introduction of purified bacteriorhodopsin (bR) and the human type 3 serotonin receptor (5HT3R) are investigated focusing on the resulting protein function, diffusion, orientation, and incorporation efficiency. In both cases, functional proteins are observed; however, the reconstitution efficiency and orientation selectivity are significantly enhanced through detergent-mediated protein reconstitution. The results of these experiments provide a basis for bulk ionic and fluorescent dye-based compartmentalization assays as well as single-molecule optical and single-channel electrochemical interrogation of transmembrane proteins in a biomimetic platform.

  18. Kinetic Stability of Proteins in Beans and Peas: Implications for Protein Digestibility, Seed Germination, and Plant Adaptation.

    Science.gov (United States)

    Xia, Ke; Pittelli, Sandy; Church, Jennifer; Colón, Wilfredo

    2016-10-12

    Kinetically stable proteins (KSPs) are resistant to the denaturing detergent sodium dodecyl sulfate (SDS). Such resilience makes KSPs resistant to proteolytic degradation and may have arisen in nature as a mechanism for organismal adaptation and survival against harsh conditions. Legumes are well-known for possessing degradation-resistant proteins that often diminish their nutritional value. Here we applied diagonal two-dimensional (D2D) SDS-polyacrylamide gel electrophoresis (PAGE), a method that allows for the proteomics-level identification of KSPs, to a group of 12 legumes (mostly beans and peas) of agricultural and nutritional importance. Our proteomics results show beans that are more difficult to digest, such as soybean, lima beans, and various common beans, have high contents of KSPs. In contrast, mung bean, red lentil, and various peas that are highly digestible contain low amounts of KSPs. Identified proteins with high kinetic stability are associated with warm-season beans, which germinate at higher temperatures. In contrast, peas and red lentil, which are cool-season legumes, contain low levels of KSPs. Thus, our results show protein kinetic stability is an important factor in the digestibility of legume proteins and may relate to nutrition efficiency, timing of seed germination, and legume resistance to biotic stressors. Furthermore, we show D2D SDS-PAGE is a powerful method that could be applied for determining the abundance and identity of KSPs in engineered and wild legumes and for advancing basic research and associated applications.

  19. Interactions of cullin3/KCTD5 complexes with both cytoplasmic and nuclear proteins: Evidence for a role in protein stabilization

    Energy Technology Data Exchange (ETDEWEB)

    Rutz, Natalja; Heilbronn, Regine; Weger, Stefan, E-mail: stefan.weger@charite.de

    2015-08-28

    Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 induced polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability. - Highlights: • KCTD5 nuclear translocation depends upon M phase and protein oligomerization. • Identification of MCM7, ZNF711 and FAM193 as KCTD5 interaction partners. • Formation of trimeric complexes of KCTD5/cullin3 with MCM7, ZNF711 and FAM193B. • KCTD5 is not involved in polyubiquitylation of MCM7 replication factor. • The KCTD5/cullin3 complex stabilizes ZNF711 transcription factor.

  20. Rapid directed evolution of stabilized proteins with cellular high-throughput encapsulation solubilization and screening (CHESS).

    Science.gov (United States)

    Yong, K J; Scott, D J

    2015-03-01

    Directed evolution is a powerful method for engineering proteins towards user-defined goals and has been used to generate novel proteins for industrial processes, biological research and drug discovery. Typical directed evolution techniques include cellular display, phage display, ribosome display and water-in-oil compartmentalization, all of which physically link individual members of diverse gene libraries to their translated proteins. This allows the screening or selection for a desired protein function and subsequent isolation of the encoding gene from diverse populations. For biotechnological and industrial applications there is a need to engineer proteins that are functional under conditions that are not compatible with these techniques, such as high temperatures and harsh detergents. Cellular High-throughput Encapsulation Solubilization and Screening (CHESS), is a directed evolution method originally developed to engineer detergent-stable G proteins-coupled receptors (GPCRs) for structural biology. With CHESS, library-transformed bacterial cells are encapsulated in detergent-resistant polymers to form capsules, which serve to contain mutant genes and their encoded proteins upon detergent mediated solubilization of cell membranes. Populations of capsules can be screened like single cells to enable rapid isolation of genes encoding detergent-stable protein mutants. To demonstrate the general applicability of CHESS to other proteins, we have characterized the stability and permeability of CHESS microcapsules and employed CHESS to generate thermostable, sodium dodecyl sulfate (SDS) resistant green fluorescent protein (GFP) mutants, the first soluble proteins to be engineered using CHESS. © 2014 Wiley Periodicals, Inc.

  1. Ultra-High Pressure Homogenization enhances physicochemical properties of soy protein isolate-stabilized emulsions.

    Science.gov (United States)

    Fernández-Ávila, C; Escriu, R; Trujillo, A J

    2015-09-01

    The effect of Ultra-High Pressure Homogenization (UHPH, 100-300MPa) on the physicochemical properties of oil-in-water emulsions prepared with 4.0% (w/v) of soy protein isolate (SPI) and soybean oil (10 and 20%, v/v) was studied and compared to emulsions treated by conventional homogenization (CH, 15MPa). CH emulsions were prepared with non-heated and heated (95°C for 15min) SPI dispersions. Emulsions were characterized by particle size determination with laser diffraction, rheological properties using a rotational rheometer by applying measurements of flow curve and by transmission electron microscopy. The variation on particle size and creaming was assessed by Turbiscan® analysis, and visual observation of the emulsions was also carried out. UHPH emulsions showed much smaller d 3.2 values and greater physical stability than CH emulsions. The thermal treatment of SPI prior CH process did not improve physical stability properties. In addition, emulsions containing 20% of oil exhibited greater physical stability compared to emulsions containing 10% of oil. Particularly, UHPH emulsions treated at 100 and 200MPa with 20% of oil were the most stable due to low particle size values (d 3.2 and Span), greater viscosity and partial protein denaturation. These results address the physical stability improvement of protein isolate-stabilized emulsions by using the emerging UHPH technology. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Crystal structure and conformational flexibility of the unligated FK506-binding protein FKBP12.6

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Hui; Mustafi, Sourajit M. [New York State Department of Health, Empire State Plaza, Albany, NY 12201 (United States); LeMaster, David M. [New York State Department of Health, Empire State Plaza, Albany, NY 12201 (United States); University at Albany – SUNY, Empire State Plaza, Albany, NY 12201 (United States); Li, Zhong [New York State Department of Health, Empire State Plaza, Albany, NY 12201 (United States); Héroux, Annie [Brookhaven National Laboratory, Upton, NY 11973 (United States); Li, Hongmin; Hernández, Griselda, E-mail: griselda@wadsworth.org [New York State Department of Health, Empire State Plaza, Albany, NY 12201 (United States); University at Albany – SUNY, Empire State Plaza, Albany, NY 12201 (United States)

    2014-03-01

    Two crystal forms of unligated FKBP12.6 exhibit multiple conformations in the active site and in the 80s loop, the primary site for known protein-recognition interactions. The previously unreported NMR backbone assignment of FKBP12.6 revealed extensive doubling of amide resonances, which reflects a slow conformational transition centered in the 80s loop. The primary known physiological function of FKBP12.6 involves its role in regulating the RyR2 isoform of ryanodine receptor Ca{sup 2+} channels in cardiac muscle, pancreatic β islets and the central nervous system. With only a single previously reported X-ray structure of FKBP12.6, bound to the immunosuppressant rapamycin, structural inferences for this protein have been drawn from the more extensive studies of the homologous FKBP12. X-ray structures at 1.70 and 1.90 Å resolution from P2{sub 1} and P3{sub 1}21 crystal forms are reported for an unligated cysteine-free variant of FKBP12.6 which exhibit a notable diversity of conformations. In one monomer from the P3{sub 1}21 crystal form, the aromatic ring of Phe59 at the base of the active site is rotated perpendicular to its typical orientation, generating a steric conflict for the immunosuppressant-binding mode. The peptide unit linking Gly89 and Val90 at the tip of the protein-recognition ‘80s loop’ is flipped in the P2{sub 1} crystal form. Unlike the >30 reported FKBP12 structures, the backbone conformation of this loop closely follows that of the first FKBP domain of FKBP51. The NMR resonances for 21 backbone amides of FKBP12.6 are doubled, corresponding to a slow conformational transition centered near the tip of the 80s loop, as recently reported for 31 amides of FKBP12. The comparative absence of doubling for residues along the opposite face of the active-site pocket in FKBP12.6 may in part reflect attenuated structural coupling owing to increased conformational plasticity around the Phe59 ring.

  3. Carotenoid crystal formation in Arabidopsis and carrot roots caused by increased phytoene synthase protein levels.

    Directory of Open Access Journals (Sweden)

    Dirk Maass

    Full Text Available BACKGROUND: As the first pathway-specific enzyme in carotenoid biosynthesis, phytoene synthase (PSY is a prime regulatory target. This includes a number of biotechnological approaches that have successfully increased the carotenoid content in agronomically relevant non-green plant tissues through tissue-specific PSY overexpression. We investigated the differential effects of constitutive AtPSY overexpression in green and non-green cells of transgenic Arabidopsis lines. This revealed striking similarities to the situation found in orange carrot roots with respect to carotenoid amounts and sequestration mechanism. METHODOLOGY/PRINCIPAL FINDINGS: In Arabidopsis seedlings, carotenoid content remained unaffected by increased AtPSY levels although the protein was almost quantitatively imported into plastids, as shown by western blot analyses. In contrast, non-photosynthetic calli and roots overexpressing AtPSY accumulated carotenoids 10 and 100-fold above the corresponding wild-type tissues and contained 1800 and 500 microg carotenoids per g dry weight, respectively. This increase coincided with a change of the pattern of accumulated carotenoids, as xanthophylls decreased relative to beta-carotene and carotene intermediates accumulated. As shown by polarization microscopy, carotenoids were found deposited in crystals, similar to crystalline-type chromoplasts of non-green tissues present in several other taxa. In fact, orange-colored carrots showed a similar situation with increased PSY protein as well as carotenoid levels and accumulation patterns whereas wild white-rooted carrots were similar to Arabidopsis wild type roots in this respect. Initiation of carotenoid crystal formation by increased PSY protein amounts was further confirmed by overexpressing crtB, a bacterial PSY gene, in white carrots, resulting in increased carotenoid amounts deposited in crystals. CONCLUSIONS: The sequestration of carotenoids into crystals can be driven by the

  4. CAN THE STABILITY OF PROTEIN MUTANTS BE PREDICTED BY FREE-ENERGY CALCULATIONS

    NARCIS (Netherlands)

    YUNYU, S; MARK, AE; WANG, CX; HUANG, FH; BERENDSEN, HJC; VANGUNSTEREN, WF

    The use of free energy simulation techniques in the study of protein stability is critically evaluated. Results from two simulations of the thermostability mutation Asn218 to Ser218 in Subtilisin are presented. It is shown that components of the free energy change can be highly sensitive to the

  5. Ectodomain of plasmodesmata-localized protein 5 in Arabidopsis: expression, purification, crystallization and crystallographic analysis.

    Science.gov (United States)

    Wang, Xiaocui; Zhu, Peiyan; Qu, Shanshan; Zhao, Jie; Singh, Prashant K; Wang, Wei

    2017-09-01

    Plasmodesmata-localized protein 5 (PDLP5) is a cysteine-rich receptor-like protein which is localized on the plasmodesmata of Arabidopsis thaliana. Overexpression of PDLP5 can reduce the permeability of the plasmodesmata and further affect the cell-to-cell movement of viruses and macromolecules in plants. The ectodomain of PDLP5 contains two DUF26 domains; however, the function of these domains is still unknown. Here, the ectodomain of PDLP5 from Arabidopsis was cloned and overexpressed using an insect expression system and was then purified and crystallized. X-ray diffraction data were collected to 1.90 Å resolution and were indexed in space group P1, with unit-cell parameters a = 41.9, b = 48.1, c = 62.2 Å, α = 97.3, β = 103.1, γ = 99.7°. Analysis of the crystal content indicated that there are two molecules in the asymmetric unit, with a Matthews coefficient of 2.51 Å 3  Da -1 and a solvent content of 50.97%.

  6. Routine phasing of coiled-coil protein crystal structures with AMPLE

    Directory of Open Access Journals (Sweden)

    Jens M. H. Thomas

    2015-03-01

    Full Text Available Coiled-coil protein folds are among the most abundant in nature. These folds consist of long wound α-helices and are architecturally simple, but paradoxically their crystallographic structures are notoriously difficult to solve with molecular-replacement techniques. The program AMPLE can solve crystal structures by molecular replacement using ab initio search models in the absence of an existent homologous protein structure. AMPLE has been benchmarked on a large and diverse test set of coiled-coil crystal structures and has been found to solve 80% of all cases. Successes included structures with chain lengths of up to 253 residues and resolutions down to 2.9 Å, considerably extending the limits on size and resolution that are typically tractable by ab initio methodologies. The structures of two macromolecular complexes, one including DNA, were also successfully solved using their coiled-coil components. It is demonstrated that both the ab initio modelling and the use of ensemble search models contribute to the success of AMPLE by comparison with phasing attempts using single structures or ideal polyalanine helices. These successes suggest that molecular replacement with AMPLE should be the method of choice for the crystallographic elucidation of a coiled-coil structure. Furthermore, AMPLE may be able to exploit the presence of a coiled coil in a complex to provide a convenient route for phasing.

  7. Evaluation of Semi-supervised Learning for Classification of Protein Crystallization Imagery.

    Science.gov (United States)

    Sigdel, Madhav; Dinç, İmren; Dinç, Semih; Sigdel, Madhu S; Pusey, Marc L; Aygün, Ramazan S

    2014-03-01

    In this paper, we investigate the performance of two wrapper methods for semi-supervised learning algorithms for classification of protein crystallization images with limited labeled images. Firstly, we evaluate the performance of semi-supervised approach using self-training with naïve Bayesian (NB) and sequential minimum optimization (SMO) as the base classifiers. The confidence values returned by these classifiers are used to select high confident predictions to be used for self-training. Secondly, we analyze the performance of Yet Another Two Stage Idea (YATSI) semi-supervised learning using NB, SMO, multilayer perceptron (MLP), J48 and random forest (RF) classifiers. These results are compared with the basic supervised learning using the same training sets. We perform our experiments on a dataset consisting of 2250 protein crystallization images for different proportions of training and test data. Our results indicate that NB and SMO using both self-training and YATSI semi-supervised approaches improve accuracies with respect to supervised learning. On the other hand, MLP, J48 and RF perform better using basic supervised learning. Overall, random forest classifier yields the best accuracy with supervised learning for our dataset.

  8. Crystallization and preliminary characterization of a novel haem-binding protein of Streptomyces reticuli

    International Nuclear Information System (INIS)

    Zou, Peijian; Groves, Matthew R.; Viale-Bouroncle, Sandra D.; Ortiz de Orué Lucana, Darío

    2008-01-01

    The haem-binding protein HbpS from Streptomyces reticuli was crystallized and diffraction data were collected to a maximal resolution of 2.25 Å. Streptomyces reticuli is a soil-growing Gram-positive bacteria that has been shown to secrete a novel haem-binding protein known as HbpS. Sequence analysis reveals that homologues of HbpS are found in a wide variety of bacteria, including different Actinobacteria and the Gram-negative Vibrio cholera and Klebsiella pneumoniae. The in vivo production of HbpS is greatly increased when S. reticuli is cultured in the presence of the natural antibiotic haemin (Fe 3+ oxidized form of haem). Mutational analysis demonstrated that HbpS significantly increases the resistance of S. reticuli to toxic concentrations of haemin. Previous data show that the presence of the newly identified two-component sensor system SenS–SenR also considerably enhances the resistance of S. reticuli to haemin and the redox-cycling compound plumbagin, suggesting a role in the sensing of redox changes. Specific interaction between HbpS and SenS–SenR, which regulates the expression of the catalase–peroxidase CpeB, as well as HbpS, has been demonstrated in vitro. HbpS has been recombinantly overexpressed, purified and crystallized in space group P2 1 3, with a cell edge of 152.5 Å. Diffraction data were recorded to a maximal resolution of 2.25 Å and phases were obtained using the SAD method from crystals briefly soaked in high concentrations of sodium bromide

  9. Crystallization and preliminary characterization of a novel haem-binding protein of Streptomyces reticuli

    Energy Technology Data Exchange (ETDEWEB)

    Zou, Peijian [EMBL Outstation Hamburg, c/o DESY, Notkestrasse 85, 22607 Hamburg (Germany); Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstrasse 1, 85764 Neuherberg (Germany); Groves, Matthew R. [EMBL Outstation Hamburg, c/o DESY, Notkestrasse 85, 22607 Hamburg (Germany); Viale-Bouroncle, Sandra D.; Ortiz de Orué Lucana, Darío, E-mail: ortiz@biologie.uni-osnabrueck.de [Universität Osnabrück, FB Biologie/Chemie, Angewandte Genetik der Mikroorganismen, Barbarastrasse 13, 49069 Osnabrück (Germany); EMBL Outstation Hamburg, c/o DESY, Notkestrasse 85, 22607 Hamburg (Germany)

    2008-05-01

    The haem-binding protein HbpS from Streptomyces reticuli was crystallized and diffraction data were collected to a maximal resolution of 2.25 Å. Streptomyces reticuli is a soil-growing Gram-positive bacteria that has been shown to secrete a novel haem-binding protein known as HbpS. Sequence analysis reveals that homologues of HbpS are found in a wide variety of bacteria, including different Actinobacteria and the Gram-negative Vibrio cholera and Klebsiella pneumoniae. The in vivo production of HbpS is greatly increased when S. reticuli is cultured in the presence of the natural antibiotic haemin (Fe{sup 3+} oxidized form of haem). Mutational analysis demonstrated that HbpS significantly increases the resistance of S. reticuli to toxic concentrations of haemin. Previous data show that the presence of the newly identified two-component sensor system SenS–SenR also considerably enhances the resistance of S. reticuli to haemin and the redox-cycling compound plumbagin, suggesting a role in the sensing of redox changes. Specific interaction between HbpS and SenS–SenR, which regulates the expression of the catalase–peroxidase CpeB, as well as HbpS, has been demonstrated in vitro. HbpS has been recombinantly overexpressed, purified and crystallized in space group P2{sub 1}3, with a cell edge of 152.5 Å. Diffraction data were recorded to a maximal resolution of 2.25 Å and phases were obtained using the SAD method from crystals briefly soaked in high concentrations of sodium bromide.

  10. Carotenoid-protein complexes and their stability towards oxygen and radiation

    International Nuclear Information System (INIS)

    Ramakrishnan, T.V.; Francis, F.J.

    1980-01-01

    Carotenoid-protein complexes isolated from fresh mangoes were found to be more stable to oxygen and radiation when dissolved in water as compared with β-carotene in petroleum ether. Part of the pigment could be released from the complex by gamma irradiation. Observations on the stability of the carotenoid (98% β-carotene) in the complex indicated that the pigment is either associated with the lipid prosthetic group of the protein or loosely attached to the protein by weak hydrophobic bonds. (author)

  11. Influence of Pea Protein Aggregates on the Structure and Stability of Pea Protein/Soybean Polysaccharide Complex Emulsions

    Directory of Open Access Journals (Sweden)

    Baoru Yin

    2015-03-01

    Full Text Available The applications of plant proteins in the food and beverage industry have been hampered by their precipitation in acidic solution. In this study, pea protein isolate (PPI with poor dispersibility in acidic solution was used to form complexes with soybean soluble polysaccharide (SSPS, and the effects of PPI aggregates on the structure and stability of PPI/SSPS complex emulsions were investigated. Under acidic conditions, high pressure homogenization disrupts the PPI aggregates and the electrostatic attraction between PPI and SSPS facilitates the formation of dispersible PPI/SSPS complexes. The PPI/SSPS complex emulsions prepared from the PPI containing aggregates prove to possess similar droplet structure and similar stability compared with the PPI/SSPS emulsions produced from the PPI in which the aggregates have been previously removed by centrifugation. The oil droplets are protected by PPI/SSPS complex interfacial films and SSPS surfaces. The emulsions show long-term stability against pH and NaCl concentration changes. This study demonstrates that PPI aggregates can also be used to produce stable complex emulsions, which may promote the applications of plant proteins in the food and beverage industry.

  12. Influence of pea protein aggregates on the structure and stability of pea protein/soybean polysaccharide complex emulsions.

    Science.gov (United States)

    Yin, Baoru; Zhang, Rujing; Yao, Ping

    2015-03-20

    The applications of plant proteins in the food and beverage industry have been hampered by their precipitation in acidic solution. In this study, pea protein isolate (PPI) with poor dispersibility in acidic solution was used to form complexes with soybean soluble polysaccharide (SSPS), and the effects of PPI aggregates on the structure and stability of PPI/SSPS complex emulsions were investigated. Under acidic conditions, high pressure homogenization disrupts the PPI aggregates and the electrostatic attraction between PPI and SSPS facilitates the formation of dispersible PPI/SSPS complexes. The PPI/SSPS complex emulsions prepared from the PPI containing aggregates prove to possess similar droplet structure and similar stability compared with the PPI/SSPS emulsions produced from the PPI in which the aggregates have been previously removed by centrifugation. The oil droplets are protected by PPI/SSPS complex interfacial films and SSPS surfaces. The emulsions show long-term stability against pH and NaCl concentration changes. This study demonstrates that PPI aggregates can also be used to produce stable complex emulsions, which may promote the applications of plant proteins in the food and beverage industry.

  13. Increased Protein Stability and Decreased Protein Turnover in the Caenorhabditis elegans Ins/IGF-1 daf-2 Mutant.

    Science.gov (United States)

    Depuydt, Geert; Shanmugam, Nilesh; Rasulova, Madina; Dhondt, Ineke; Braeckman, Bart P

    2016-12-01

    In Caenorhabditis elegans, cellular proteostasis is likely essential for longevity. Autophagy has been shown to be essential for lifespan extension of daf-2 insulin/IGF mutants. Therefore, it can be hypothesized that daf-2 mutants achieve this phenotype by increasing protein turnover. However, such a mechanism would exert a substantial energy cost. By using classical 35 S pulse-chase labeling, we observed that protein synthesis and degradation rates are decreased in young adults of the daf-2 insulin/IGF mutants. Although reduction of protein turnover may be energetically favorable, it may lead to accumulation and aggregation of damaged proteins. As this has been shown not to be the case in daf-2 mutants, another mechanism must exist to maintain proteostasis in this strain. We observed that proteins isolated from daf-2 mutants are more soluble in acidic conditions due to increased levels of trehalose. This suggests that trehalose may decrease the potential for protein aggregation and increases proteostasis in the daf-2 mutants. We postulate that daf-2 mutants save energy by decreasing protein turnover rates and instead stabilize their proteome by trehalose. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America.

  14. Two new coordination polymers based on tartaric acid ligand: Syntheses, crystal structure and thermal stability

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Fei-Yan; Lan, You-Zhao, E-mail: sky37@zjnu.cn; Han, Min-Min; Feng, Yun-Long, E-mail: lyzhao@zjnu.cn [Zhejiang Normal University, Zhejiang Key Laboratory for Reactive Chemistry on Solid Surfaces, Institute of Physical Chemistry (China)

    2016-09-15

    Two new coordination polymers [Cd{sub 3}(D-Tar){sub 3}]{sub n} (1) and [Pb(meso-Tar)]{sub n} (2) (H{sub 2}Tar = tartaric acid) have been synthesized by hydrothermal reaction and characterized by single crystal X-ray diffraction analysis and IR spectra. 1 crystallizes in the C222{sub 1} chiral space group and shows a 3D (4,4)-connected net with the (4.6.8{sup 4}){sub 4}(4.6{sup 2}.8{sup 2}.10)(4.6{sup 2}.8{sup 3})(4.6{sup 3}.8{sup 2})(4.6{sup 3}.8{sup 2}){sub 4}(4.8{sup 5}){sub 2} topology. 2 possesses a 3D (4,4)-connected net with the (4{sup 3}.6{sup 2}.8) topology. In addition, the thermogravimetric analyses (TGA) results for polymers are discussed.

  15. Stability to irradiation of SiGe whisker crystals used for sensors of physical values

    Directory of Open Access Journals (Sweden)

    Druzhinin A. A.

    2011-04-01

    Full Text Available An influence of g-irradiation (Co60 with doze up to 1—1018 сm–2 and magnetic field with induction up to 14 T on conduction of 1–xGex (х = 0,03 whisker crystals with resistivity of 0,08—0,025 Оhm·сm in temperature range 4,2—300 K have been studied. It is shown that whisker crystals resistance faintly varies under irradiation with doze 2·1017 сm–2, while their magnetoresistance substantially changes. The strain sensors stable to irradiation action operating in high magnetic fields on the base of the whiskers have been designed.

  16. The purification, crystallization and preliminary structural characterization of human MAWDBP, a member of the phenazine biosynthesis-like protein family

    International Nuclear Information System (INIS)

    Herde, Petra; Blankenfeldt, Wulf

    2006-01-01

    The purification, crystallization and preliminary structural characterization of human MAWD-binding protein (MAWDBP) are described. MAWDBP is the only representative of the phenazine biosynthesis-like protein family in the human genome. Its expression is elevated in several disease processes, including insulin resistance, folate deficiency and hypotension, and it may also be involved in carcinogenesis. The exact molecular function of MAWDBP is unknown. Native and seleno-l-methionine-labelled MAWDBP were expressed in Escherichia coli and crystallized at room temperature from precipitants containing 10 mM KF, 14%(w/v) PEG 3350 and 0.1 M sodium citrate pH 5.4. Crystals belong to space group H32, with unit-cell parameters a = b = 187, c = 241 Å, indicative of three to five monomers per asymmetric unit. Crystals were cryoprotected with 15%(v/v) glycerol and data have been collected to 2.7 Å resolution

  17. The purification, crystallization and preliminary structural characterization of human MAWDBP, a member of the phenazine biosynthesis-like protein family

    Energy Technology Data Exchange (ETDEWEB)

    Herde, Petra; Blankenfeldt, Wulf, E-mail: wulf.blankenfeldt@mpi-dortmund.mpg.de [Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)

    2006-06-01

    The purification, crystallization and preliminary structural characterization of human MAWD-binding protein (MAWDBP) are described. MAWDBP is the only representative of the phenazine biosynthesis-like protein family in the human genome. Its expression is elevated in several disease processes, including insulin resistance, folate deficiency and hypotension, and it may also be involved in carcinogenesis. The exact molecular function of MAWDBP is unknown. Native and seleno-l-methionine-labelled MAWDBP were expressed in Escherichia coli and crystallized at room temperature from precipitants containing 10 mM KF, 14%(w/v) PEG 3350 and 0.1 M sodium citrate pH 5.4. Crystals belong to space group H32, with unit-cell parameters a = b = 187, c = 241 Å, indicative of three to five monomers per asymmetric unit. Crystals were cryoprotected with 15%(v/v) glycerol and data have been collected to 2.7 Å resolution.

  18. Large-scale purification and crystallization of the endoribonuclease XendoU: troubleshooting with His-tagged proteins

    International Nuclear Information System (INIS)

    Renzi, Fabiana; Panetta, Gianna; Vallone, Beatrice; Brunori, Maurizio; Arceci, Massimo; Bozzoni, Irene; Laneve, Pietro; Caffarelli, Elisa

    2006-01-01

    Recombinant His-tagged XendoU, a eukaryotic endoribonuclease, appeared to aggregate in the presence of divalent cations. Monodisperse protein which yielded crystals diffracting to 2.2 Å was obtained by addition of EDTA. XendoU is the first endoribonuclease described in higher eukaryotes as being involved in the endonucleolytic processing of intron-encoded small nucleolar RNAs. It is conserved among eukaryotes and its viral homologue is essential in SARS replication and transcription. The large-scale purification and crystallization of recombinant XendoU are reported. The tendency of the recombinant enzyme to aggregate could be reversed upon the addition of chelating agents (EDTA, imidazole): aggregation is a potential drawback when purifying and crystallizing His-tagged proteins, which are widely used, especially in high-throughput structural studies. Purified monodisperse XendoU crystallized in two different space groups: trigonal P3 1 21, diffracting to low resolution, and monoclinic C2, diffracting to higher resolution

  19. A critical switch in the enzymatic properties of the Cid1 protein deciphered from its product-bound crystal structure.

    Science.gov (United States)

    Munoz-Tello, Paola; Gabus, Caroline; Thore, Stéphane

    2014-03-01

    The addition of uridine nucleotide by the poly(U) polymerase (PUP) enzymes has a demonstrated impact on various classes of RNAs such as microRNAs (miRNAs), histone-encoding RNAs and messenger RNAs. Cid1 protein is a member of the PUP family. We solved the crystal structure of Cid1 in complex with non-hydrolyzable UMPNPP and a short dinucleotide compound ApU. These structures revealed new residues involved in substrate/product stabilization. In particular, one of the three catalytic aspartate residues explains the RNA dependence of its PUP activity. Moreover, other residues such as residue N165 or the β-trapdoor are shown to be critical for Cid1 activity. We finally suggest that the length and sequence of Cid1 substrate RNA influence the balance between Cid1's processive and distributive activities. We propose that particular processes regulated by PUPs require the enzymes to switch between the two types of activity as shown for the miRNA biogenesis where PUPs can either promote DICER cleavage via short U-tail or trigger miRNA degradation by adding longer poly(U) tail. The enzymatic properties of these enzymes may be critical for determining their particular function in vivo.

  20. Microstructural evolution and stability of tetragonal precipitates in Y2O3 partially-stabilized ZrO2 single crystals

    International Nuclear Information System (INIS)

    Martinez-Fernandez, J.; Jimenez-Melendo, M.; Dominguez-Rodriguez, A.

    1995-01-01

    The microstructure and morphology of tetragonal precipitates (t-ZrO 2 ) in yttria partially-stabilized zirconia single crystals containing various amounts of Y 2 O 3 (3, 4, 4.7 and 5.8 mol%) have been studied as a function of aging time at 1,600 C in air. The precipitate size and volume fraction of t-ZrO 2 phase were determined using transmission electron microscopy. The evolution of the precipitate volume fraction with aging time indicated that the precipitation reaction was completed after 24 h of annealing, in agreement with the values of the Y 2 O 3 composition in the matrix measured by analytical electron microscopy. Further aging coarsened the precipitates which joined together forming fiber-like particles of several micrometers in length and remaining untransformed despite their large size. The stability of the t-ZrO 2 precipitates against the tetragonal to monoclinic transformation in the ZrO 2 -Y 2 O 3 system seems to be related to interactions between precipitates due to coherency stresses rather than with their morphological characteristics

  1. Polymer stabilization of the smectic C-alpha* liquid crystal phase—Over tenfold thermal stabilization by confining networks of photo-polymerized reactive mesogens

    Energy Technology Data Exchange (ETDEWEB)

    Labeeb, A. [Liquid Crystal Institute, Chemical Physics Interdisciplinary Program, Kent State University, Kent, Ohio 44242 (United States); Microwave Physics and Dielectrics, National Research Center, Dokki 12622 (Egypt); Gleeson, H. F. [School of Physics and Astronomy, University of Leeds, Leeds LS2 9JT (United Kingdom); Hegmann, T., E-mail: thegmann@kent.edu [Liquid Crystal Institute, Chemical Physics Interdisciplinary Program, Kent State University, Kent, Ohio 44242 (United States)

    2015-12-07

    The smectic C*-alpha (SmC{sub α}*) phase is one of the sub-phases of ferroelectric liquid crystals that has drawn much interest due to its electro-optical properties and ultrafast switching. Generally observed above the ferroelectric SmC* phase in temperature, the SmC{sub α}* commonly shows only very narrow phase temperature range of a few degree Celsius. To broaden the SmC{sub α}* phase, polymer stabilization was investigated for thermal phase stabilization. Two different reactive monomers were tested in three mixtures, and all three broadened the temperature range of the SmC{sub α}* phase from 3 °C to 39 °C. The current reversal method was used to determine the phase existence versus temperature. Moreover, the texture and network structure was studied by polarized optical microscopy and scanning electron microscopy, with the latter revealing the confinement of the smectic layer structure within the porous polymer network.

  2. Cysteine residue is not essential for CPM protein thermal-stability assay.

    Science.gov (United States)

    Wang, Zhaoshuai; Ye, Cui; Zhang, Xinyi; Wei, Yinan

    2015-05-01

    A popular thermal-stability assay developed especially for the study of membrane proteins uses a thiol-specific probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). The fluorescence emission of CPM surges when it forms a covalent bond with the side chain of a free Cys, which becomes more readily accessible upon protein thermal denaturation. Interestingly, the melting temperatures of membrane proteins determined using the CPM assay in literature are closely clustered in the temperature range 45-55 °C. A thorough understanding of the mechanism behind the observed signal change is critical for the accurate interpretation of the protein unfolding. Here we used two α-helical membrane proteins, AqpZ and AcrB, as model systems to investigate the nature of the fluorescence surge in the CPM assay. We found that the transition temperatures measured using circular-dichroism (CD) spectroscopy and the CPM assay were significantly different. To eliminate potential artifact that might arise from the presence of detergent, we monitored the unfolding of two soluble proteins. We found that, contrary to current understanding, the presence of a sulfhydryl group was not a prerequisite for the CPM thermal-stability assay. The observed fluorescence increase is probably caused by binding of the fluorophore to hydrophobic patches exposed upon protein unfolding.

  3. Increased helix and protein stability through the introduction of a new tertiary hydrogen bond.

    Science.gov (United States)

    Peterson, R W; Nicholson, E M; Thapar, R; Klevit, R E; Scholtz, J M

    1999-03-12

    In an effort to quantify the importance of hydrogen bonding and alpha-helix formation to protein stability, a capping box motif was introduced into the small phosphocarrier protein HPr. Previous studies had confirmed that Ser46, at the N-cap position of the short helix-B in HPr, serves as an N-cap in solution. Thus, only a single-site mutation was required to produce a canonical S-X-X-E capping box: Lys49 at the N3 position was substituted with a glutamic acid residue. Thermal and chemical denaturation studies on the resulting K49E HPr show that the designed variant is approximately 2 kcal mol-1 more stable than the wild-type protein. However, NMR studies indicate that the side-chain of Glu49 does not participate in the expected capping H-bond interaction, but instead forms a new tertiary H-bond that links helix-B to the four-stranded beta-sheet of HPr. Here, we demonstrate that a strategy in which new non-native H-bonds are introduced can generate proteins with increased stability. We discuss why the original capping box design failed, and compare the energetic consequences of the new tertiary side-chain to main-chain H-bond with a local (helix-capping) side-chain to main-chain H-bond on the protein's global stability. Copyright 1999 Academic Press.

  4. Stability of spray-dried beetroot extract using oligosaccharides and whey proteins.

    Science.gov (United States)

    Carmo, Eloá Lourenço do; Teodoro, Rhana Amanda Ribeiro; Félix, Pedro Henrique Campelo; Fernandes, Regiane Victória de Barros; Oliveira, Érica Resende de; Veiga, Taís Regina Lima Abreu; Borges, Soraia Vilela; Botrel, Diego Alvarenga

    2018-05-30

    The properties and stability of spray-dried beetroot extract using maltodextrin (MD), inulin (IN), and whey protein isolate (WPI) as carrier agents were evaluated. The values of moisture, betalains content, and retention were 3.33-4.24%, 348.79-385.47 mg/100 g (dry-basis), and 88.45-95.69%, respectively. Higher values of antioxidant activity were observed for the treatments using WPI. The treatment with inulin alone presented higher hygroscopicity in the moisture adsorption isotherms at 25 °C and lower thermal stability when evaluating the thermogravimetric curves. When stored at 60 °C, the use of WPI alone conferred lower stability to the beetroot extract powder. In general, the simultaneous use of IN and WPI as carrier agents resulted in good stability of the beetroot extract powder, representing an opportunity for innovation in food products. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Crystal structure and thermal stability of AgIn(MoO4)2

    International Nuclear Information System (INIS)

    Klevtsov, P.V.; Solodovnikov, S.F.; Perepelitsa, A.P.; Klevtsova, R.F.

    1984-01-01

    Tetragonal crystals of double molybdate AgIn(MoO 4 ) 2 are prepared bi crystallization from solution in Ag 2 Mo 2 O 7 melt (a=4.998, c=36.725 A, space group I4 1 , Z=6). Its crystal structure is determined (autodaffractometer ''Syntex P2 1 '', MoKsub(α)-radiation, 876 reflections, R=0.054) in which along with Mo-tetrahedrons Mo-octahedrons are present. By mutual edges latter are united into bands forming fragments of wolframite structure alonside with (In, Ag) octahedrons. In the direction of c axis wolframite fragments alternate with scheelite fragments consisting of Mo-tetrahedrons and Ag-octavertices. The crystallochemical formula of the compound is Ag(Insub(0.75)Agsub(0.25))sub(2)Mosub(2)Osub(8) [MoO 4 ]. At a temperature of about 600 deq C AgIn-molybdate transforms into modification with NaIn(MoO 4 ) 2 structure NaIn(MoO 4 ) 2 and melts at 650 deg C decomposing into In 2 (MoO 4 ) 3 solid phase and Ag 2 MoO 4 melt

  6. Protein Adsorption to Titanium and Zirconia Using a Quartz Crystal Microbalance Method

    Directory of Open Access Journals (Sweden)

    You Kusakawa

    2017-01-01

    Full Text Available Protein adsorption onto titanium (Ti or zirconia (ZrO2 was evaluated using a 27 MHz quartz crystal microbalance (QCM. As proteins, fibronectin (Fn, a cell adhesive protein, and albumin (Alb, a cell adhesion-inhibiting protein, were evaluated. The Ti and ZrO2 sensors for QCM were characterized by atomic force microscopy and electron probe microanalysis observation, measurement of contact angle against water, and surface roughness. The amounts of Fn and Alb adsorbed onto the Ti and ZrO2 sensors and apparent reaction rate were obtained using QCM measurements. Ti sensor showed greater adsorption of Fn and Alb than the ZrO2 sensor. In addition, amount of Fn adsorbed onto the Ti or ZrO2 sensors was higher than that of Alb. The surface roughness and hydrophilicity of Ti or ZrO2 may influence the adsorption of Fn or Alb. With regard to the adsorption rate, Alb adsorbed more rapidly than Fn onto Ti. Comparing Ti and ZrO2, Alb adsorption rate to Ti was faster than that to ZrO2. Fn adsorption will be effective for cell activities, but Alb adsorption will not. QCM method could simulate in vivo Fn and Alb adsorption to Ti or ZrO2.

  7. Protein NMR Structures Refined with Rosetta Have Higher Accuracy Relative to Corresponding X-ray Crystal Structures

    Science.gov (United States)

    2014-01-01

    We have found that refinement of protein NMR structures using Rosetta with experimental NMR restraints yields more accurate protein NMR structures than those that have been deposited in the PDB using standard refinement protocols. Using 40 pairs of NMR and X-ray crystal structures determined by the Northeast Structural Genomics Consortium, for proteins ranging in size from 5–22 kDa, restrained Rosetta refined structures fit better to the raw experimental data, are in better agreement with their X-ray counterparts, and have better phasing power compared to conventionally determined NMR structures. For 37 proteins for which NMR ensembles were available and which had similar structures in solution and in the crystal, all of the restrained Rosetta refined NMR structures were sufficiently accurate to be used for solving the corresponding X-ray crystal structures by molecular replacement. The protocol for restrained refinement of protein NMR structures was also compared with restrained CS-Rosetta calculations. For proteins smaller than 10 kDa, restrained CS-Rosetta, starting from extended conformations, provides slightly more accurate structures, while for proteins in the size range of 10–25 kDa the less CPU intensive restrained Rosetta refinement protocols provided equally or more accurate structures. The restrained Rosetta protocols described here can improve the accuracy of protein NMR structures and should find broad and general for studies of protein structure and function. PMID:24392845

  8. Size and temperature dependent stability and phase transformation in single-crystal zirconium nanowire

    International Nuclear Information System (INIS)

    Sutrakar, Vijay Kumar; Roy Mahapatra, D.

    2011-01-01

    A novel size dependent FCC (face-centered-cubic) → HCP (hexagonally-closed-pack) phase transformation and stability of an initial FCC zirconium nanowire are studied. FCC zirconium nanowires with cross-sectional dimensions 20 Å, in which surface stresses are not enough to drive the phase transformation, show meta-stability. In such a case, an external kinetic energy in the form of thermal heating is required to overcome the energy barrier and achieve FCC → HCP phase transformation. The FCC-HCP transition pathway is also studied using Nudged Elastic Band (NEB) method, to further confirm the size dependent stability/metastability of Zr nanowires. We also show size dependent critical temperature, which is required for complete phase transformation of a metastable-FCC nanowire.

  9. Influence of encapsulated functional lipids on crystal structure and chemical stability in solid lipid nanoparticles: Towards bioactive-based design of delivery systems.

    Science.gov (United States)

    Salminen, Hanna; Gömmel, Christina; Leuenberger, Bruno H; Weiss, Jochen

    2016-01-01

    We investigated the influence of physicochemical properties of encapsulated functional lipids--vitamin A, β-carotene and ω-3 fish oil--on the structural arrangement of solid lipid nanoparticles (SLN). The relationship between the crystal structure and chemical stability of the incorporated bioactive lipids was evaluated with different emulsifier compositions of a saponin-rich, food-grade Quillaja extract alone or combined with high-melting or low-melting lecithins. The major factors influencing the structural arrangement and chemical stability of functional lipids in solid lipid dispersions were their solubility in the aqueous phase and their crystallization temperature in relation to that of the carrier lipid. The results showed that the stabilization of the α-subcell crystals in the lattice of the carrier lipid is a key parameter for forming stable solid lipid dispersions. This study contributes to a better understanding of SLN as a function of the bioactive lipid. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of a cyclophilin A-like protein from Piriformospora indica

    International Nuclear Information System (INIS)

    Bhatt, Harshesh; Trivedi, Dipesh Kumar; Pal, Ravi Kant; Johri, Atul Kumar; Tuteja, Narendra; Bhavesh, Neel Sarovar

    2012-01-01

    Cyclophin A like protein from Piriformospora indica involved in salt-stress tolerance was cloned, overexpressed, purified and crystallized. The crystals obtained diffracted X-rays to 2 Å resolution and belonged to space group C222 1 . Cyclophilins are widely distributed both in eukaryotes and prokaryotes and have a primary role as peptidyl-prolyl cis–trans isomerases (PPIases). This study focuses on the cloning, expression, purification and crystallization of a salinity-stress-induced cyclophilin A (CypA) homologue from the symbiotic fungus Piriformospora indica. Crystallization experiments in the presence of 56 mM sodium phosphate monobasic monohydrate, 1.34 M potassium phosphate dibasic pH 8.2 yielded crystals that were suitable for X-ray diffraction analysis. The crystals belonged to the orthorhombic space group C222 1 , with unit-cell parameters a = 121.15, b = 144.12, c = 110.63 Å. The crystals diffracted to a resolution limit of 2.0 Å. Analysis of the diffraction data indicated the presence of three molecules of the protein per asymmetric unit (V M = 4.48 Å 3 Da −1 , 72.6% solvent content)

  11. Crystallization of protein–ligand complexes

    International Nuclear Information System (INIS)

    Hassell, Anne M.; An, Gang; Bledsoe, Randy K.; Bynum, Jane M.; Carter, H. Luke III; Deng, Su-Jun J.; Gampe, Robert T.; Grisard, Tamara E.; Madauss, Kevin P.; Nolte, Robert T.; Rocque, Warren J.; Wang, Liping; Weaver, Kurt L.; Williams, Shawn P.; Wisely, G. Bruce; Xu, Robert; Shewchuk, Lisa M.

    2007-01-01

    Methods presented for growing protein–ligand complexes fall into the categories of co-expression of the protein with the ligands of interest, use of the ligands during protein purification, cocrystallization and soaking the ligands into existing crystals. Obtaining diffraction-quality crystals has long been a bottleneck in solving the three-dimensional structures of proteins. Often proteins may be stabilized when they are complexed with a substrate, nucleic acid, cofactor or small molecule. These ligands, on the other hand, have the potential to induce significant conformational changes to the protein and ab initio screening may be required to find a new crystal form. This paper presents an overview of strategies in the following areas for obtaining crystals of protein–ligand complexes: (i) co-expression of the protein with the ligands of interest, (ii) use of the ligands during protein purification, (iii) cocrystallization and (iv) soaks

  12. Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

    Science.gov (United States)

    Xu, Qifang; Malecka, Kimberly L.; Fink, Lauren; Jordan, E. Joseph; Duffy, Erin; Kolander, Samuel; Peterson, Jeffrey; Dunbrack, Roland L.

    2016-01-01

    Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Crystal structures of several homomeric protein kinase complexes have a serine, threonine, or tyrosine autophosphorylation site of one kinase monomer located in the active site of another monomer, a structural complex that we call an “autophosphorylation complex.” We developed and applied a structural bioinformatics method to identify all such autophosphorylation kinase complexes in X-ray crystallographic structures in the Protein Data Bank (PDB). We identified 15 autophosphorylation complexes in the PDB, of which 5 complexes had not previously been described in the publications describing the crystal structures. These 5 consist of tyrosine residues in the N-terminal juxtamembrane regions of colony stimulating factor 1 receptor (CSF1R, Tyr561) and EPH receptor A2 (EPHA2, Tyr594), tyrosine residues in the activation loops of the SRC kinase family member LCK (Tyr394) and insulin-like growth factor 1 receptor (IGF1R, Tyr1166), and a serine in a nuclear localization signal region of CDC-like kinase 2 (CLK2, Ser142). Mutations in the complex interface may alter autophosphorylation activity and contribute to disease; therefore we mutated residues in the autophosphorylation complex interface of LCK and found that two mutations impaired autophosphorylation (T445V and N446A) and mutation of Pro447 to Ala, Gly, or Leu increased autophosphorylation. The identified autophosphorylation sites are conserved in many kinases, suggesting that, by homology, these complexes may provide insight into autophosphorylation complex interfaces of kinases that are relevant drug targets. PMID:26628682

  13. Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae

    International Nuclear Information System (INIS)

    Yamada, Mototsugu; Watanabe, Takashi; Baba, Nobuyoshi; Miyara, Takako; Saito, Jun; Takeuchi, Yasuo

    2008-01-01

    The selenomethionyl-substituted transpeptidase domain of penicillin-binding protein (PBP) 2B from S. pneumoniae was isolated from a limited proteolysis digest of the soluble form of recombinant PBP 2B and then crystallized. MAD data were collected to 2.4 Å resolution. Penicillin-binding protein (PBP) 2B from Streptococcus pneumoniae catalyzes the cross-linking of peptidoglycan precursors that occurs during bacterial cell-wall biosynthesis. A selenomethionyl (SeMet) substituted PBP 2B transpeptidase domain was isolated from a limited proteolysis digest of a soluble form of recombinant PBP 2B and then crystallized. The crystals belonged to space group P4 3 2 1 2, with unit-cell parameters a = b = 86.39, c = 143.27 Å. Diffraction data were collected to 2.4 Å resolution using the BL32B2 beamline at SPring-8. The asymmetric unit contains one protein molecule and 63.7% solvent

  14. Cloning, purification, crystallization and preliminary X-ray analysis of the Burkholderia pseudomallei L1 ribosomal protein

    International Nuclear Information System (INIS)

    Abd Aziz, Abd Ghani; Ruzheinikov, Sergey N.; Sedelnikova, Svetlana E.; Mohamed, Rahmah; Nathan, Sheila; Baker, Patrick J.; Rice, David W.

    2012-01-01

    The L1 ribosomal protein from B. pseudomallei has been overexpressed, purified and crystallized in a form suitable for X-ray analysis. The gene encoding the L1 ribosomal protein from Burkholderia pseudomallei strain D286 has been cloned into the pETBLUE-1 vector system, overexpressed in Escherichia coli and purified. Crystals of the native protein were grown by the hanging-drop vapour-diffusion technique using PEG 3350 as a precipitant and diffracted to beyond 1.65 Å resolution. The crystals belonged to space group P2 1 2 1 2, with unit-cell parameters a = 53.6, b = 127.1, c = 31.8 Å and with a single molecule in the asymmetric unit

  15. Molecular Dynamics Driven Design of pH-Stabilized Mutants of MNEI, a Sweet Protein.

    Directory of Open Access Journals (Sweden)

    Serena Leone

    Full Text Available MNEI is a single chain derivative of monellin, a plant protein that can interact with the human sweet taste receptor, being therefore perceived as sweet. This unusual physiological activity makes MNEI a potential template for the design of new sugar replacers for the food and beverage industry. Unfortunately, applications of MNEI have been so far limited by its intrinsic sensitivity to some pH and temperature conditions, which could occur in industrial processes. Changes in physical parameters can, in fact, lead to irreversible protein denaturation, as well as aggregation and precipitation. It has been previously shown that the correlation between pH and stability in MNEI derives from the presence of a single glutamic residue in a hydrophobic pocket of the protein. We have used molecular dynamics to study the consequences, at the atomic level, of the protonation state of such residue and have identified the network of intramolecular interactions responsible for MNEI stability at acidic pH. Based on this information, we have designed a pH-independent, stabilized mutant of MNEI and confirmed its increased stability by both molecular modeling and experimental techniques.

  16. Molecular Dynamics Driven Design of pH-Stabilized Mutants of MNEI, a Sweet Protein.

    Science.gov (United States)

    Leone, Serena; Picone, Delia

    2016-01-01

    MNEI is a single chain derivative of monellin, a plant protein that can interact with the human sweet taste receptor, being therefore perceived as sweet. This unusual physiological activity makes MNEI a potential template for the design of new sugar replacers for the food and beverage industry. Unfortunately, applications of MNEI have been so far limited by its intrinsic sensitivity to some pH and temperature conditions, which could occur in industrial processes. Changes in physical parameters can, in fact, lead to irreversible protein denaturation, as well as aggregation and precipitation. It has been previously shown that the correlation between pH and stability in MNEI derives from the presence of a single glutamic residue in a hydrophobic pocket of the protein. We have used molecular dynamics to study the consequences, at the atomic level, of the protonation state of such residue and have identified the network of intramolecular interactions responsible for MNEI stability at acidic pH. Based on this information, we have designed a pH-independent, stabilized mutant of MNEI and confirmed its increased stability by both molecular modeling and experimental techniques.

  17. The stability of PEMFC electrodes : platinum dissolution vs potential and temperature investigated by quartz crystal microbalance

    NARCIS (Netherlands)

    Dam, V.A.T.; Bruijn, de F.A.

    2007-01-01

    The stability of platinum in proton exchange membrane fuel cell (PEMFC) electrodes has been investigated by determining the dissolution of platinum from a thin platinum film deposited on a gold substrate in 1 M HClO4 at different temperatures ranging between 40 and 80°C and potentials between 0.85

  18. Stress and stability of sputter deposited A-15 and bcc crystal structure tungsten thin films

    Energy Technology Data Exchange (ETDEWEB)

    O' Keefe, M.J.; Stutz, C.E.

    1997-07-01

    Magnetron sputter deposition was used to fabricate body centered cubic (bcc) and A-15 crystal structure W thin films. Previous work demonstrated that the as-deposited crystal structure of the films was dependent on the deposition parameters and that the formation of a metastable A-15 structure was favored over the thermodynamically stable bcc phase when the films contained a few atomic percent oxygen. However, the A-15 phase was shown to irreversibly transform into the bcc phase between 500 C and 650 C and that a significant decrease in the resistivity of the metallic films was measured after the transformation. The current investigation of 150 nm thick, sputter deposited A-15 and bcc tungsten thin films on silicon wafers consisted of a series of experiments in which the stress, resistivity and crystal structure of the films was measured as a function of temperatures cycles in a Flexus 2900 thin film stress measurement system. The as-deposited film stress was found to be a function of the sputtering pressure and presputter time; under conditions in which the as-deposited stress of the film was {approximately}1.5 GPa compressive delamination of the W film from the substrate was observed. Data from the thermal studies indicated that bcc film stress was not affected by annealing but transformation of the A-15 structure resulted in a large tensile increase in the stress of the film, regardless of the as-deposited stress of the film. In several instances, complete transformation of the A-15 structure into the bcc phase resulted in {ge}1 GPa tensile increase in film stress.

  19. Stress and stability of sputter deposited A-15 and bcc crystal structure tungsten thin films

    International Nuclear Information System (INIS)

    O'Keefe, M.J.; Stutz, C.E.

    1997-01-01

    Magnetron sputter deposition was used to fabricate body centered cubic (bcc) and A-15 crystal structure W thin films. Previous work demonstrated that the as-deposited crystal structure of the films was dependent on the deposition parameters and that the formation of a metastable A-15 structure was favored over the thermodynamically stable bcc phase when the films contained a few atomic percent oxygen. However, the A-15 phase was shown to irreversibly transform into the bcc phase between 500 C and 650 C and that a significant decrease in the resistivity of the metallic films was measured after the transformation. The current investigation of 150 nm thick, sputter deposited A-15 and bcc tungsten thin films on silicon wafers consisted of a series of experiments in which the stress, resistivity and crystal structure of the films was measured as a function of temperatures cycles in a Flexus 2900 thin film stress measurement system. The as-deposited film stress was found to be a function of the sputtering pressure and presputter time; under conditions in which the as-deposited stress of the film was approximately1.5 GPa compressive delamination of the W film from the substrate was observed. Data from the thermal studies indicated that bcc film stress was not affected by annealing but transformation of the A-15 structure resulted in a large tensile increase in the stress of the film, regardless of the as-deposited stress of the film. In several instances, complete transformation of the A-15 structure into the bcc phase resulted in ge1 GPa tensile increase in film stress

  20. Purification and crystallization of the ABC-type transport substrate-binding protein OppA from Thermoanaerobacter tengcongensis

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Jinlan; Li, Xiaolu [State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Tsinghua University, Beijing 100005, People' s Republic of China (China); Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People' s Republic of China (China); Feng, Yue; Zhang, Bo [Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People' s Republic of China (China); Miao, Shiying [State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Tsinghua University, Beijing 100005, People' s Republic of China (China); Wang, Linfang, E-mail: lfwangz@yahoo.com [State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Tsinghua University, Beijing 100005, People' s Republic of China (China); Wang, Na, E-mail: nawang@tsinghua.edu.cn [Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People' s Republic of China (China)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We truncated the signal peptide of OppA{sub TTE0054} to make it express in Escherichia coli as a soluble protein. Black-Right-Pointing-Pointer Crystals of OppA{sub TTE0054} were grown by sitting-drop vapor diffusion method. Black-Right-Pointing-Pointer The crystal of OppA{sub TTE0054} diffracted to 2.25 A. -- Abstract: Di- and oligopeptide- binding protein OppAs play important roles in solute and nutrient uptake, sporulation, biofilm formation, cell wall muropeptides recycling, peptide-dependent quorum-sensing responses, adherence to host cells, and a variety of other biological processes. Soluble OppA from Thermoanaerobacter tengcongensis was expressed in Escherichia coli. The protein was found to be >95% pure with SDS-PAGE after a series of purification steps and the purity was further verified by mass spectrometry. The protein was crystallized using the sitting-drop vapour-diffusion method with PEG 400 as the precipitant. Crystal diffraction extended to 2.25 A. The crystal belonged to space group C222{sub 1}, with unit-cell parameters of a = 69.395, b = 199.572, c = 131.673 A, and {alpha} = {beta} = {gamma} = 90 Degree-Sign .

  1. Crystallization and preliminary crystallographic analysis of a calcineurin B-like protein 1 (CBL1) mutant from Ammopiptanthus mongolicus

    International Nuclear Information System (INIS)

    Shang, Guijun; Cang, Huaixing; Liu, Zhijie; Gao, Wei; Bi, Ruchang

    2010-01-01

    Recombinant calcineurin B-like protein 1 from Ammopiptanthus mongolicus (AmCBL1) was overexpressed, purified and crystallized. Calcineurin B-like protein 1 (CBL1) is a calcium sensor in plants. It transmits the calcium signal through the downstream protein CBL-interacting protein kinase (CIPK). CBL1 and CIPK play crucial roles in the response to environmental stresses such as low K + , osmotic shock, high salt, cold and drought. Recombinant CBL1 from Ammopiptanthus mongolicus (AmCBL1) was overexpressed, purified and crystallized. However, the crystal did not diffract well. A mutant prepared using the surface-entropy method and crystallized using the hanging-drop method at 298 K with PEG 2000 MME as a precipitant diffracted to 2.90 Å resolution. The crystal belonged to space group P2 1 2 1 2, with unit-cell parameters a = 99.87, b = 114.42, c = 63.80 Å, α = β = γ = 90.00° and three molecules per asymmetric unit

  2. Long Non-coding RNA, PANDA, Contributes to the Stabilization of p53 Tumor Suppressor Protein.

    Science.gov (United States)

    Kotake, Yojiro; Kitagawa, Kyoko; Ohhata, Tatsuya; Sakai, Satoshi; Uchida, Chiharu; Niida, Hiroyuki; Naemura, Madoka; Kitagawa, Masatoshi

    2016-04-01

    P21-associated noncoding RNA DNA damage-activated (PANDA) is induced in response to DNA damage and represses apoptosis by inhibiting the function of nuclear transcription factor Y subunit alpha (NF-YA) transcription factor. Herein, we report that PANDA affects regulation of p53 tumor-suppressor protein. U2OS cells were transfected with PANDA siRNAs. At 72 h post-transfection, cells were subjected to immunoblotting and quantitative reverse transcription-polymerase chain reaction. Depletion of PANDA was associated with decreased levels of p53 protein, but not p53 mRNA. The stability of p53 protein was markedly reduced by PANDA silencing. Degradation of p53 protein by silencing PANDA was prevented by treatment of MG132, a proteasome inhibitor. Moreover, depletion of PANDA prevented accumulation of p53 protein, as a result of DNA damage, induced by the genotoxic agent etoposide. These results suggest that PANDA stabilizes p53 protein in response to DNA damage, and provide new insight into the regulatory mechanisms of p53. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  3. Effect of hydrogen peroxide on improving the heat stability of whey protein isolate solutions.

    Science.gov (United States)

    Sutariya, Suresh; Patel, Hasmukh

    2017-05-15

    Whey protein isolate (WPI) solutions (12.8%w/w protein) were treated with varying concentrations of H 2 O 2 in the range of 0-0.144 H 2 O 2 to protein ratios (HTPR) by the addition of the required quantity of H 2 O 2 and deionized water. The samples were analyzed for heat stability, rheological properties, denaturation level of β-lactoglobulin (β-LG) and α-lactalbumin (α-LA). The samples treated with H 2 O 2 concentration >0.072 (HTPR) showed significant improvement in the heat stability, and decreased whey protein denaturation and aggregation. The WPI solution treated with H 2 O 2 (>0.072 HTPR) remained in the liquid state after heat treatment at 120°C, whereas the control samples formed gel upon heat treatment. Detailed analysis of these samples suggested that the improvement in the heat stability of H 2 O 2 treated WPI solution was attributed to the significant reduction in the sulfhydryl-disulfide interchange reaction during denaturation of β-LG and α-LA. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Weak Links: Stabilizers of Complex Systems from Proteins to Social Networks

    Science.gov (United States)

    Csermely, Peter

    Why do women stabilize our societies? Why can we enjoy and understand Shakespeare? Why are fruitflies uniform? Why do omnivorous eating habits aid our survival? Why is Mona Lisa's smile beautiful? -- Is there any answer to these questions? This book shows that the statement: "weak links stabilize complex systems" holds the answers to all of the surprising questions above. The author (recipientof several distinguished science communication prizes) uses weak (low affinity, low probability) interactions as a thread to introduce a vast varietyof networks from proteins to ecosystems.

  5. Protein adsorption at the electrified air-water interface: implications on foam stability.

    Science.gov (United States)

    Engelhardt, Kathrin; Rumpel, Armin; Walter, Johannes; Dombrowski, Jannika; Kulozik, Ulrich; Braunschweig, Björn; Peukert, Wolfgang

    2012-05-22

    The surface chemistry of ions, water molecules, and proteins as well as their ability to form stable networks in foams can influence and control macroscopic properties such as taste and texture of dairy products considerably. Despite the significant relevance of protein adsorption at liquid interfaces, a molecular level understanding on the arrangement of proteins at interfaces and their interactions has been elusive. Therefore, we have addressed the adsorption of the model protein bovine serum albumin (BSA) at the air-water interface with vibrational sum-frequency generation (SFG) and ellipsometry. SFG provides specific information on the composition and average orientation of molecules at interfaces, while complementary information on the thickness of the adsorbed layer can be obtained with ellipsometry. Adsorption of charged BSA proteins at the water surface leads to an electrified interface, pH dependent charging, and electric field-induced polar ordering of interfacial H(2)O and BSA. Varying the bulk pH of protein solutions changes the intensities of the protein related vibrational bands substantially, while dramatic changes in vibrational bands of interfacial H(2)O are simultaneously observed. These observations have allowed us to determine the isoelectric point of BSA directly at the electrolyte-air interface for the first time. BSA covered air-water interfaces with a pH near the isoelectric point form an amorphous network of possibly agglomerated BSA proteins. Finally, we provide a direct correlation of the molecular structure of BSA interfaces with foam stability and new information on the link between microscopic properties of BSA at water surfaces and macroscopic properties such as the stability of protein foams.

  6. Crystallization of the novel flavodoxine-like protein, WrbA, - on the way to three-dimensional structure

    Czech Academy of Sciences Publication Activity Database

    Kutá-Smatanová, Ivana; Wolfová, J.; Grandori, R.; Chatterjee, N.; Carey, J.

    2004-01-01

    Roč. 54, č. 2004 (2004), s. 35 ISSN 1210-8529. [European Crystallographic Meeting /22./. Budapešť, 26.08.2004-31.08.2004] R&D Projects: GA MŠk ME 640; GA MŠk LN00A141 Grant - others:GA ÚV1 INT-03-09049 Keywords : flavodoxine-like protein, tryprophan-repressor binding protein, WrbA apoprotein, WrbA holoprotein, FMN, standard crystallization techniques, advanced crystallization techniques Subject RIV: BO - Biophysics

  7. Characterization of the Protein Crystal Growth Apparatus for Microgravity Aboard the Space Station

    Science.gov (United States)

    Kundrot, Craig E.; Roeber, D.; Achari, A.; Stinson, Thomas N. (Technical Monitor)

    2002-01-01

    We have conducted experiments to determine the equilibration rates of some major precipitants used in protein crystallography aboard the International Space Station (ISS). The solutions were placed in the Protein Crystallization Apparatus for Microgravity (PCAM) which mimic Cryschem sitting drop trays. The trays were placed in cylinders. These cylinders were placed inside a Single locker Thermal Enclosure System (STES), and were activated for different durations during the flight. Bumpers pressed against elastomers seal drops in a deactivated state during pre-flight and prior to transfer to the ISS. Activation occurs while in flight on the ISS by releasing the bumpers allowing the drops to be exposed to the reservoir. PCAM was flown to the ISS on STS 100, Flight 6A, on April 19, 2001. Six series of equilibration experiments were tested for each precipitant with a small amount of Green Fluorescent Protein (GFP). Cylinder 10 was never activated, 7 was activated for 40 days, 8 was activated for 20 days, 9 was activated for 10 days, 11 was activated for 4 days and 12 was activated for 2 days. Upon the return to Earth by STS 104 on July 24,2001 the samples were transferred to Marshall Space Flight Center. The samples were then brought to the lab and the volumes of each sample were measured.

  8. Influence of additive L-phenylalanine on stabilization of metastable α-form of L-glutamic acid in cooling crystallization

    Science.gov (United States)

    Quang, Khuu Chau; Nhan, Le Thi Hong; Huyen, Trinh Thi Thanh; Tuan, Nguyen Anh

    2017-09-01

    The influence of additive amino acid L-phenylalanine on stabilization of metastable α-form of L-glutamic acid was investigated in cooling crystallization. The present study found that the additive L-phenylalanine could be used to stabilize the pure metastable α-form in L-glutamic acid crystallization, where the additive concentration of 0.05-0.1 (g/L) was sufficient to stabilize the 100% wt metastable α-form in solid product at L-glutamic acid concentration of 30-45 (g/L). Additionally, the present results indicated that the adsorption of additive L-phenylalanine on the (001) surface of α-form was more favorable than that of the β-form molecular, so the nucleation sites of stable β-form was occupied by additive molecular, which resulted in inhibition of nucleation and growth of β-form, allowing stabilization of metastable α-form.

  9. Crystallization of FcpA from Leptospira, a novel flagellar protein that is essential for pathogenesis.

    Science.gov (United States)

    San Martin, Fabiana; Mechaly, Ariel E; Larrieux, Nicole; Wunder, Elsio A; Ko, Albert I; Picardeau, Mathieu; Trajtenberg, Felipe; Buschiazzo, Alejandro

    2017-03-01

    The protein FcpA is a unique component of the flagellar filament of spirochete bacteria belonging to the genus Leptospira. Although it plays an essential role in translational motility and pathogenicity, no structures of FcpA homologues are currently available in the PDB. Its three-dimensional structure will unveil the novel motility mechanisms that render pathogenic Leptospira particularly efficient at invading and disseminating within their hosts, causing leptospirosis in humans and animals. FcpA from L. interrogans was purified and crystallized, but despite laborious attempts no useful X ray diffraction data could be obtained. This challenge was solved by expressing a close orthologue from the related saprophytic species L. biflexa. Three different crystal forms were obtained: a primitive and a centred monoclinic form, as well as a hexagonal variant. All forms diffracted X-rays to suitable resolutions for crystallographic analyses, with the hexagonal type typically reaching the highest limits of 2.0 Å and better. A variation of the quick-soaking procedure resulted in an iodide derivative that was instrumental for single-wavelength anomalous diffraction methods.

  10. Competition between crystallization and glassification for particles with short-ranged attraction. Possible applications to protein crystallization

    Science.gov (United States)

    Zaccarelli, E.; Sciortino, F.; Tartaglia, P.; Foffi, G.; McCullagh, G. D.; Lawlor, A.; Dawson, K. A.

    2002-11-01

    We discuss the phase behaviour of spherical hard-core particles, with an attractive potential, as described by a hard-core Yukawa model. The ratio of the range of the attraction to the diameter of the particles is an important control parameter of the problem. Upon decreasing the range of the attraction, the phase diagram changes quite significantly, with the liquid-gas transition becoming metastable, and the crystal being in equilibrium with the fluid, with no intervening liquid. We also study the glass transition lines and, crucially, find that the situation, being very simple for pure repulsive potentials, becomes much richer in competition between glass and crystal phases for short-range attractions. Also a transition between attractive and repulsive glass appears somewhat in analogy with the isostructural equilibrium transition between two crystals.

  11. A Medipix quantum area detector allows rotation electron diffraction data collection from submicrometre three-dimensional protein crystals

    Energy Technology Data Exchange (ETDEWEB)

    Nederlof, Igor; Genderen, Eric van; Li, Yao-Wang; Abrahams, Jan Pieter, E-mail: abrahams@chem.leidenuniv.nl [Leiden University, Einsteinweg 55, 2333 CC Leiden (Netherlands)

    2013-07-01

    An ultrasensitive Medipix2 detector allowed the collection of rotation electron-diffraction data from single three-dimensional protein nanocrystals for the first time. The data could be analysed using the standard X-ray crystallography programs MOSFLM and SCALA. When protein crystals are submicrometre-sized, X-ray radiation damage precludes conventional diffraction data collection. For crystals that are of the order of 100 nm in size, at best only single-shot diffraction patterns can be collected and rotation data collection has not been possible, irrespective of the diffraction technique used. Here, it is shown that at a very low electron dose (at most 0.1 e{sup −} Å{sup −2}), a Medipix2 quantum area detector is sufficiently sensitive to allow the collection of a 30-frame rotation series of 200 keV electron-diffraction data from a single ∼100 nm thick protein crystal. A highly parallel 200 keV electron beam (λ = 0.025 Å) allowed observation of the curvature of the Ewald sphere at low resolution, indicating a combined mosaic spread/beam divergence of at most 0.4°. This result shows that volumes of crystal with low mosaicity can be pinpointed in electron diffraction. It is also shown that strategies and data-analysis software (MOSFLM and SCALA) from X-ray protein crystallography can be used in principle for analysing electron-diffraction data from three-dimensional nanocrystals of proteins.

  12. A Medipix quantum area detector allows rotation electron diffraction data collection from submicrometre three-dimensional protein crystals

    International Nuclear Information System (INIS)

    Nederlof, Igor; Genderen, Eric van; Li, Yao-Wang; Abrahams, Jan Pieter

    2013-01-01

    An ultrasensitive Medipix2 detector allowed the collection of rotation electron-diffraction data from single three-dimensional protein nanocrystals for the first time. The data could be analysed using the standard X-ray crystallography programs MOSFLM and SCALA. When protein crystals are submicrometre-sized, X-ray radiation damage precludes conventional diffraction data collection. For crystals that are of the order of 100 nm in size, at best only single-shot diffraction patterns can be collected and rotation data collection has not been possible, irrespective of the diffraction technique used. Here, it is shown that at a very low electron dose (at most 0.1 e − Å −2 ), a Medipix2 quantum area detector is sufficiently sensitive to allow the collection of a 30-frame rotation series of 200 keV electron-diffraction data from a single ∼100 nm thick protein crystal. A highly parallel 200 keV electron beam (λ = 0.025 Å) allowed observation of the curvature of the Ewald sphere at low resolution, indicating a combined mosaic spread/beam divergence of at most 0.4°. This result shows that volumes of crystal with low mosaicity can be pinpointed in electron diffraction. It is also shown that strategies and data-analysis software (MOSFLM and SCALA) from X-ray protein crystallography can be used in principle for analysing electron-diffraction data from three-dimensional nanocrystals of proteins

  13. High-resolution X-ray crystal structure of bovine H-protein using the high-pressure cryocooling method

    International Nuclear Information System (INIS)

    Higashiura, Akifumi; Ohta, Kazunori; Masaki, Mika; Sato, Masaru; Inaka, Koji; Tanaka, Hiroaki; Nakagawa, Atsushi

    2013-01-01

    Using the high-pressure cryocooling method, the high-resolution X-ray crystal structure of bovine H-protein was determined at 0.86 Å resolution. This is the first ultra-high-resolution structure obtained from a high-pressure cryocooled crystal. Recently, many technical improvements in macromolecular X-ray crystallography have increased the number of structures deposited in the Protein Data Bank and improved the resolution limit of protein structures. Almost all high-resolution structures have been determined using a synchrotron radiation source in conjunction with cryocooling techniques, which are required in order to minimize radiation damage. However, optimization of cryoprotectant conditions is a time-consuming and difficult step. To overcome this problem, the high-pressure cryocooling method was developed (Kim et al., 2005 ▶) and successfully applied to many protein-structure analyses. In this report, using the high-pressure cryocooling method, the X-ray crystal structure of bovine H-protein was determined at 0.86 Å resolution. Structural comparisons between high- and ambient-pressure cryocooled crystals at ultra-high resolution illustrate the versatility of this technique. This is the first ultra-high-resolution X-ray structure obtained using the high-pressure cryocooling method

  14. Effects of lysine residues on structural characteristics and stability of tau proteins

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Myeongsang; Baek, Inchul; Choi, Hyunsung; Kim, Jae In; Na, Sungsoo, E-mail: nass@korea.ac.kr

    2015-10-23

    Pathological amyloid proteins have been implicated in neuro-degenerative diseases, specifically Alzheimer's, Parkinson's, Lewy-body diseases and prion related diseases. In prion related diseases, functional tau proteins can be transformed into pathological agents by environmental factors, including oxidative stress, inflammation, Aβ-mediated toxicity and covalent modification. These pathological agents are stable under physiological conditions and are not easily degraded. This un-degradable characteristic of tau proteins enables their utilization as functional materials to capturing the carbon dioxides. For the proper utilization of amyloid proteins as functional materials efficiently, a basic study regarding their structural characteristic is necessary. Here, we investigated the basic tau protein structure of wild-type (WT) and tau proteins with lysine residues mutation at glutamic residue (Q2K) on tau protein at atomistic scale. We also reported the size effect of both the WT and Q2K structures, which allowed us to identify the stability of those amyloid structures. - Highlights: • Lysine mutation effect alters the structure conformation and characteristic of tau. • Over the 15 layers both WT and Q2K models, both tau proteins undergo fractions. • Lysine mutation causes the increment of non-bonded energy and solvent accessible surface area. • Structural instability of Q2K model was proved by the number of hydrogen bonds analysis.

  15. Effects of lysine residues on structural characteristics and stability of tau proteins

    International Nuclear Information System (INIS)

    Lee, Myeongsang; Baek, Inchul; Choi, Hyunsung; Kim, Jae In; Na, Sungsoo

    2015-01-01

    Pathological amyloid proteins have been implicated in neuro-degenerative diseases, specifically Alzheimer's, Parkinson's, Lewy-body diseases and prion related diseases. In prion related diseases, functional tau proteins can be transformed into pathological agents by environmental factors, including oxidative stress, inflammation, Aβ-mediated toxicity and covalent modification. These pathological agents are stable under physiological conditions and are not easily degraded. This un-degradable characteristic of tau proteins enables their utilization as functional materials to capturing the carbon dioxides. For the proper utilization of amyloid proteins as functional materials efficiently, a basic study regarding their structural characteristic is necessary. Here, we investigated the basic tau protein structure of wild-type (WT) and tau proteins with lysine residues mutation at glutamic residue (Q2K) on tau protein at atomistic scale. We also reported the size effect of both the WT and Q2K structures, which allowed us to identify the stability of those amyloid structures. - Highlights: • Lysine mutation effect alters the structure conformation and characteristic of tau. • Over the 15 layers both WT and Q2K models, both tau proteins undergo fractions. • Lysine mutation causes the increment of non-bonded energy and solvent accessible surface area. • Structural instability of Q2K model was proved by the number of hydrogen bonds analysis.

  16. Evaporation kinetics in the hanging drop method of protein crystal growth

    Science.gov (United States)

    Baird, James K.; Frieden, Richard W.; Meehan, E. J., Jr.; Twigg, Pamela J.; Howard, Sandra B.; Fowlis, William A.

    1987-01-01

    An engineering analysis of the rate of evaporation of solvent in the hanging drop method of protein crystal growth is presented; these results are applied to 18 different drop and well arrangements commonly encountered in the laboratory, taking into account the chemical nature of the salt, the drop size and shape, the drop concentration, the well size, the well concentration, and the temperature. It is found that the rate of evaporation increases with temperature, drop size, and with the salt concentration difference between the drop and the well. The evaporation possesses no unique half-life. Once the salt in the drop achieves about 80 percent of its final concentration, further evaporation suffers from the law of diminishing returns.

  17. A New Generation Fiber Optic Probe: Characterization of Biological Fluids, Protein Crystals and Ophthalmic Diseases

    Science.gov (United States)

    Ansari, Rafat R.; Suh, Kwang I.

    1996-01-01

    A new fiber optic probe developed for determining transport properties of sub-micron particles in fluids experiments in a microgravity environment has been applied to characterize particulate dispersions/suspensions in various challenging environments which have been hitherto impossible. The probe positioned in front of a sample delivers a low power light (few nW - 3mW) from a laser and guides the light which is back scattered by the suspended particles through a receiving optical fiber to a photo detector and to a digital correlator. The probe provides rapid determination of macromolecular diffusivities and their respective size distributions. It has been applied to characterize various biological fluids, protein crystals, and ophthalmic diseases.

  18. New aspects of protein stability and turnover in the regulation of genome integrity

    DEFF Research Database (Denmark)

    Gallina, Irene

    of DNA repair is the control of protein abundance, both at a global cellular level, and locally at the site of damage. This is achieved through transcriptional regulation of protein synthesis and through the control of protein stability and turnover. In this study, we investigate the role of Rad56...... sensitivity when mutant. Prior to the work presented here,all these loci have been mapped to a specific gene except RAD56. We map the rad56-1 mutation to the NAT3 gene, which encodes the catalytic subunit of the NatB N-terminal acetyltransferase in yeast. Deletion of RAD56 causes sensitivity to X-rays, methyl......-scale studies investigating factors involved in DNA metabolism, but no specific function has been assigned to Cmr1. Taking advantage of a series of high-throughput screens we characterize Cmr1 as a chromatinassociated protein, involved in the regulation of fork progression in the presence of replication stress...

  19. Bacterial Genome Editing Strategy for Control of Transcription and Protein Stability

    DEFF Research Database (Denmark)

    Lauritsen, Ida; Martinez, Virginia; Ronda, Carlotta

    2018-01-01

    In molecular biology and cell factory engineering, tools that enable control of protein production and stability are highly important. Here, we describe protocols for tagging genes in Escherichia coli allowing for inducible degradation and transcriptional control of any soluble protein of interest....... The underlying molecular biology is based on the two cross-kingdom tools CRISPRi and the N-end rule for protein degradation. Genome editing is performed with the CRMAGE technology and randomization of the translational initiation region minimizes the polar effects of tag insertion. The approach has previously...... been applied for targeting proteins originating from essential operon-located genes and has potential to serve as a universal synthetic biology tool....

  20. Stability of some Cactaceae proteins based on fluorescence, circular dichroism, and differential scanning calorimetry measurements.

    Science.gov (United States)

    Gorinstein, S; Zemser, M; Vargas-Albores, F; Ochoa, J L; Paredes-Lopez, O; Scheler, C; Aksu, S; Salnikow, J

    1999-02-01

    Characterization of three cactus proteins (native and denatured) from Machaerocereus gummosus (Pitahaya agria), Lophocereu schottii (Garambullo), and Cholla opuntia (Cholla), was based on electrophoretic, fluorescence, CD (circular dichroism), DSC (differential scanning calorimetry), and FT-IR (Fourier transform infrared) measurements. The obtained results of intrinsic fluorescence, DSC, and CD were dissimilar for the three species of cactus, providing evidence of differences in secondary and tertiary structures. Cactus proteins may be situated in the following order corresponding to their relative stability: Machaerocereus gummosus (Pitahaya agria) > Cholla opuntia (Cholla) > Lophocereu schottii (Garambullo). Thermodynamic properties of proteins and their changes upon denaturation (temperature of denaturation, enthalphy, and the number of ruptured hydrogen bonds) were correlated with the secondary structure of proteins and disappearance of alpha-helix.

  1. The influence of magnetic fields on protein crystal growth and quality; Zum Einfluss magnetischer Felder auf das Wachstum und die Qualitaet von Proteinkristallen

    Energy Technology Data Exchange (ETDEWEB)

    Meents, Alke

    2005-08-01

    Magnetic fields can affect protein crystal growth in several ways. In homogeneous magnetic fields molecules and crystallites line up themselves along the magnetic field direction due to their magnetic anisotropy. Inhomogeneous magnetic fields exert a force on diamagnetic and paramagnetic compounds towards regions of lower or higher field strength. This effect can be used to create a microgravity-like environment for diamagnetic proteins and an environment comparable to hypergravity for paramagnetic proteins. Crystallization in homogeneous magnetic fields and a microgravity-like environment are reported to have a positive effect on crystal quality. The aim of this work was to systematically investigate the effect of protein crystallization in magnetic fields on the crystal quality by comparing a large number of crystals grown under identical conditions with- and without magnetic fields. Crystal quality was determined by means of high resolution rocking-curve measurements. Furthermore in certain cases complete diffraction datasets were collected. Any possible influence of magnetic fields on the mosaicity and the quality of the diffraction data was evaluated statistically by applying Wilcoxon-Ranksum tests. To investigate the effect of protein crystallization in homogeneous magnetic fields the diamagnetic proteins Thaumatin, Trypsin, and Lysozyme and paramagnetic Myoglobin were crystallized in magnetic fields of 5 T, 8.8 T, and 15.8 T. The analysis of crystal mosaicity and quality of the diffraction data of the diamagnetic proteins did not reveal a significant influence on the crystal quality. In contrast the crystals of paramagnetic Myoglobin grew up to 14 times larger than the ones in the control experiment. In addition they had a significant lower mosaicity, and diffracted to a higher resolution than ever reported before. Special pole pieces for an existing magnet were designed and build to grow protein crystals in an inhomogeneous magnetic field The experimental

  2. Two dynamin-like proteins stabilize FtsZ rings during Streptomyces sporulation.

    Science.gov (United States)

    Schlimpert, Susan; Wasserstrom, Sebastian; Chandra, Govind; Bibb, Maureen J; Findlay, Kim C; Flärdh, Klas; Buttner, Mark J

    2017-07-25

    During sporulation, the filamentous bacteria Streptomyces undergo a massive cell division event in which the synthesis of ladders of sporulation septa convert multigenomic hyphae into chains of unigenomic spores. This process requires cytokinetic Z-rings formed by the bacterial tubulin homolog FtsZ, and the stabilization of the newly formed Z-rings is crucial for completion of septum synthesis. Here we show that two dynamin-like proteins, DynA and DynB, play critical roles in this process. Dynamins are a family of large, multidomain GTPases involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. Using a cell biological approach, we show that the two Streptomyces dynamins specifically localize to sporulation septa in an FtsZ-dependent manner. Moreover, dynamin mutants have a cell division defect due to the decreased stability of sporulation-specific Z-rings, as demonstrated by kymographs derived from time-lapse images of FtsZ ladder formation. This defect causes the premature disassembly of individual Z-rings, leading to the frequent abortion of septum synthesis, which in turn results in the production of long spore-like compartments with multiple chromosomes. Two-hybrid analysis revealed that the dynamins are part of the cell division machinery and that they mediate their effects on Z-ring stability during developmentally controlled cell division via a network of protein-protein interactions involving DynA, DynB, FtsZ, SepF, SepF2, and the FtsZ-positioning protein SsgB.

  3. Structure and stability insights into tumour suppressor p53 evolutionary related proteins.

    Directory of Open Access Journals (Sweden)

    Bruno Pagano

    Full Text Available The p53 family of genes and their protein products, namely, p53, p63 and p73, have over one billion years of evolutionary history. Advances in computational biology and genomics are enabling studies of the complexities of the molecular evolution of p53 protein family to decipher the underpinnings of key biological conditions spanning from cancer through to various metabolic and developmental disorders and facilitate the design of personalised medicines. However, a complete understanding of the inherent nature of the thermodynamic and structural stability of the p53 protein family is still lacking. This is due, to a degree, to the lack of comprehensive structural information for a large number of homologous proteins and to an incomplete knowledge of the intrinsic factors responsible for their stability and how these might influence function. Here we investigate the thermal stability, secondary structure and folding properties of the DNA-binding domains (DBDs of a range of proteins from the p53 family using biophysical methods. While the N- and the C-terminal domains of the p53 family show sequence diversity and are normally targets for post-translational modifications and alternative splicing, the central DBD is highly conserved. Together with data obtained from Molecular Dynamics simulations in solution and with structure based homology modelling, our results provide further insights into the molecular properties of evolutionary related p53 proteins. We identify some marked structural differences within the p53 family, which could account for the divergence in biological functions as well as the subtleties manifested in the oligomerization properties of this family.

  4. Crystal Structure of the N-terminal Domain of the Group B Streptococcus Alpha C Protein

    Energy Technology Data Exchange (ETDEWEB)

    Auperin,T.; Bolduc, G.; Baron, M.; Heroux, A.; Filman, D.; Madoff, L.; Hogle, J.

    2005-01-01

    Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis, and meningitis among neonates and an important cause of morbidity among pregnant women and immunocompromised adults. Invasive diseases due to GBS are attributed to the ability of the pathogen to translocate across human epithelial surfaces. The alpha C protein (ACP) has been identified as an invasin that plays a role in internalization and translocation of GBS across epithelial cells. The soluble N-terminal domain of ACP (NtACP) blocks the internalization of GBS. We determined the 1.86-{angstrom} resolution crystal structure of NtACP comprising residues Ser{sup 52} through Leu{sup 225} of the full-length ACP. NtACP has two domains, an N-terminal {beta}-sandwich and a C-terminal three-helix bundle. Structural and topological alignments reveal that the {beta}-sandwich shares structural elements with the type III fibronectin fold (FnIII), but includes structural elaborations that make it unique. We have identified a potential integrin-binding motif consisting of Lys-Thr-Asp{sup 146}, Arg{sup 110}, and Asp{sup 118}. A similar arrangement of charged residues has been described in other invasins. ACP shows a heparin binding activity that requires NtACP. We propose a possible heparin-binding site, including one surface of the three-helix bundle, and nearby portions of the sandwich and repeat domains. We have validated this prediction using assays of the heparin binding and cell-adhesion properties of engineered fragments of ACP. This is the first crystal structure of a member of the highly conserved Gram-positive surface alpha-like protein family, and it will enable the internalization mechanism of GBS to be dissected at the atomic level.

  5. Protein Glycosylation in Archaea: A Post-Translational Modification to Enhance Extremophilic Protein Stability

    Science.gov (United States)

    2010-01-15

    Analysis of the chemical composition of the Asn-linked polysaccharides decorating many archaeal proteins has revealed the use of a wider variety of sugar...reminiscent of the eukaryal glycan-charged lipid, linked to a variety of monosaccharides , including glucose, mannose, and N-acetylglucosamine (GlcNAc

  6. Folding 19 proteins to their native state and stability of large proteins from a coarse-grained model.

    Science.gov (United States)

    Kapoor, Abhijeet; Travesset, Alex

    2014-03-01

    We develop an intermediate resolution model, where the backbone is modeled with atomic resolution but the side chain with a single bead, by extending our previous model (Proteins (2013) DOI: 10.1002/prot.24269) to properly include proline, preproline residues and backbone rigidity. Starting from random configurations, the model properly folds 19 proteins (including a mutant 2A3D sequence) into native states containing β sheet, α helix, and mixed α/β. As a further test, the stability of H-RAS (a 169 residue protein, critical in many signaling pathways) is investigated: The protein is stable, with excellent agreement with experimental B-factors. Despite that proteins containing only α helices fold to their native state at lower backbone rigidity, and other limitations, which we discuss thoroughly, the model provides a reliable description of the dynamics as compared with all atom simulations, but does not constrain secondary structures as it is typically the case in more coarse-grained models. Further implications are described. Copyright © 2013 Wiley Periodicals, Inc.

  7. The role of PEG conformation in mixed layers: from protein corona substrate to steric stabilization avoiding protein adsorption

    Directory of