The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae.

Science.gov (United States)

Galvão, C W; Souza, E M; Etto, R M; Pedrosa, F O; Chubatsu, L S; Yates, M G; Schumacher, J; Buck, M; Steffens, M B R

2012-12-01

DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs) can interact with the H. seropedicaeRecA protein (RecA Hs) and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs. RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions.

Mycobacterium tuberculosis UvrD1 and UvrA proteins suppress DNA strand exchange promoted by cognate and noncognate RecA proteins.

Science.gov (United States)

Singh, Pawan; Patil, K Neelakanteshwar; Khanduja, Jasbeer Singh; Kumar, P Sanjay; Williams, Alan; Rossi, Franca; Rizzi, Menico; Davis, Elaine O; Muniyappa, K

2010-06-15

DNA helicases are present in all kingdoms of life and play crucial roles in processes of DNA metabolism such as replication, repair, recombination, and transcription. To date, however, the role of DNA helicases during homologous recombination in mycobacteria remains unknown. In this study, we show that Mycobacterium tuberculosis UvrD1 more efficiently inhibited the strand exchange promoted by its cognate RecA, compared to noncognate Mycobacterium smegmatis or Escherichia coli RecA proteins. The M. tuberculosis UvrD1(Q276R) mutant lacking the helicase and ATPase activities was able to block strand exchange promoted by mycobacterial RecA proteins but not of E. coli RecA. We observed that M. tuberculosis UvrA by itself has no discernible effect on strand exchange promoted by E. coli RecA but impedes the reaction catalyzed by the mycobacterial RecA proteins. Our data also show that M. tuberculosis UvrA and UvrD1 can act together to inhibit strand exchange promoted by mycobacterial RecA proteins. Taken together, these findings raise the possibility that UvrD1 and UvrA might act together in vivo to counter the deleterious effects of RecA nucleoprotein filaments and/or facilitate the dissolution of recombination intermediates. Finally, we provide direct experimental evidence for a physical interaction between M. tuberculosis UvrD1 and RecA on one hand and RecA and UvrA on the other hand. These observations are consistent with a molecular mechanism, whereby M. tuberculosis UvrA and UvrD1, acting together, block DNA strand exchange promoted by cognate and noncognate RecA proteins.

Equilibrium amide hydrogen exchange and protein folding kinetics

International Nuclear Information System (INIS)

Bai Yawen

1999-01-01

The classical Linderstrom-Lang hydrogen exchange (HX) model is extended to describe the relationship between the HX behaviors (EX1 and EX2) and protein folding kinetics for the amide protons that can only exchange by global unfolding in a three-state system including native (N), intermediate (I), and unfolded (U) states. For these slowly exchanging amide protons, it is shown that the existence of an intermediate (I) has no effect on the HX behavior in an off-pathway three-state system (I↔U↔N). On the other hand, in an on-pathway three-state system (U↔I↔N), the existence of a stable folding intermediate has profound effect on the HX behavior. It is shown that fast refolding from the unfolded state to the stable intermediate state alone does not guarantee EX2 behavior. The rate of refolding from the intermediate state to the native state also plays a crucial role in determining whether EX1 or EX2 behavior should occur. This is mainly due to the fact that only amide protons in the native state are observed in the hydrogen exchange experiment. These new concepts suggest that caution needs to be taken if one tries to derive the kinetic events of protein folding from equilibrium hydrogen exchange experiments

Exchange protein directly activated by cAMP mediates slow delayed-rectifier current remodeling by sustained β-adrenergic activation in guinea pig hearts.

Science.gov (United States)

Aflaki, Mona; Qi, Xiao-Yan; Xiao, Ling; Ordog, Balazs; Tadevosyan, Artavazd; Luo, Xiaobin; Maguy, Ange; Shi, Yanfen; Tardif, Jean-Claude; Nattel, Stanley

2014-03-14

β-Adrenoceptor activation contributes to sudden death risk in heart failure. Chronic β-adrenergic stimulation, as occurs in patients with heart failure, causes potentially arrhythmogenic reductions in slow delayed-rectifier K(+) current (IKs). To assess the molecular mechanisms of IKs downregulation caused by chronic β-adrenergic activation, particularly the role of exchange protein directly activated by cAMP (Epac). Isolated guinea pig left ventricular cardiomyocytes were incubated in primary culture and exposed to isoproterenol (1 μmol/L) or vehicle for 30 hours. Sustained isoproterenol exposure decreased IKs density (whole cell patch clamp) by 58% (P<0.0001), with corresponding decreases in potassium voltage-gated channel subfamily E member 1 (KCNE1) mRNA and membrane protein expression (by 45% and 51%, respectively). Potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1) mRNA expression was unchanged. The β1-adrenoceptor antagonist 1-[2-((3-Carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol dihydrochloride (CGP-20712A) prevented isoproterenol-induced IKs downregulation, whereas the β2-antagonist ICI-118551 had no effect. The selective Epac activator 8-pCPT-2'-O-Me-cAMP decreased IKs density to an extent similar to isoproterenol exposure, and adenoviral-mediated knockdown of Epac1 prevented isoproterenol-induced IKs/KCNE1 downregulation. In contrast, protein kinase A inhibition with a cell-permeable highly selective peptide blocker did not affect IKs downregulation. 1,2-Bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate-AM acetoxymethyl ester (BAPTA-AM), cyclosporine, and inhibitor of nuclear factor of activated T cell (NFAT)-calcineurin association-6 (INCA6) prevented IKs reduction by isoproterenol and INCA6 suppressed isoproterenol-induced KCNE1 downregulation, consistent with signal-transduction via the Ca(2+)/calcineurin/NFAT pathway. Isoproterenol induced nuclear NFATc3/c4

The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

Energy Technology Data Exchange (ETDEWEB)

Galvão, C.W. [Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR (Brazil); Souza, E.M. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil); Etto, R.M. [Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR (Brazil); Pedrosa, F.O.; Chubatsu, L.S.; Yates, M.G. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil); Schumacher, J.; Buck, M. [Department of Life Sciences, Imperial College London, London (United Kingdom); Steffens, M.B.R. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil)

2012-10-15

DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX{sub Hs}) can interact with the H. seropedicae RecA protein (RecA{sub Hs}) and that RecA{sub Hs} possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX{sub Hs} inhibited 90% of the RecA{sub Hs} DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA{sub Hs}. RecA{sub Hs} ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX{sub Hs} was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX{sub Hs} protein negatively modulates the RecA{sub Hs} activities by protein-protein interactions and also by DNA-protein interactions.

Renal epithelial cell growth can occur in absence of Na+-H+ exchanger activity

International Nuclear Information System (INIS)

Mohrmann, M.; Cantiello, H.F.; Ausiello, D.A.

1987-01-01

An electroneutral Na+-H+ exchange system has been described in a variety of tissues and cell types, including those of renal origin, and has been proposed to play a role in the activation of growth. We have recently characterized the presence of this ubiquitous transporter in the apical domain of confluent epithelial LLC-PK1 cells. Because most apical membrane proteins appear late in cell growth, accompanying epithelial cell polarization, we determined whether the Na+-H+ exchanger is required for the growth of LLC-PK1 cells. The studies reported here show that there is no obligatory requirement for increased H+ efflux or Na+ entry via the Na+-H+ exchanger for the initiation of cell growth in this epithelial cell line. We used 22 Na+ influx, acid extrusion, and intracellular pH determinations to show that onset of cell growth, as measured by DNA content, precedes the activity of the Na+-H+ exchanger in exponentially growing cells, whereas confluent monolayers express Na+-H+ exchanger activity. When confluent cells are replated at low density, Na+-H+ exchanger activity disappears within 8 h in contrast to high-density replated cells. The fact that Na+-H+ exchanger activity is only present in confluent monolayers suggests that the development of tight junctions and polar differentiation play a role in the expression of the Na+-H+ exchanger and that this exchanger is more important to the polar epithelial cell for transepithelial transport than for the maintenance of intracellular pH

Light activation of the LOV protein vivid generates a rapidly exchanging dimer.

Science.gov (United States)

Zoltowski, Brian D; Crane, Brian R

2008-07-08

The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (light, oxygen, voltage) protein, couples light-induced cysteinyl adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal beta-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap-PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of N. crassa and indicate that LOV-LOV homo- or heterodimerization may be a mechanism for regulating light-activated gene expression.

How amide hydrogens exchange in native proteins.

Science.gov (United States)

Persson, Filip; Halle, Bertil

2015-08-18

Amide hydrogen exchange (HX) is widely used in protein biophysics even though our ignorance about the HX mechanism makes data interpretation imprecise. Notably, the open exchange-competent conformational state has not been identified. Based on analysis of an ultralong molecular dynamics trajectory of the protein BPTI, we propose that the open (O) states for amides that exchange by subglobal fluctuations are locally distorted conformations with two water molecules directly coordinated to the N-H group. The HX protection factors computed from the relative O-state populations agree well with experiment. The O states of different amides show little or no temporal correlation, even if adjacent residues unfold cooperatively. The mean residence time of the O state is ∼100 ps for all examined amides, so the large variation in measured HX rate must be attributed to the opening frequency. A few amides gain solvent access via tunnels or pores penetrated by water chains including native internal water molecules, but most amides access solvent by more local structural distortions. In either case, we argue that an overcoordinated N-H group is necessary for efficient proton transfer by Grotthuss-type structural diffusion.

How amide hydrogens exchange in native proteins

Science.gov (United States)

Persson, Filip; Halle, Bertil

2015-01-01

Amide hydrogen exchange (HX) is widely used in protein biophysics even though our ignorance about the HX mechanism makes data interpretation imprecise. Notably, the open exchange-competent conformational state has not been identified. Based on analysis of an ultralong molecular dynamics trajectory of the protein BPTI, we propose that the open (O) states for amides that exchange by subglobal fluctuations are locally distorted conformations with two water molecules directly coordinated to the N–H group. The HX protection factors computed from the relative O-state populations agree well with experiment. The O states of different amides show little or no temporal correlation, even if adjacent residues unfold cooperatively. The mean residence time of the O state is ∼100 ps for all examined amides, so the large variation in measured HX rate must be attributed to the opening frequency. A few amides gain solvent access via tunnels or pores penetrated by water chains including native internal water molecules, but most amides access solvent by more local structural distortions. In either case, we argue that an overcoordinated N–H group is necessary for efficient proton transfer by Grotthuss-type structural diffusion. PMID:26195754

Reversals and collisions optimize protein exchange in bacterial swarms

Energy Technology Data Exchange (ETDEWEB)

Amiri, Aboutaleb; Harvey, Cameron; Buchmann, Amy; Christley, Scott; Shrout, Joshua D.; Aranson, Igor S.; Alber, Mark

2017-03-01

Swarming groups of bacteria coordinate their behavior by self-organizing as a population to move over surfaces in search of nutrients and optimal niches for colonization. Many open questions remain about the cues used by swarming bacteria to achieve this self-organization. While chemical cue signaling known as quorum sensing is well-described, swarming bacteria often act and coordinate on time scales that could not be achieved via these extracellular quorum sensing cues. Here, cell-cell contact-dependent protein exchange is explored as amechanism of intercellular signaling for the bacterium Myxococcus xanthus. A detailed biologically calibrated computational model is used to study how M. xanthus optimizes the connection rate between cells and maximizes the spread of an extracellular protein within the population. The maximum rate of protein spreading is observed for cells that reverse direction optimally for swarming. Cells that reverse too slowly or too fast fail to spread extracellular protein efficiently. In particular, a specific range of cell reversal frequencies was observed to maximize the cell-cell connection rate and minimize the time of protein spreading. Furthermore, our findings suggest that predesigned motion reversal can be employed to enhance the collective behavior of biological synthetic active systems.

Ectopic expression of the erythrocyte band 3 anion exchange protein, using a new avian retrovirus vector

DEFF Research Database (Denmark)

Fuerstenberg, S; Beug, H; Introna, M

1990-01-01

into protein. Using the Escherichia coli beta-galactosidase gene cloned into the vector as a test construct, expression of enzyme activity could be detected in 90 to 95% of transfected target cells and in 80 to 85% of subsequently infected cells. In addition, a cDNA encoding the avian erythrocyte band 3 anion...... exchange protein has been expressed from the vector in both chicken embryo fibroblasts and QT6 cells and appears to function as an active, plasma membrane-based anion transporter. The ectopic expression of band 3 protein provides a visual marker for vector function in these cells....

Hydration shells exchange charge with their protein

DEFF Research Database (Denmark)

Abitan, Haim; Lindgård, Per-Anker; Nielsen, Bjørn Gilbert

2010-01-01

. In our experiments, the amplitude of an ultrasonic pressure wave is gradually increased (0–20 atm) while we simultaneously measure the Raman spectra from the hydrated protein (β-lactoglobulin and lysozyme). We detected two types of spectral changes: first, up to 70% increase in the intensity......Investigation of the interaction between a protein and its hydration shells is an experimental and theoretical challenge. Here, we used ultrasonic pressure waves in aqueous solutions of a protein to explore the conformational states of the protein and its interaction with its hydration shells...... the presence of an ultrasonic pressure, a protein and its hydration shells are in thermodynamic and charge equilibrium, i.e. a protein and its hydration shells exchange charges. The ultrasonic wave disrupts these equilibria which are regained within 30–45 min after the ultrasonic pressure is shut off....

Dynamics and energetics of the mammalian phosphatidylinositol transfer protein phospholipid exchange cycle.

Science.gov (United States)

Grabon, Aby; Orłowski, Adam; Tripathi, Ashutosh; Vuorio, Joni; Javanainen, Matti; Róg, Tomasz; Lönnfors, Max; McDermott, Mark I; Siebert, Garland; Somerharju, Pentti; Vattulainen, Ilpo; Bankaitis, Vytas A

2017-09-01

Phosphatidylinositol-transfer proteins (PITPs) regulate phosphoinositide signaling in eukaryotic cells. The defining feature of PITPs is their ability to exchange phosphatidylinositol (PtdIns) molecules between membranes, and this property is central to PITP-mediated regulation of lipid signaling. However, the details of the PITP-mediated lipid exchange cycle remain entirely obscure. Here, all-atom molecular dynamics simulations of the mammalian StART-like PtdIns/phosphatidylcholine (PtdCho) transfer protein PITPα, both on membrane bilayers and in solvated systems, informed downstream biochemical analyses that tested key aspects of the hypotheses generated by the molecular dynamics simulations. These studies provided five key insights into the PITPα lipid exchange cycle: (i) interaction of PITPα with the membrane is spontaneous and mediated by four specific protein substructures; (ii) the ability of PITPα to initiate closure around the PtdCho ligand is accompanied by loss of flexibility of two helix/loop regions, as well as of the C-terminal helix; (iii) the energy barrier of phospholipid extraction from the membrane is lowered by a network of hydrogen bonds between the lipid molecule and PITPα; (iv) the trajectory of PtdIns or PtdCho into and through the lipid-binding pocket is chaperoned by sets of PITPα residues conserved throughout the StART-like PITP family; and (v) conformational transitions in the C-terminal helix have specific functional involvements in PtdIns transfer activity. Taken together, these findings provide the first mechanistic description of key aspects of the PITPα PtdIns/PtdCho exchange cycle and offer a rationale for the high conservation of particular sets of residues across evolutionarily distant members of the metazoan StART-like PITP family. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Light activation of the LOV protein Vivid generates a rapidly exchanging dimer†‡

Science.gov (United States)

Zoltowski, Brian D.; Crane, Brian R.

2009-01-01

The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (Light, Oxygen, Voltage) protein, couples light-induced cysteinyl-adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal β-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap-to-PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of Neurospora crassa, and indicate that LOV:LOV homo or hetero dimerization may be a mechanism for regulating light-activated gene expression. PMID:18553928

Function of the Nucleotide Exchange Activity of Vav1 in T cell Development and Activation*

Science.gov (United States)

Saveliev, Alexander; Vanes, Lesley; Ksionda, Olga; Rapley, Jonathan; Smerdon, Stephen J.; Rittinger, Katrin; Tybulewicz, Victor L. J.

2012-01-01

The guanine nucleotide exchange factor (GEF) Vav1 is essential for transducing T cell antigen receptor (TCR) signals and therefore plays a critical role in the development and activation of T cells. It has been presumed that the GEF activity of Vav1 is important for its function; however, there has been no direct demonstration of this. Here, we generated mice expressing enzymatically inactive, but normally folded, Vav1 protein. Analysis of these mice showed that the GEF activity of Vav1 was necessary for the selection of thymocytes and for the optimal activation of T cells, including signal transduction to Rac1, Akt, and integrins. In contrast, the GEF activity of Vav1 was not required for TCR-induced calcium flux, activation of extracellular signal–regulated kinase (ERK) and protein kinase D1 (PKD1), and cell polarization. Thus, in T cells, the GEF activity of Vav1 is essential for some, but not all, of its functions. PMID:20009105

Function of the nucleotide exchange activity of vav1 in T cell development and activation.

Science.gov (United States)

Saveliev, Alexander; Vanes, Lesley; Ksionda, Olga; Rapley, Jonathan; Smerdon, Stephen J; Rittinger, Katrin; Tybulewicz, Victor L J

2009-12-15

The guanine nucleotide exchange factor (GEF) Vav1 is essential for transducing T cell antigen receptor (TCR) signals and therefore plays a critical role in the development and activation of T cells. It has been presumed that the GEF activity of Vav1 is important for its function; however, there has been no direct demonstration of this. Here, we generated mice expressing enzymatically inactive, but normally folded, Vav1 protein. Analysis of these mice showed that the GEF activity of Vav1 was necessary for the selection of thymocytes and for the optimal activation of T cells, including signal transduction to Rac1, Akt, and integrins. In contrast, the GEF activity of Vav1 was not required for TCR-induced calcium flux, activation of extracellular signal-regulated kinase and protein kinase D1, and cell polarization. Thus, in T cells, the GEF activity of Vav1 is essential for some, but not all, of its functions.

Dynamics and energetics of the mammalian phosphatidylinositol transfer protein phospholipid exchange cycle

DEFF Research Database (Denmark)

Grabon, Aby; Orłowski, Adam; Tripathi, Ashutosh

2017-01-01

. However, the details of the PITP-mediated lipid exchange cycle remain entirely obscure. Here, all-atom molecular dynamics simulations of the mammalian StART-like PtdIns/phosphatidylcholine (PtdCho) transfer protein PITPα, both on membrane bilayers and in solvated systems, informed downstream biochemical...... analyses that tested key aspects of the hypotheses generated by the molecular dynamics simulations. These studies provided five key insights into the PITPα lipid exchange cycle: (i) interaction of PITPα with the membrane is spontaneous and mediated by four specific protein substructures; (ii) the ability......Phosphatidylinositol-transfer proteins (PITPs) regulate phosphoinositide signaling in eukaryotic cells. The defining feature of PITPs is their ability to exchange phosphatidylinositol (PtdIns) molecules between membranes, and this property is central to PITP-mediated regulation of lipid signaling...

Release of proteins via ion exchange from albumin-heparin microspheres

NARCIS (Netherlands)

Kwon, Glen S.; Bae, You Han; Cremers, H.F.M.; Cremers, Harry; Feijen, Jan; Kim, Sung Wan

1992-01-01

Albumin-heparin and albumin microspheres were prepared as ion exchange gels for the controlled release of positively charged polypeptides and proteins. The adsorption isotherms of chicken egg and human lysozyme, as model proteins, on microspheres were obtained. An adsorption isotherm of chicken egg

Bovine plasma protein fractionation by ion exchange chromatography.

Science.gov (United States)

Moure, F; Rendueles, M; Díaz, M

2004-12-01

An ion exchange chromatography process was developed to separate the main protein fractions of bovine blood plasma using a composite material, Q-HyperD resin, and a gel material, DEAE-Sepharose. The experiments were carried out at semipreparative scale. It was necessary to establish analytical methods of electrophoresis and HPLC to identify the fractionated proteins. Results show that these materials are able to adequately fractionate different protein groups from the raw blood plasma. This method may be used to avoid chemical fractionation using agents such as ethanol or PEG and, thus, decrease protein denaturation of the different fractions to be used for research or pharmaceutical purposes. The Q-HyperD resin presents a better retention capacity for plasma protein than DEAE-Sepharose under the experimental conditions employed.

A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells

Science.gov (United States)

Stech, Marlitt; Quast, Robert B.; Sachse, Rita; Schulze, Corina; Wüstenhagen, Doreen A.; Kubick, Stefan

2014-01-01

In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds. PMID:24804975

Continuous desalting of refolded protein solution improves capturing in ion exchange chromatography: A seamless process.

Science.gov (United States)

Walch, Nicole; Jungbauer, Alois

2017-06-01

Truly continuous biomanufacturing processes enable an uninterrupted feed stream throughout the whole production without the need for holding tanks. We have utilized microporous anion and cation exchangers into which only salts, but not proteins, can penetrate into the pores for desalting of protein solutions, while diafiltration or dilution is usually employed for feed adjustments. Anion exchange and cation exchange chromatography columns were connected in series to remove both anions and cations. To increase operation performance, a continuous process was developed comprised of four columns. Continuous mode was achieved by staggered cycle operation, where one set of columns, consisting of one anion exchange and one cation exchange column, was loaded during the regeneration of the second set. Refolding, desalting and subsequent ion exchange capturing with a scFv as the model protein was demonstrated. The refolding solution was successfully desalted resulting in a consistent conductivity below 0.5 mS/cm from initial values of 10 to 11 mS/cm. With continuous operation process time could be reduced by 39% while productivity was increased to 163% compared to batch operation. Desalting of the protein solution resulted in up to 7-fold higher binding capacities in the subsequent ion exchange capture step with conventional protein binding resins. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Role of Detergents in Conformational Exchange of a G Protein-coupled Receptor*

Science.gov (United States)

Chung, Ka Young; Kim, Tae Hun; Manglik, Aashish; Alvares, Rohan; Kobilka, Brian K.; Prosser, R. Scott

2012-01-01

The G protein-coupled β2-adrenoreceptor (β2AR) signals through the heterotrimeric G proteins Gs and Gi and β-arrestin. As such, the energy landscape of β2AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β2AR with a trifluoromethyl probe, 19F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β2AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of β2AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor. PMID:22893704

Role of detergents in conformational exchange of a G protein-coupled receptor.

Science.gov (United States)

Chung, Ka Young; Kim, Tae Hun; Manglik, Aashish; Alvares, Rohan; Kobilka, Brian K; Prosser, R Scott

2012-10-19

The G protein-coupled β(2)-adrenoreceptor (β(2)AR) signals through the heterotrimeric G proteins G(s) and G(i) and β-arrestin. As such, the energy landscape of β(2)AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β(2)AR with a trifluoromethyl probe, (19)F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β(2)AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of β(2)AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor.

Non-equilibrium hydrogen exchange for determination of H-bond strength and water accessibility in solid proteins.

Science.gov (United States)

Grohe, Kristof; Movellan, Kumar Tekwani; Vasa, Suresh Kumar; Giller, Karin; Becker, Stefan; Linser, Rasmus

2017-05-01

We demonstrate measurement of non-equilibrium backbone amide hydrogen-deuterium exchange rates (HDX) for solid proteins. The target of this study are the slowly exchanging residues in solid samples, which are associated with stable secondary-structural elements of proteins. These hydrogen exchange processes escape methods measuring equilibrium exchange rates of faster processes. The method was applied to a micro-crystalline preparation of the SH3 domain of chicken α-spectrin. Therefore, from a 100% back-exchanged micro-crystalline protein preparation, the supernatant buffer was exchanged by a partially deuterated buffer to reach a final protonation level of approximately 20% before packing the sample in a 1.3 mm rotor. Tracking of the HN peak intensities for 2 weeks reports on site-specific hydrogen bond strength and also likely reflects water accessibility in a qualitative manner. H/D exchange can be directly determined for hydrogen-bonded amides using 1 H detection under fast magic angle spinning. This approach complements existing methods and provides the means to elucidate interesting site-specific characteristics for protein functionality in the solid state.

Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase

International Nuclear Information System (INIS)

Witters, L.A.; Bacon, G.W.

1985-01-01

The protein phosphatases in rat liver cytosol, active on rat liver acetyl-CoA carboxylase (ACC) phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase, have been partially purified by anion-exchange and gel filtration chromatography. The major phosphatase activities against all three substrates copurify through fractionation and appear to be identical to protein phosphatases 2A1 and 2A2. No unique protein phosphatase active on 32 P-ACC phosphorylated by the casein kinases was identified

Rapid and accurate processing method for amide proton exchange rate measurement in proteins

International Nuclear Information System (INIS)

Koskela, Harri; Heikkinen, Outi; Kilpelaeinen, Ilkka; Heikkinen, Sami

2007-01-01

Exchange between protein backbone amide hydrogen and water gives relevant information about solvent accessibility and protein secondary structure stability. NMR spectroscopy provides a convenient tool to study these dynamic processes with saturation transfer experiments. Processing of this type of NMR spectra has traditionally required peak integration followed by exponential fitting, which can be tedious with large data sets. We propose here a computer-aided method that applies inverse Laplace transform in the exchange rate measurement. With this approach, the determination of exchange rates can be automated, and reliable results can be acquired rapidly without a need for manual processing

Membrane Recruitment of the Non-receptor Protein GIV/Girdin (Gα-interacting, Vesicle-associated Protein/Girdin) Is Sufficient for Activating Heterotrimeric G Protein Signaling.

Science.gov (United States)

Parag-Sharma, Kshitij; Leyme, Anthony; DiGiacomo, Vincent; Marivin, Arthur; Broselid, Stefan; Garcia-Marcos, Mikel

2016-12-30

GIV (aka Girdin) is a guanine nucleotide exchange factor that activates heterotrimeric G protein signaling downstream of RTKs and integrins, thereby serving as a platform for signaling cascade cross-talk. GIV is recruited to the cytoplasmic tail of receptors upon stimulation, but the mechanism of activation of its G protein regulatory function is not well understood. Here we used assays in humanized yeast models and G protein activity biosensors in mammalian cells to investigate the role of GIV subcellular compartmentalization in regulating its ability to promote G protein signaling. We found that in unstimulated cells GIV does not co-fractionate with its substrate G protein Gα i3 on cell membranes and that constitutive membrane anchoring of GIV in yeast cells or rapid membrane translocation in mammalian cells via chemically induced dimerization leads to robust G protein activation. We show that membrane recruitment of the GIV "Gα binding and activating" motif alone is sufficient for G protein activation and that it does not require phosphomodification. Furthermore, we engineered a synthetic protein to show that recruitment of the GIV "Gα binding and activating" motif to membranes via association with active RTKs, instead of via chemically induced dimerization, is also sufficient for G protein activation. These results reveal that recruitment of GIV to membranes in close proximity to its substrate G protein is a major mechanism responsible for the activation of its G protein regulatory function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of poly(allylamine) as a polymeric ligand for ion-exchange protein chromatography.

Science.gov (United States)

Li, Ming; Li, Yanying; Yu, Linling; Sun, Yan

2017-02-24

This work reports poly(allylamine) (PAA), as a polymeric ion-exchange ligand for protein chromatography. Sepharose FF was modified with PAA, and six anion exchangers with ionic capacities (ICs) from 165 to 618mmol/L were prepared. Inverse size exclusion chromatography, adsorption equilibrium, uptake kinetics and column elution were performed. It was found that both the adsorption capacity and effective diffusivity maintained low values in the IC range of 165-373mmol/L, but they started to increase beyond 373mmol/L, and increased by 80% and 23 times, respectively, when the IC reached 618mmol/L. Interestingly, a drastic decrease of pore size was observed around the IC of 373mmol/L. The results suggest that the PAA chains played an important role in protein adsorption by altering the inner pore structure of the gels. It is considered that, PAA chains turn from inextensible states with multipoint-grafting on the pore surface at low coupling densities (IC373mmol/L). These characters of the grafted chains at higher IC values benefit in protein adsorption by three-dimensional binding and encouraged the happening of "chain delivery" of bound proteins on the chains. Besides, the ion exchangers showed favorable adsorption and uptake properties in a wide ionic strength range, 0-500mmol/L NaCl, indicating much better salt tolerance feature than the so-far reported ion exchangers. Moreover, a mild condition of pH 5.0 offered effective recovery of bound proteins in elution chromatography. The results indicate that the PAA-based anion exchanger of a high IC value is promising for high-capacity protein chromatography dealing with feedstock of a wide range of ionic strengths. Copyright © 2016 Elsevier B.V. All rights reserved.

Delipidation of the phosphatidylcholine exchange protein from beef liver by detergents

NARCIS (Netherlands)

Kamp, H.H.; Wirtz, K.W.A.; Deenen, L.L.M. van

1975-01-01

1. 1. A method is described to introduce [14C]phosphatidylcholine into the phosphatidylcholine exchange protein from beef liver. The effects of various detergents on this 14C-labelled phospholipid · protein complex are considered. 2. 2. As shown by spectrophotometric and radioactivity analysis of

Action of phospholipases on the phosphatidylcholine exchange protein from beef liver

NARCIS (Netherlands)

Kamp, H.H.; Sprengers, E.D.; Westerman, J.; Wirtz, K.W.A.; Deenen, L.L.M. van

1975-01-01

Abstract The phospholipases A2, C and D have been used to investigate the localization of phosphatidylcholine in the phosphatidylcholine exchange protein from beef liver. The rate of enzymatic hydrolysis of the protein-bound phosphatidylcholine was found to be very low. Addition of deoxycholate,

When Heterotrimeric G Proteins Are Not Activated by G Protein-Coupled Receptors: Structural Insights and Evolutionary Conservation.

Science.gov (United States)

DiGiacomo, Vincent; Marivin, Arthur; Garcia-Marcos, Mikel

2018-01-23

Heterotrimeric G proteins are signal-transducing switches conserved across eukaryotes. In humans, they work as critical mediators of intercellular communication in the context of virtually any physiological process. While G protein regulation by G protein-coupled receptors (GPCRs) is well-established and has received much attention, it has become recently evident that heterotrimeric G proteins can also be activated by cytoplasmic proteins. However, this alternative mechanism of G protein regulation remains far less studied than GPCR-mediated signaling. This Viewpoint focuses on recent advances in the characterization of a group of nonreceptor proteins that contain a sequence dubbed the "Gα-binding and -activating (GBA) motif". So far, four proteins present in mammals [GIV (also known as Girdin), DAPLE, CALNUC, and NUCB2] and one protein in Caenorhabditis elegans (GBAS-1) have been described as possessing a functional GBA motif. The GBA motif confers guanine nucleotide exchange factor activity on Gαi subunits in vitro and activates G protein signaling in cells. The importance of this mechanism of signal transduction is highlighted by the fact that its dysregulation underlies human diseases, such as cancer, which has made the proteins attractive new candidates for therapeutic intervention. Here we discuss recent discoveries on the structural basis of GBA-mediated activation of G proteins and its evolutionary conservation and compare them with the better-studied mechanism mediated by GPCRs.

Investigation of isolation conditions and ion-exchange purification of protein coagulation components from common bean seed

Directory of Open Access Journals (Sweden)

Antov Mirjana G.

2007-01-01

Full Text Available Investigation of an extraction procedure of protein coagulants from common bean seed regarding concentration of NaCl and pH was performed. High values of protein concentration and coagulation activity in crude extract (9.19 g/l and 23.9%, respectively were obtained when the extraction was performed using 0.5 mol/l NaCl and water as solvent, which represents an advantage for economic and environmental reasons. Crude extract of common bean seed was purified by precipitation at two different percentages of (NH42SO4 saturation, followed by batch ion-exchange chromatography. The highest obtained coagulation activity, 45%, was determined in fraction that was eluated at 1.75 mol/l NaCl from resin loaded with proteins precipitated upon 80-100% (NH42SO4 saturation. High values of coagulation activity showed by some eluates suggest their application as natural coagulant for water purification. .

Ion-Exchange Sample Displacement Chromatography as a Method for Fast and Simple Isolation of Low- and High-Abundance Proteins from Complex Biological Mixtures

Directory of Open Access Journals (Sweden)

Martina Srajer Gajdosik

2014-01-01

Full Text Available Sample displacement chromatography (SDC in reversed phase and ion-exchange modes was introduced at the end of 1980s. This chromatographic method was first used for preparative purification of synthetic peptides, and subsequently adapted for protein fractionation, mainly in anion-exchange mode. In the past few years, SDC has been successfully used for enrichment of low- and medium-abundance proteins from complex biological fluids on both monolithic and bulk chromatographic supports. If aqueous mobile phase is used with the application of mild chromatographic conditions, isolated proteins are not denatured and can also keep their biological activity. In this paper, the use of SDC in anion-exchange mode on a high-capacity chromatographic resin for separation of proteins from complex biological mixtures such as human plasma is demonstrated. By use of three and more columns coupled in series during sample application, and subsequent parallel elution of detached columns, additional separation of bound proteins was achieved. Highly enriched human serum albumin fraction and a number of physiologically active medium- and low-abundance proteins could be fractionated and detected by electrospray ionization tandem mass spectrometry (ESI-MS/MS and matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS. The use of the aforementioned columns that can be sanitized with 1 M sodium hydroxide for further application of SDC in biotechnology and food technology was discussed.

Probing protein interactions with hydrogen/deuterium exchange and mass spectrometry—A review

International Nuclear Information System (INIS)

Percy, Andrew J.; Rey, Martial; Burns, Kyle M.; Schriemer, David C.

2012-01-01

Highlights: ► Protein chemistry generates mass shifts useful for structure–function studies. ► H/DX supports a powerful mass shift method for protein interaction analysis. ► H/DX mass shifts are useful for determining binding data (K d , off-rates). ► Improved H/DX–MS workflows can accommodate complex protein systems. - Abstract: Assessing the functional outcome of protein interactions in structural terms is a goal of structural biology, however most techniques have a limited capacity for making structure–function determinations with both high resolution and high throughput. Mass spectrometry can be applied as a reader of protein chemistries in order to fill this void, and enable methodologies whereby protein structure–function determinations may be made on a proteome-wide level. Protein hydrogen/deuterium exchange (H/DX) offers a chemical labeling strategy suitable for tracking changes in “dynamic topography” and thus represents a powerful means of monitoring protein structure–function relationships. This review presents the exchange method in the context of interaction analysis. Applications involving interface detection, quantitation of binding, and conformational responses to ligation are discussed, and commentary on recent analytical developments is provided.

Structure of the cold-shock domain protein from Neisseria meningitidis reveals a strand-exchanged dimer

Energy Technology Data Exchange (ETDEWEB)

Ren, Jingshan [The Oxford Protein Production Facility, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Division of Structural Biology, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Nettleship, Joanne E.; Sainsbury, Sarah [The Oxford Protein Production Facility, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom); Saunders, Nigel J. [Bacterial Pathogenesis and Functional Genomics Group, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE (United Kingdom); Owens, Raymond J., E-mail: ray@strubi.ox.ac.uk [The Oxford Protein Production Facility, Henry Wellcome Building for Genomic Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (United Kingdom)

2008-04-01

The X-ray crystal structure of the cold-shock domain protein from N. meningitidis reveals a strand-exchanged dimer. The structure of the cold-shock domain protein from Neisseria meningitidis has been solved to 2.6 Å resolution and shown to comprise a dimer formed by the exchange of two β-strands between protein monomers. The overall fold of the monomer closely resembles those of other bacterial cold-shock proteins. The neisserial protein behaved as a monomer in solution and was shown to bind to a hexathymidine oligonucleotide with a stoichiometry of 1:1 and a K{sub d} of 1.25 µM.

Affinity capture of biotinylated proteins at acidic conditions to facilitate hydrogen/deuterium exchange mass spectrometry analysis of multimeric protein complexes

DEFF Research Database (Denmark)

Jensen, Pernille Foged; Jørgensen, Thomas J. D.; Koefoed, Klaus

2013-01-01

Characterization of conformational and dynamic changes associated with protein interactions can be done by hydrogen/deuterium exchange mass spectrometry (HDX-MS) by comparing the deuterium uptake in the bound and unbound state of the proteins. Investigation of local hydrogen/deuterium exchange...... in heteromultimeric protein complexes poses a challenge for the method due to the increased complexity of the mixture of peptides originating from all interaction partners in the complex. Previously, interference of peptides from one interaction partner has been removed by immobilizing the intact protein on beads...... complexes without interference of peptides originating from other interaction partners in the complex. The biotin-streptavidin strategy has been successfully implemented in a model system with two recombinant monoclonal antibodies that target nonoverlapping epitopes on the human epidermal growth factor...

Slow Histidine H/D Exchange Protocol for Thermodynamic Analysis of Protein Folding and Stability using Mass Spectrometry

OpenAIRE

Tran, Duc T.; Banerjee, Sambuddha; Alayash, Abdu I.; Crumbliss, Alvin L.; Fitzgerald, Michael C.

2012-01-01

Described here is a mass spectrometry based protocol to study the thermodynamic stability of proteins and protein-ligand complexes using the slow H/D exchange reaction of the imidazole C2 proton in histidine side chains. The protocol, which involves evaluating the denaturant dependence of this slow H/D exchange reaction in proteins, allows the global and/or subglobal unfolding/refolding properties of proteins and protein-ligand complexes to be probed. The protocol is developed using several m...

Collagen proteins exchange O with demineralisation and gelatinisation reagents and also with atmospheric moisture.

Science.gov (United States)

von Holstein, Isabella; von Tersch, Matthew; Coutu, Ashley N; Penkman, Kirsty E H; Makarewicz, Cheryl A; Collins, Matthew J

2018-01-23

The oxygen isotope composition of collagen proteins is a potential indicator of adult residential location, useful for provenancing in ecology, archaeology and forensics. In acidic solution, proteins can exchange O from carboxylic acid moieties with reagent O. This study investigated whether this exchange occurs during demineralisation and gelatinisation preparation of bone/ivory collagen. EDTA and HCl demineralisation or gelatinisation reagents were made up in waters with different δ 18 O values, and were used to extract collagen from four skeletal tissue samples. Aliquots of extracted collagen were exposed to two different atmospheric waters, at 120°C and ambient temperature, and subsequently dried in a vacuum oven at 40°C or by freeze drying. Sample δ 18 O values were measured by HT/EA pyrolysis-IRMS using a zero-blank autosampler. Collagen samples exchanged O with both reagent waters and atmospheric water, which altered sample δ 18 O values. Exchange with reagent waters occurred in all extraction methods, but was greater at lower pH. Damage to the collagen samples during extraction increased O exchange. The nature of exchange of O with atmospheric water depended on the temperature of exposure: kinetic fractionation of O was identified at 120°C but not at ambient temperature. Exchange was difficult to quantify due to high variability of δ 18 O value between experimental replicates. Studies of δ 18 O values in collagen proteins should avoid extraction methods using acid solutions. This article is protected by copyright. All rights reserved.

Quantification of protein backbone hydrogen-deuterium exchange rates by solid state NMR spectroscopy

International Nuclear Information System (INIS)

Lopez del Amo, Juan-Miguel; Fink, Uwe; Reif, Bernd

2010-01-01

We present the quantification of backbone amide hydrogen-deuterium exchange rates (HDX) for immobilized proteins. The experiments make use of the deuterium isotope effect on the amide nitrogen chemical shift, as well as on proton dilution by deuteration. We find that backbone amides in the microcrystalline α-spectrin SH3 domain exchange rather slowly with the solvent (with exchange rates negligible within the individual 15 N-T 1 timescales). We observed chemical exchange for 6 residues with HDX exchange rates in the range from 0.2 to 5 s -1 . Backbone amide 15 N longitudinal relaxation times that we determined previously are not significantly affected for most residues, yielding no systematic artifacts upon quantification of backbone dynamics (Chevelkov et al. 2008b). Significant exchange was observed for the backbone amides of R21, S36 and K60, as well as for the sidechain amides of N38, N35 and for W41ε. These residues could not be fit in our previous motional analysis, demonstrating that amide proton chemical exchange needs to be considered in the analysis of protein dynamics in the solid-state, in case D 2 O is employed as a solvent for sample preparation. Due to the intrinsically long 15 N relaxation times in the solid-state, the approach proposed here can expand the range of accessible HDX rates in the intermediate regime that is not accessible so far with exchange quench and MEXICO type experiments.

Incorporating water-release and lateral protein interactions in modeling equilibrium adsorption for ion-exchange chromatography.

Science.gov (United States)

Thrash, Marvin E; Pinto, Neville G

2006-09-08

The equilibrium adsorption of two albumin proteins on a commercial ion exchanger has been studied using a colloidal model. The model accounts for electrostatic and van der Waals forces between proteins and the ion exchanger surface, the energy of interaction between adsorbed proteins, and the contribution of entropy from water-release accompanying protein adsorption. Protein-surface interactions were calculated using methods previously reported in the literature. Lateral interactions between adsorbed proteins were experimentally measured with microcalorimetry. Water-release was estimated by applying the preferential interaction approach to chromatographic retention data. The adsorption of ovalbumin and bovine serum albumin on an anion exchanger at solution pH>pI of protein was measured. The experimental isotherms have been modeled from the linear region to saturation, and the influence of three modulating alkali chlorides on capacity has been evaluated. The heat of adsorption is endothermic for all cases studied, despite the fact that the net charge on the protein is opposite that of the adsorbing surface. Strong repulsive forces between adsorbed proteins underlie the endothermic heat of adsorption, and these forces intensify with protein loading. It was found that the driving force for adsorption is the entropy increase due to the release of water from the protein and adsorbent surfaces. It is shown that the colloidal model predicts protein adsorption capacity in both the linear and non-linear isotherm regions, and can account for the effects of modulating salt.

Phisiological and biochemical characteristics of protein and lipid exchanges of maple and chestnut seeds from different regions of Dnepropetrovsk city technogenic pollution

Directory of Open Access Journals (Sweden)

I. O. Filonik

2015-04-01

Full Text Available The indexes of protein and lipid exchanges - the content of proteins, lipase activity, level of lipids and their composition, component composition of free fatty acids in the maple and chestnut seeds from several sites of Dnepropetrovsk technical pollution were investigated. The revealed figures can be used as biomarkers of anthropogenic pollution in industrial region.

Side-chain dynamics of a detergent-solubilized membrane protein: Measurement of tryptophan and glutamine hydrogen-exchange rates in M13 coat protein by 1H NMR spectroscopy

International Nuclear Information System (INIS)

O'Neil, J.D.J.; Sykes, B.D.

1989-01-01

M13 coat protein is a small (50 amino acids) lipid-soluble protein that becomes an integral membrane protein during the infection stage of the life cycle of the M13 phage and is therefore used as a model membrane protein. To study side-chain dynamics in the protein, the authors have measured individual hydrogen-exchange rates for a primary amide in the side chain of glutamine-15 and for the indole amine of tryptophan-26. The protein was solubilized with the use of perdeuteriated sodium dodecyl sulfate (SDS), and hydrogen-exchange rates were measured by using 1 H nuclear magnetic resonance spectroscopy. The glutamine-15 syn proton exchanged at a rate identical with that in glutamine model peptides except that the pH corresponding to minimum exchange was elevated by about 1.5 pH units. The tryptophan-26 indole amine proton exchange was biphasic, suggesting that two populations of tryptophan-26 exist. It is suggested that the two populations may reflect protein dimerization or aggregation in the SDS micelles. The pH values of minimum exchange for tryptophan-26 in both environments were also elevated by 1.3-1.9 pH units. This phenomenon is reproduced when small tryptophan- and glutamine-containing hydrophobic peptides are dissolved in the presence of SDS micelles. The electrostatic nature of this phenomenon is proven by showing that the minimum pH for exchange can be reduced by dissolving the hydrophobic peptides in the positively charged detergent micelle dodecyltrimethylammonium bromide

Differential activation of G-proteins by μ-opioid receptor agonists

Science.gov (United States)

Saidak, Zuzana; Blake-Palmer, Katherine; Hay, Debbie L; Northup, John K; Glass, Michelle

2006-01-01

We investigated the ability of the activated μ-opioid receptor (MOR) to differentiate between myristoylated Gαi1 and GαoA type Gα proteins, and the maximal activity of a range of synthetic and endogenous agonists to activate each Gα protein. Membranes from HEK293 cells stably expressing transfected MOR were chaotrope extracted to denature endogenous G-proteins and reconstituted with specific purified G-proteins. The Gα subunits were generated in bacteria and were demonstrated to be recognised equivalently to bovine brain purified Gα protein by CB1 cannabinoid receptors. The ability of agonists to catalyse the MOR-dependent GDP/[35S]GTPγS exchange was then compared for Gαi1 and GαoA. Activation of MOR by DAMGO produced a high-affinity saturable interaction for GαoA (Km=20±1 nM) but a low-affinity interaction with Gαi1 (Km=116±12 nM). DAMGO, met-enkephalin and leucine-enkephalin displayed maximal Gα activation among the agonists evaluated. Endomorphins 1 and 2, methadone and β-endorphin activated both Gα to more than 75% of the maximal response, whereas fentanyl partially activated both G-proteins. Buprenorphine and morphine demonstrated a statistically significant difference between the maximal activities between Gαi1 and GαoA. Interestingly, DAMGO, morphine, endomorphins 1 and 2, displayed significant differences in the potencies for the activation of the two Gα. Differences in maximal activity and potency, for Gαi1 versus GαoA, are both indicative of agonist selective activation of G-proteins in response to MOR activation. These findings may provide a starting point for the design of drugs that demonstrate greater selectivity between these two G-proteins and therefore produce a more limited range of effects. PMID:16415903

Transbilayer distribution and mobility of phosphatidylcholine in intact erythrocyte membranes. A study with phosphatidylcholine exchange protein

NARCIS (Netherlands)

van Meer, G.|info:eu-repo/dai/nl/068570368; Poorthuis, B.J.H.M.; Wirtz, K.W.A.|info:eu-repo/dai/nl/068427956; op den Kamp, J.A.F.; van Deenen, L.L.M.

1980-01-01

The exchange of phosphatidylcholine between intact human or rat erythrocytes and rat liver microsomes was greatly stimulated by phosphatidylcholine-specific exchange proteins from rat liver and beef liver. It was found, however, that compared to the exchange reaction between phospholipid vesicles

Transbilayer distribution and mobility of phosphatidylcholine in intact erythrocyte membranes. A study with phosphatidylcholine exchange protein

NARCIS (Netherlands)

van Meer, G.; Poorthuis, B. J.; Wirtz, K. W.; Op den Kamp, J. A.; van Deenen, L. L.

1980-01-01

1. The exchange of phosphatidylcholine between intact human or rat erythrocytes and rat liver microsomes was greatly stimulated by phosphatidylcholine-specific exchange proteins from rat liver and beef liver. It was found, however, that compared to the exchange reaction between phospholipid vesicles

Measuring the hydrogen/deuterium exchange of proteins at high spatial resolution by mass spectrometry: overcoming gas-phase hydrogen/deuterium scrambling.

Science.gov (United States)

Rand, Kasper D; Zehl, Martin; Jørgensen, Thomas J D

2014-10-21

Proteins are dynamic molecules that exhibit conformational flexibility to function properly. Well-known examples of this are allosteric regulation of protein activity and ligand-induced conformational changes in protein receptors. Detailed knowledge of the conformational properties of proteins is therefore pertinent to both basic and applied research, including drug development, since the majority of drugs target protein receptors and a growing number of drugs introduced to the market are therapeutic peptides or proteins. X-ray crystallography provides a static picture at atomic resolution of the lowest-energy structure of the native ensemble. There is a growing need for sensitive analytical tools to explore all of the significant molecular structures in the conformational landscape of proteins. Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has recently emerged as a powerful method for characterizing protein conformational dynamics. The basis of this method is the fact that backbone amides in stable hydrogen-bonded structures (e.g., α-helices and β-sheets) are protected against exchange with the aqueous solvent. All protein structures are dynamic, however, and eventually all of the protecting hydrogen bonds will transiently break as the protein--according to thermodynamic principles--cycles through partially unfolded states that correspond to excited free energy levels. As a result, all of the backbone amides will eventually become temporarily solvent-exposed and exchange-competent over time. Consequently, a folded protein in D2O will gradually incorporate deuterium into its backbone amides, and the kinetics of the process can be readily monitored by mass spectrometry. The deuterium uptake kinetics for the intact protein (global exchange kinetics) represents the sum of the exchange kinetics for the individual backbone amides. Local exchange kinetics is typically achieved by using pepsin digestion under quench conditions (i.e., under cold

Contribution to the study of proteins and peptides structure by hydrogen isotopic exchange

International Nuclear Information System (INIS)

Nabedryk-Viala, Eliane.

1978-01-01

Development of hydrogen exchange measurement methods to study the structure and the molecular interaction of globular protein molecules in aqueous solution (ribonuclease A, cytochrome c, coupling factors of chloroplasts), in peptide hormones in trifluoroethanol solution (angiotensin II, corticotropin) and in proteins of membranes (rhodopsin) [fr

Improved protein hydrogen/deuterium exchange mass spectrometry platform with fully automated data processing.

Science.gov (United States)

Zhang, Zhongqi; Zhang, Aming; Xiao, Gang

2012-06-05

Protein hydrogen/deuterium exchange (HDX) followed by protease digestion and mass spectrometric (MS) analysis is accepted as a standard method for studying protein conformation and conformational dynamics. In this article, an improved HDX MS platform with fully automated data processing is described. The platform significantly reduces systematic and random errors in the measurement by introducing two types of corrections in HDX data analysis. First, a mixture of short peptides with fast HDX rates is introduced as internal standards to adjust the variations in the extent of back exchange from run to run. Second, a designed unique peptide (PPPI) with slow intrinsic HDX rate is employed as another internal standard to reflect the possible differences in protein intrinsic HDX rates when protein conformations at different solution conditions are compared. HDX data processing is achieved with a comprehensive HDX model to simulate the deuterium labeling and back exchange process. The HDX model is implemented into the in-house developed software MassAnalyzer and enables fully unattended analysis of the entire protein HDX MS data set starting from ion detection and peptide identification to final processed HDX output, typically within 1 day. The final output of the automated data processing is a set (or the average) of the most possible protection factors for each backbone amide hydrogen. The utility of the HDX MS platform is demonstrated by exploring the conformational transition of a monoclonal antibody by increasing concentrations of guanidine.

Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations.

Science.gov (United States)

Kisley, Lydia; Chen, Jixin; Mansur, Andrea P; Shuang, Bo; Kourentzi, Katerina; Poongavanam, Mohan-Vivekanandan; Chen, Wen-Hsiang; Dhamane, Sagar; Willson, Richard C; Landes, Christy F

2014-02-11

Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally.

Lipid Exchange Mechanism of the Cholesteryl Ester Transfer Protein Clarified by Atomistic and Coarse-grained Simulations

DEFF Research Database (Denmark)

Koivuniemi, A.; Vuorela, T.; Kovanen, P. T.

2012-01-01

molecular dynamics simulations to unravel the mechanisms associated with the CETP-mediated lipid exchange. To this end we used both atomistic and coarse-grained models whose results were consistent with each other. We found CETP to bind to the surface of high density lipoprotein (HDL) -like lipid droplets......Cholesteryl ester transfer protein (CETP) transports cholesteryl esters, triglycerides, and phospholipids between different lipoprotein fractions in blood plasma. The inhibition of CETP has been shown to be a sound strategy to prevent and treat the development of coronary heart disease. We employed...... evidence that helix X acts as a lid which conducts lipid exchange by alternating the open and closed states. The findings have potential for the design of novel molecular agents to inhibit the activity of CETP....

The rapid and direct determination of ATPase activity by ion exchange chromatography and the application to the activity of heat shock protein-90.

Science.gov (United States)

Bartolini, Manuela; Wainer, Irving W; Bertucci, Carlo; Andrisano, Vincenza

2013-01-25

Adenosine nucleotides are involved as substrates or co-factors in several biochemical reactions, catalyzed by enzymes, which modulate energy production, signal transduction and cell proliferation. We here report the development and optimization of an ion exchange liquid chromatography (LC) method for the determination of ATP, ADP and AMP. This method is specifically aimed at the determination of the ATP-ase activity of human heat shock protein 90 (Hsp90), a molecular chaperone that has emerged as target enzyme in cancer therapy. Separation of the three nucleotides was achieved in a 15-min run by using a disk shaped monolithic ethylene diamine stationary phase of small dimensions (2mm×6mm i.d.), under a three-solvent gradient elution mode and UV detection at 256nm. The described direct LC method resulted highly specific as a consequence of the baseline separation of the three adenosine nucleotides and could be applied to the determination of the enzymatic activity of ADP/ATP generating or consuming enzymes (such as kinases). Furthermore, comparison of the LOD and LOQ values of the LC method with those obtained with the malachite green assay, which is one of the most used indirect screening methodologies for ATP-ase activity, showed that the LC method has a similar range of application without presenting the drawbacks related to contamination by inorganic phosphate ions and glycerol, which are present in Hsp90 commercial samples. Copyright © 2012 Elsevier B.V. All rights reserved.

Backbone dynamics of a model membrane protein: measurement of individual amide hydrogen-exchange rates in detergent-solubilized M13 coat protein using 13C NMR hydrogen/deuterium isotope shifts

International Nuclear Information System (INIS)

Henry, G.D.; Weiner, J.H.; Sykes, B.D.

1987-01-01

Hydrogen-exchange rates have been measured for individual assigned amide protons in M13 coat protein, a 50-residue integral membrane protein, using a 13 C nuclear magnetic resonance (NMR) equilibrium isotope shift technique. The locations of the more rapidly exchanging amides have been determined. In D 2 O solutions, a peptide carbonyl resonance undergoes a small upfield isotope shift (0.08-0.09 ppm) from its position in H 2 O solutions; in 1:1 H 2 O/D 2 O mixtures, the carbonyl line shape is determined by the exchange rate at the adjacent nitrogen atom. M13 coat protein was labeled biosynthetically with 13 C at the peptide carbonyls of alanine, glycine, phenylalanine, proline, and lysine, and the exchange rates of 12 assigned amide protons in the hydrophilic regions were measured as a function of pH by using the isotope shift method. This equilibrium technique is sensitive to the more rapidly exchanging protons which are difficult to measure by classical exchange-out experiments. In proteins, structural factors, notably H bonding, can decrease the exchange rate of an amide proton by many orders of magnitude from that observed in the freely exposed amides of model peptides such as poly(DL-alanine). With corrections for sequence-related inductive effects, the retardation of amide exchange in sodium dodecyl sulfate solubilized coat protein has been calculated with respect to poly(DL-alanine). The most rapidly exchanging protons, which are retarded very little or not at all, are shown to occur at the N- and C-termini of the molecule. A model of the detergent-solubilized coat protein is constructed from these H-exchange data which is consistent with circular dichroism and other NMR results

The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf

Directory of Open Access Journals (Sweden)

Geldner Niko

2005-02-01

Full Text Available Abstract Background Small G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs that stimulate the exchange of the tightly bound GDP nucleotide by GTP. The catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf subfamily, which are major regulators of membrane trafficking, GEFs form a heterogeneous family whose only common characteristic is the well-characterized Sec7 catalytic domain. In contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown. Results Based on Sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from Paramecium, we have investigated the domain architecture of large ArfGEFs of the BIG and GBF subfamilies, which are involved in Golgi traffic. Multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. We also report a novel ArfGEF subfamily with a domain organization so far unique to alveolates, which we name TBS (TBC-Sec7. Conclusion Our analysis unifies the BIG and GBF subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific ArfGEFs have subsequently evolved. Our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains.

Divided-evolution-based pulse scheme for quantifying exchange processes in proteins: powerful complement to relaxation dispersion experiments.

Science.gov (United States)

Bouvignies, Guillaume; Hansen, D Flemming; Vallurupalli, Pramodh; Kay, Lewis E

2011-02-16

A method for quantifying millisecond time scale exchange in proteins is presented based on scaling the rate of chemical exchange using a 2D (15)N, (1)H(N) experiment in which (15)N dwell times are separated by short spin-echo pulse trains. Unlike the popular Carr-Purcell-Meiboom-Gill (CPMG) experiment where the effects of a radio frequency field on measured transverse relaxation rates are quantified, the new approach measures peak positions in spectra that shift as the effective exchange time regime is varied. The utility of the method is established through an analysis of data recorded on an exchanging protein-ligand system for which the exchange parameters have been accurately determined using alternative approaches. Computations establish that a combined analysis of CPMG and peak shift profiles extends the time scale that can be studied to include exchanging systems with highly skewed populations and exchange rates as slow as 20 s(-1).

Isolation and characterization of biologically active venom protein from sea snake Enhydrina schistosa.

Science.gov (United States)

Damotharan, Palani; Veeruraj, Anguchamy; Arumugam, Muthuvel; Balasubramanian, Thangavel

2015-03-01

The present study is designed to investigate the isolation and characterization of biological and biochemical active venom protein from sea snake, Enhydrina schistosa. The highest purification peaks in ion-exchange chromatography on DEAE-cellulose column were obtained for fraction numbers 39-49 when eluted with 0.35-0.45 M NaCl. Eighty per cent purity was obtained in the final stage of purification, and a single protein band of about 44 kDa was visualized in SDS-polyacrylamide gel under reducing condition. Purified venom protein expressed as haemolytic, cytotoxicity and proteolytic activities with lethal concentration (LC50 ) at 2.0 μg/mL. Venom protein exhibits enzymatic activity and hydrolyzed casein and gelatin. Gelatinolytic activity was optimal at pH 5-9. In conclusion, the present results suggested that the sea snake venom might be feasible sources for biologically active substances. Thus, this low molecular weight component of the venom protein could be used in potentially serve biological and pharmaceutical aspects. © 2014 Wiley Periodicals, Inc.

Noncoded amino acids in protein engineering: Structure-activity relationship studies of hirudin-thrombin interaction.

Science.gov (United States)

De Filippis, Vincenzo; Acquasaliente, Laura; Pontarollo, Giulia; Peterle, Daniele

2018-01-01

The advent of recombinant DNA technology allowed to site-specifically insert, delete, or mutate almost any amino acid in a given protein, significantly improving our knowledge of protein structure, stability, and function. Nevertheless, a quantitative description of the physical and chemical basis that makes a polypeptide chain to efficiently fold into a stable and functionally active conformation is still elusive. This mainly originates from the fact that nature combined, in a yet unknown manner, different properties (i.e., hydrophobicity, conformational propensity, polarizability, and hydrogen bonding capability) into the 20 standard natural amino acids, thus making difficult, if not impossible, to univocally relate the change in protein stability or function to the alteration of physicochemical properties caused by amino acid exchange(s). In this view, incorporation of noncoded amino acids with tailored side chains, allowing to finely tune the structure at a protein site, would facilitate to dissect the effects of a given mutation in terms of one or a few physicochemical properties, thus much expanding the scope of physical organic chemistry in the study of proteins. In this review, relevant applications from our laboratory will be presented on the use of noncoded amino acids in structure-activity relationships studies of hirudin binding to thrombin. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

NMR Analysis of Amide Hydrogen Exchange Rates in a Pentapeptide-Repeat Protein from A. thaliana.

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Xu, Shenyuan; Ni, Shuisong; Kennedy, Michael A

2017-05-23

At2g44920 from Arabidopsis thaliana is a pentapeptide-repeat protein (PRP) composed of 25 repeats capped by N- and C-terminal α-helices. PRP structures are dominated by four-sided right-handed β-helices typically consisting of mixtures of type II and type IV β-turns. PRPs adopt repeated five-residue (Rfr) folds with an Rfr consensus sequence (STAV)(D/N)(L/F)(S/T/R)(X). Unlike other PRPs, At2g44920 consists exclusively of type II β-turns. At2g44920 is predicted to be located in the thylakoid lumen although its biochemical function remains unknown. Given its unusual structure, we investigated the biophysical properties of At2g44920 as a representative of the β-helix family to determine if it had exceptional global stability, backbone dynamics, or amide hydrogen exchange rates. Circular dichroism measurements yielded a melting point of 62.8°C, indicating unexceptional global thermal stability. Nuclear spin relaxation measurements indicated that the Rfr-fold core was rigid with order parameters ranging from 0.7 to 0.9. At2g44920 exhibited a striking range of amide hydrogen exchange rates spanning 10 orders of magnitude, with lifetimes ranging from minutes to several months. A weak correlation was found among hydrogen exchange rates, hydrogen bonding energies, and amino acid solvent-accessible areas. Analysis of contributions from fast (approximately picosecond to nanosecond) backbone dynamics to amide hydrogen exchange rates revealed that the average order parameter of amides undergoing fast exchange was significantly smaller compared to those undergoing slow exchange. Importantly, the activation energies for amide hydrogen exchange were found to be generally higher for the slowest exchanging amides in the central Rfr coil and decreased toward the terminal coils. This could be explained by assuming that the concerted motions of two preceding or following coils required for hydrogen bond disruption and amide hydrogen exchange have a higher activation energy

Detergent activation of the binding protein in the folate radioassay

International Nuclear Information System (INIS)

Hansen, S.I.; Holm, J.; Lyngbye, J.

1982-01-01

A minor cow's whey protein associated with β-lactoglobulin is used as binding protein in the competitive radioassay for serum and erythrocyte folate. Seeking to optimize the assay, we tested the performance of binder solutions of increasing purity. The folate binding protein was isolated from cow's whey by means of CM-Sepharose CL-6B cation-exchange chromatography, and further purified on a methotrexate-AH-Sepharose 4B affinity matrix. In contrast to β-lactoglobulin, the purified protein did not bind folate unless the detergents cetyltrimethylammonium (10 mmol/Ll) or Triton X-100 (1 g/L) were present. Such detergent activation was not needed in the presence of serum. There seems to be a striking analogy between these phenomena and the well-known reactivation of certain purified membrane-derived enzymes by surfactants

Role of Subunit Exchange and Electrostatic Interactions on the Chaperone Activity of Mycobacterium leprae HSP18

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Nandi, Sandip Kumar; Panda, Alok Kumar; Chakraborty, Ayon; Ray, Sougata Sinha; Biswas, Ashis

2015-01-01

Mycobacterium leprae HSP18, a major immunodominant antigen of M. leprae pathogen, is a small heat shock protein. Previously, we reported that HSP18 is a molecular chaperone that prevents aggregation of different chemically and thermally stressed client proteins and assists refolding of denatured enzyme at normal temperature. We also demonstrated that it can efficiently prevent the thermal killing of E. coli at higher temperature. However, molecular mechanism behind the chaperone function of HSP18 is still unclear. Therefore, we studied the structure and chaperone function of HSP18 at normal temperature (25°C) as well as at higher temperatures (31–43°C). Our study revealed that the chaperone function of HSP18 is enhanced significantly with increasing temperature. Far- and near-UV CD experiments suggested that its secondary and tertiary structure remain intact in this temperature range (25–43°C). Besides, temperature has no effect on the static oligomeric size of this protein. Subunit exchange study demonstrated that subunits of HSP18 exchange at 25°C with a rate constant of 0.018 min-1. Both rate of subunit exchange and chaperone activity of HSP18 is found to increase with rise in temperature. However, the surface hydrophobicity of HSP18 decreases markedly upon heating and has no correlation with its chaperone function in this temperature range. Furthermore, we observed that HSP18 exhibits diminished chaperone function in the presence of NaCl at 25°C. At elevated temperatures, weakening of interactions between HSP18 and stressed client proteins in the presence of NaCl results in greater reduction of its chaperone function. The oligomeric size, rate of subunit exchange and structural stability of HSP18 were also found to decrease when electrostatic interactions were weakened. These results clearly indicated that subunit exchange and electrostatic interactions play a major role in the chaperone function of HSP18. PMID:26098662

Role of Subunit Exchange and Electrostatic Interactions on the Chaperone Activity of Mycobacterium leprae HSP18.

Science.gov (United States)

Nandi, Sandip Kumar; Panda, Alok Kumar; Chakraborty, Ayon; Sinha Ray, Sougata; Biswas, Ashis

2015-01-01

Mycobacterium leprae HSP18, a major immunodominant antigen of M. leprae pathogen, is a small heat shock protein. Previously, we reported that HSP18 is a molecular chaperone that prevents aggregation of different chemically and thermally stressed client proteins and assists refolding of denatured enzyme at normal temperature. We also demonstrated that it can efficiently prevent the thermal killing of E. coli at higher temperature. However, molecular mechanism behind the chaperone function of HSP18 is still unclear. Therefore, we studied the structure and chaperone function of HSP18 at normal temperature (25°C) as well as at higher temperatures (31-43°C). Our study revealed that the chaperone function of HSP18 is enhanced significantly with increasing temperature. Far- and near-UV CD experiments suggested that its secondary and tertiary structure remain intact in this temperature range (25-43°C). Besides, temperature has no effect on the static oligomeric size of this protein. Subunit exchange study demonstrated that subunits of HSP18 exchange at 25°C with a rate constant of 0.018 min(-1). Both rate of subunit exchange and chaperone activity of HSP18 is found to increase with rise in temperature. However, the surface hydrophobicity of HSP18 decreases markedly upon heating and has no correlation with its chaperone function in this temperature range. Furthermore, we observed that HSP18 exhibits diminished chaperone function in the presence of NaCl at 25°C. At elevated temperatures, weakening of interactions between HSP18 and stressed client proteins in the presence of NaCl results in greater reduction of its chaperone function. The oligomeric size, rate of subunit exchange and structural stability of HSP18 were also found to decrease when electrostatic interactions were weakened. These results clearly indicated that subunit exchange and electrostatic interactions play a major role in the chaperone function of HSP18.

Activation of exchange protein activated by cAMP in the rat basolateral amygdala impairs reconsolidation of a memory associated with self-administered cocaine.

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Wan, Xun; Torregrossa, Mary M; Sanchez, Hayde; Nairn, Angus C; Taylor, Jane R

2014-01-01

The intracellular mechanisms underlying memory reconsolidation critically involve cAMP signaling. These events were originally attributed to PKA activation by cAMP, but the identification of Exchange Protein Activated by cAMP (Epac), as a distinct mediator of cAMP signaling, suggests that cAMP-regulated processes that subserve memory reconsolidation are more complex. Here we investigated how activation of Epac with 8-pCPT-cAMP (8-CPT) impacts reconsolidation of a memory that had been associated with cocaine self-administration. Rats were trained to lever press for cocaine on an FR-1 schedule, in which each cocaine delivery was paired with a tone+light cue. Lever pressing was then extinguished in the absence of cue presentations and cocaine delivery. Following the last day of extinction, rats were put in a novel context, in which the conditioned cue was presented to reactivate the cocaine-associated memory. Immediate bilateral infusions of 8-CPT into the basolateral amygdala (BLA) following reactivation disrupted subsequent cue-induced reinstatement in a dose-dependent manner, and modestly reduced responding for conditioned reinforcement. When 8-CPT infusions were delayed for 3 hours after the cue reactivation session or were given after a cue extinction session, no effect on cue-induced reinstatement was observed. Co-administration of 8-CPT and the PKA activator 6-Bnz-cAMP (10 nmol/side) rescued memory reconsolidation while 6-Bnz alone had no effect, suggesting an antagonizing interaction between the two cAMP signaling substrates. Taken together, these studies suggest that activation of Epac represents a parallel cAMP-dependent pathway that can inhibit reconsolidation of cocaine-cue memories and reduce the ability of the cue to produce reinstatement of cocaine-seeking behavior.

Activation of exchange protein activated by cAMP in the rat basolateral amygdala impairs reconsolidation of a memory associated with self-administered cocaine.

Directory of Open Access Journals (Sweden)

Xun Wan

Full Text Available The intracellular mechanisms underlying memory reconsolidation critically involve cAMP signaling. These events were originally attributed to PKA activation by cAMP, but the identification of Exchange Protein Activated by cAMP (Epac, as a distinct mediator of cAMP signaling, suggests that cAMP-regulated processes that subserve memory reconsolidation are more complex. Here we investigated how activation of Epac with 8-pCPT-cAMP (8-CPT impacts reconsolidation of a memory that had been associated with cocaine self-administration. Rats were trained to lever press for cocaine on an FR-1 schedule, in which each cocaine delivery was paired with a tone+light cue. Lever pressing was then extinguished in the absence of cue presentations and cocaine delivery. Following the last day of extinction, rats were put in a novel context, in which the conditioned cue was presented to reactivate the cocaine-associated memory. Immediate bilateral infusions of 8-CPT into the basolateral amygdala (BLA following reactivation disrupted subsequent cue-induced reinstatement in a dose-dependent manner, and modestly reduced responding for conditioned reinforcement. When 8-CPT infusions were delayed for 3 hours after the cue reactivation session or were given after a cue extinction session, no effect on cue-induced reinstatement was observed. Co-administration of 8-CPT and the PKA activator 6-Bnz-cAMP (10 nmol/side rescued memory reconsolidation while 6-Bnz alone had no effect, suggesting an antagonizing interaction between the two cAMP signaling substrates. Taken together, these studies suggest that activation of Epac represents a parallel cAMP-dependent pathway that can inhibit reconsolidation of cocaine-cue memories and reduce the ability of the cue to produce reinstatement of cocaine-seeking behavior.

Hydrogen exchange

DEFF Research Database (Denmark)

Jensen, Pernille Foged; Rand, Kasper Dyrberg

2016-01-01

Hydrogen exchange (HX) monitored by mass spectrometry (MS) is a powerful analytical method for investigation of protein conformation and dynamics. HX-MS monitors isotopic exchange of hydrogen in protein backbone amides and thus serves as a sensitive method for probing protein conformation...... and dynamics along the entire protein backbone. This chapter describes the exchange of backbone amide hydrogen which is highly quenchable as it is strongly dependent on the pH and temperature. The HX rates of backbone amide hydrogen are sensitive and very useful probes of protein conformation......, as they are distributed along the polypeptide backbone and form the fundamental hydrogen-bonding networks of basic secondary structure. The effect of pressure on HX in unstructured polypeptides (poly-dl-lysine and oxidatively unfolded ribonuclease A) and native folded proteins (lysozyme and ribonuclease A) was evaluated...

Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament.

KAUST Repository

Fornander, Louise H

2013-12-03

The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction.

Time-resolved pulsed hydrogen/deuterium exchange mass spectrometry probes gaseous proteins structural kinetics.

Science.gov (United States)

Rajabi, Khadijeh

2015-01-01

A pulsed hydrogen/deuterium exchange (HDX) method has been developed for rapid monitoring of the exchange kinetics of protein ions with D2O a few milliseconds after electrospray ionization (ESI). The stepwise gradual evolution of HDX of multiply charged protein ions was monitored using the pulsed HDX mass spectrometry technique. Upon introducing a very short pulse of D2O (in the μs to ms time scale) into the linear ion trap (LIT) of a time-of-flight (TOF) mass spectrometer, bimodal distributions were detected for the ions of cytochrome c and ubiquitin. Mechanistic details of HDX reactions for ubiquitin and cytochrome c in the gas phase were uncovered and the structural transitions were followed by analyzing the kinetics of HDX.

Scambio, a novel guanine nucleotide exchange factor for Rho

Directory of Open Access Journals (Sweden)

Groffen John

2004-04-01

Full Text Available Abstract Background Small GTPases of the Rho family are critical regulators of various cellular functions including actin cytoskeleton organization, activation of kinase cascades and mitogenesis. For this reason, a major objective has been to understand the mechanisms of Rho GTPase regulation. Here, we examine the function of a novel protein, Scambio, which shares homology with the DH-PH domains of several known guanine nucleotide exchange factors for Rho family members. Results Scambio is located on human chromosome 14q11.1, encodes a protein of around 181 kDa, and is highly expressed in both heart and skeletal muscle. In contrast to most DH-PH-domain containing proteins, it binds the activated, GTP-bound forms of Rac and Cdc42. However, it fails to associate with V14RhoA. Immunofluorescence studies indicate that Scambio and activated Rac3 colocalize in membrane ruffles at the cell periphery. In accordance with these findings, Scambio does not activate either Rac or Cdc42 but rather, stimulates guanine nucleotide exchange on RhoA and its close relative, RhoC. Conclusion Scambio associates with Rac in its activated conformation and functions as a guanine nucleotide exchange factor for Rho.

The relationship between Na+/H+ exchanger expression and tyrosinase activity in human melanocytes

International Nuclear Information System (INIS)

Smith, Dustin R.; Spaulding, Deborah T.; Glenn, Hayden M.; Fuller, Bryan B.

2004-01-01

The activity of melanosome-associated tyrosinase in human melanocytes differs based on racial skin type. In melanocytes from Black skin, tyrosinase activity is high while in White melanocytes the activity of the enzyme is low. Recent studies suggest that low tyrosinase activity in White melanocytes may be due to an acidic pH environment within the melanosome. Because sodium/hydrogen (Na + /H + ) exchangers (NHEs) are known to regulate intracellular pH, melanocytes were treated with NHE inhibitors to determine what effect this inhibition might have on tyrosinase activity. Treatment of Black melanocytes with ethyl-isopropyl amiloride (EIPA) caused a rapid dose-dependent inhibition of tyrosinase activity. This inhibition was not due to either direct enzyme inhibition or to a decrease in tyrosinase abundance. In contrast, treatment of White melanocytes with EIPA, cimetidine, or clonidine resulted in little inhibition of tyrosinase activity. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis showed that both Black and White melanocytes expressed mRNA and protein for NHE-1, NHE-3, NHE-5, NHE-6, and NHE-7. Immunohistochemical analysis showed that NHE-7 and NHE-3 co-localized with the melanosomal protein, Tyrosinase Related Protein-1 (TRP-1). In addition, the vesicular proton pump, vesicular ATPase (V-ATPase), was found to be present in both White and Black melanosomes, indicating that organelles from both racial skin types are capable of being acidified. The results suggest that one or more NHEs may help regulate melanosome pH and tyrosinase activity in human melanocytes

Identification of individual protein-ligand NOEs in the limit of intermediate exchange

International Nuclear Information System (INIS)

Reibarkh, Mikhail; Malia, Thomas J.; Hopkins, Brian T.; Wagner, Gerhard

2006-01-01

Interactions of proteins with small molecules or other macromolecules play key roles in many biological processes and in drug action, and NMR is an excellent tool for their structural characterization. Frequently, however, line broadening due to intermediate exchange completely eliminates the signals needed for measuring specific intermolecular NOEs. This limits the use of NMR for detailed structural studies in such kinetic situations. Here we show that an optimally chosen excess of ligand over protein can reduce the extent of line broadening for both the ligand and the protein. This makes observation of ligand resonances possible but reduces the size of the measurable NOEs due to the residual line broadening and the non-stoichiometric concentrations. Because the solubility of small molecule drug leads are often limited to high micromolar concentrations, protein concentrations are restricted to even lower values in the low micromolar range. At these non-stoichiometric concentrations and in the presence of significant residual line broadening, conventional NOESY experiments very often are not sensitive enough to observe intermolecular NOEs since the signals inverted by the NOESY preparation pulse sequence relax prior to significant NOE build up. Thus, we employ methods related to driven NOE spectroscopy to investigate protein-ligand interactions in the intermediate exchange regime. In this approach, individual protein resonances are selectively irradiated for up to five seconds to build up measurable NOEs at the ligand resonances. To enable saturation of individual protein resonances we prepare deuterated protein samples selectively protonated at a few sites so that the 1D 1 H spectrum of the protein is resolved well enough to permit irradiation of individual protein signals, which do not overlap with the ligand spectrum. This approach is suitable for measuring a sufficiently large number of protein-ligand NOEs that allow calculation of initial complex structures

Evidence that Na+/H+ exchanger 1 is an ATP-binding protein.

Science.gov (United States)

Shimada-Shimizu, Naoko; Hisamitsu, Takashi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

2013-03-01

Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1. © 2013 The Authors Journal compilation © 2013 FEBS.

Transcriptional activation of peroxisome proliferator-activated receptor-γ requires activation of both protein kinase A and Akt during adipocyte differentiation

International Nuclear Information System (INIS)

Kim, Sang-pil; Ha, Jung Min; Yun, Sung Ji; Kim, Eun Kyoung; Chung, Sung Woon; Hong, Ki Whan; Kim, Chi Dae; Bae, Sun Sik

2010-01-01

Research highlights: → Elevated cAMP activates both PKA and Epac. → PKA activates CREB transcriptional factor and Epac activates PI3K/Akt pathway via Rap1. → Akt modulates PPAR-γ transcriptional activity in concert with CREB. -- Abstract: Peroxisome proliferator-activated receptor-γ (PPAR-γ) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-γ is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-γ. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-γ was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-γ transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-γ transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-γ, suggesting post-translational activation of PPAR-γ might be critical step for adipogenic gene expression.

Evidence of chemical exchange in recombinant Major Urinary Protein and quenching thereof upon pheromone binding

Energy Technology Data Exchange (ETDEWEB)

Perazzolo, Chiara, E-mail: Chiara.Perazzolo@epfl.ch; Verde, Mariachiara [Ecole Polytechnique Federale de Lausanne, Institut des Sciences et Ingenierie Chimiques (Switzerland); Homans, Steve W. [University of Leeds, Institute of Molecular and Cellular Biology (United Kingdom); Bodenhausen, Geoffrey [Ecole Polytechnique Federale de Lausanne, Institut des Sciences et Ingenierie Chimiques (Switzerland)

2007-05-15

The internal dynamics of recombinant Major Urinary Protein (rMUP) have been investigated by monitoring transverse nitrogen-15 relaxation using multiple-echo Carr-Purcell-Meiboom-Gill (CPMG) experiments. While the ligand-free protein (APO-rMUP) features extensive evidence of motions on the milliseconds time scale, the complex with 2-methoxy-3-isobutylpyrazine (HOLO-rMUP) appears to be much less mobile on this time scale. At 308 K, exchange rates k{sub ex} = 500-2000 s{sup -1} were typically observed in APO-rMUP for residues located adjacent to a {beta}-turn comprising residues 83-87. These residues occlude an entry to the binding pocket and have been proposed to be a portal for ligand entry in other members of the lipocalin family, such as the retinol binding protein and the human fatty-acid binding protein. Exchange rates and populations are largely uncorrelated, suggesting local 'breathing' motions rather than a concerted global conformational change.

Evidence of chemical exchange in recombinant Major Urinary Protein and quenching thereof upon pheromone binding

International Nuclear Information System (INIS)

Perazzolo, Chiara; Verde, Mariachiara; Homans, Steve W.; Bodenhausen, Geoffrey

2007-01-01

The internal dynamics of recombinant Major Urinary Protein (rMUP) have been investigated by monitoring transverse nitrogen-15 relaxation using multiple-echo Carr-Purcell-Meiboom-Gill (CPMG) experiments. While the ligand-free protein (APO-rMUP) features extensive evidence of motions on the milliseconds time scale, the complex with 2-methoxy-3-isobutylpyrazine (HOLO-rMUP) appears to be much less mobile on this time scale. At 308 K, exchange rates k ex = 500-2000 s -1 were typically observed in APO-rMUP for residues located adjacent to a β-turn comprising residues 83-87. These residues occlude an entry to the binding pocket and have been proposed to be a portal for ligand entry in other members of the lipocalin family, such as the retinol binding protein and the human fatty-acid binding protein. Exchange rates and populations are largely uncorrelated, suggesting local 'breathing' motions rather than a concerted global conformational change

A Novel Active-Learning Protein Purification Exercise for Large-Enrollment Introductory Biochemistry Courses Using the CHROM Web Applet

Science.gov (United States)

Barrette-Ng, Isabelle H.; Usher, Ken C.

2013-01-01

The CHROM Web applet has been used to create a new active-learning exercise in which students design a purification scheme for a recombinant protein using ion-exchange chromatography (IEC). To successfully complete the exercise, students are challenged to apply elementary concepts from acid-base chemistry as well as protein and amino acid…

Studies on the application of temperature-responsive ion exchange polymers with whey proteins.

Science.gov (United States)

Maharjan, Pankaj; Campi, Eva M; De Silva, Kirthi; Woonton, Brad W; Jackson, W Roy; Hearn, Milton T W

2016-03-18

Several new types of temperature-responsive ion exchange resins of different polymer composition have been prepared by grafting the products from the co-polymerisation of N-phenylacrylamide, N-iso-propylacrylamide and acrylic acid derivatives onto cross-linked agarose. Analysis of the binding isotherms for these different resins obtained under batch adsorption conditions indicated that the resin based on N-iso-propylacrylamide containing 5% (w/w) N-phenylacrylamide and 5% (w/w) acrylic acid resulted in the highest adsorption capacity, Bmax, for the whey protein, bovine lactoferrin, e.g. 14 mg bovine lactoferrin/mL resin at 4 °C and 62 mg bovine lactoferrin/mL resin at 40 °C, respectively. Under dynamic loading conditions at 40 °C, 94% of the loaded bovine lactoferrin on a normalised mg protein per mL resin basis was adsorbed by this new temperature-responsive ion-exchanger, and 76% was eluted by a single cycle temperature shift to 4 °C without varying the composition of the 10mM sodium dihydrogen phosphate buffer, pH 6.5, or the flow rate. The binding characteristics of these different ion exchange resins with bovine lactoferrin were also compared to results obtained using other resins based on N-isopropylacrylamide but contained N-tert-butylacrylamide rather than N-phenylacrylamide, where the corresponding dynamic capture and release properties for bovine lactoferrin required different temperature conditions of 20 °C and 50 °C, respectively for optimal desorption/adsorption. The cationic protein, bovine lactoperoxidase, was also adsorbed and desorbed with these temperature-responsive resins under similar conditions of changing temperature, whereas the anionic protein, bovine β-lactoglobulin, was not adsorbed under this regime of temperature conditions but instead eluted in the flow-through. Copyright © 2016 Elsevier B.V. All rights reserved.

Laser-based optical activity detection of amino acids and proteins

Energy Technology Data Exchange (ETDEWEB)

Reitsma, B.H.

1987-08-01

The optical activity detector (OAD) for HPLC is a selective detector for optically active substances including amino acids and proteins. Four free amino acids were resolved using cation-exchange chromatography followed by detection with refractive index detector (RI) for proline and threonine and the OAD to an ultraviolet absorbance detector (uv) for tyrosine and phenylalanine. Amino acid detection by refractive index is not sensitive and uv absorbance detects only three amino acids. Derivatization of amino acids to make them detectable by uv absorbance enhances the applicability of OA/uv for the determination of enantiomeric ratios. The separation of 16 dansyl-L-amino acids by RP-HPLC with detection by OA/uv is illustrated. Calculation of the specific rotation of 22 dansyl-L-amino acids shows that derivatization enhances the OA detectability of some amino acids but degrades that of others. RP-HPLC of proteins is a rapidly developing technique. Several researchers have reported the detection of multiple peaks when a pure protein is subjected to HPLC under certain conditions. These multiple peaks have been determined to be different conformations of the same protein. Since proteins are optically active, OA is a suitable detector. The RP-HPLC separation of conformers of soybean trypsin inhibitor is illustrated. Detection by OA/uv provides insights from the chromatogram unavailable from uv absorbance detection alone. In addition, identification of impurities is simplified with OA/uv. Specific rotations of the separated protein fractions show no significant change accompanying change in conformation. 163 refs., 13 figs., 9 tabs.

Antioxidant activity of pea protein hydrolysates produced by batch fermentation with lactic acid bacteria

Directory of Open Access Journals (Sweden)

Stanisavljević Nemanja S.

2015-01-01

Full Text Available Nine Lactobacillus strains known for surface proteinase activity were chosen from our collection and tested for their ability to grow in pea seed protein-based medium, and to hydrolyze purified pea proteins in order to produce peptides with antioxidant (AO activity. Two strains, Lactobacillus rhamnosus BGT10 and Lactobacillus zeae LMG17315, exhibited strong proteolytic activity against pea proteins. The AO activity of the pea hydrolysate fraction, MW <10 kDa, obtained by the fermentation of purified pea proteins with Lactobacillus rhamnosus BGT10, was tested by standard spectrophotometric assays (DPPH, ABTS, Fe3+-reducing capacity and the recently developed direct current (DC polarographic assay. The low molecular weight fraction of the obtained hydrolysate was separated using ion exchange chromatography, while the AO activity of eluted fractions was determined by means of a sensitive DC polarographic assay without previous concentration of samples. Results revealed that the fraction present in low abundance that contained basic peptides possessed the highest antioxidant activity. Based on the obtained results, it can be concluded that Lactobacillus rhamnosus BGT10 should be further investigated as a candidate strain for large-scale production of bioactive peptides from legume proteins. [Projekat Ministartsva nauke Republike Srbije, br. 173005 i br. 173026

Protein kinase A and Epac activation by cAMP regulates the expression of glial fibrillary acidic protein in glial cells

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Sugimoto Naotoshi

2016-01-01

Full Text Available Cyclic adenosine monophosphate (cAMP controls differentiation in several types of cells during brain development. However, the molecular mechanism of cAMP-controlled differentiation is not fully understood. We investigated the role of protein kinase A (PKA and exchange protein directly activated by cAMP (Epac on cAMP-induced glial fibrillary acidic protein (GFAP, an astrocyte marker, in cultured glial cells. B92 glial cells were treated with cAMP-elevating drugs, an activator of adenylate cyclase, phosphodiesterase inhibitor and a ß adrenal receptor agonist. These cAMP-elevating agents induced dramatic morphological changes and expression of GFAP. A cAMP analog, 8-Br-cAMP, which activates Epac as well as PKA, induced GFAP expression and morphological changes, while another cAMP analog, 8-CPT-cAMP, which activates Epac with greater efficacy when compared to PKA, induced GFAP expression but very weak morphological changes. Most importantly, the treatment with a PKA inhibitor partially reduced cAMP-induced GFAP expression. Taken together, these results indicate that cAMP-elevating drugs lead to the induction of GFAP via PKA and/or Epac activation in B92 glial cells.

Milk fouling in heat exchangers

NARCIS (Netherlands)

Jeurnink, T.J.M.

1996-01-01

The mechanisms of fouling of heat exchangers by milk were studied. Two major fouling mechanisms were indentified during the heat treatment of milk: (i) the formation and the subsequent deposition of activated serum protein molecules as a result of the heat denaturation; (ii) the

Oxysterol-binding Protein Activation at Endoplasmic Reticulum-Golgi Contact Sites Reorganizes Phosphatidylinositol 4-Phosphate Pools*

Science.gov (United States)

Goto, Asako; Charman, Mark; Ridgway, Neale D.

2016-01-01

Oxysterol-binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi/trans-Golgi network. 25-Hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to determine how OSBP controls the availability of PI-4P for this metabolic pathway. Treatment of fibroblasts or Chinese hamster ovary (CHO) cells with 25OH caused a 50–70% reduction in Golgi-associated immunoreactive PI-4P that correlated with Golgi localization of OSBP. In contrast, 25OH caused an OSBP-dependent enrichment in Golgi PI-4P that was detected with a pleckstrin homology domain probe. The cellular mass of phosphatidylinositol monophosphates and Golgi PI-4P measured with an unbiased PI-4P probe (P4M) was unaffected by 25OH and OSBP silencing, indicating that OSBP shifts the distribution of PI-4P upon localization to ER-Golgi contact sites. The PI-4P and sterol binding activities of OSBP were both required for 25OH activation of sphingomyelin synthesis, suggesting that 25OH must be exchanged for PI-4P to be concentrated at contact sites. We propose a model wherein 25OH activation of OSBP promotes the binding and retention of PI-4P at ER-Golgi contact sites. This pool of PI-4P specifically recruits pleckstrin homology domain-containing proteins involved in lipid transfer and metabolism, such as CERT. PMID:26601944

Active heat exchange system development for latent heat thermal energy storage

Science.gov (United States)

Lefrois, R. T.; Mathur, A. K.

1980-01-01

Five tasks to select, design, fabricate, test and evaluate candidate active heat exchanger modules for future applications to solar and conventional utility power plants were discussed. Alternative mechanizations of active heat exchange concepts were analyzed for use with heat of fusion phase change materials (PCMs) in the temperature range of 250 to 350 C. Twenty-six heat exchange concepts were reviewed, and eight were selected for detailed assessment. Two candidates were selected for small-scale experimentation: a coated tube and shell heat exchanger and a direct contact reflux boiler. A dilute eutectic mixture of sodium nitrate and sodium hydroxide was selected as the PCM from over 50 candidate inorganic salt mixtures. Based on a salt screening process, eight major component salts were selected initially for further evaluation. The most attractive major components in the temperature range of 250 to 350 C appeared to be NaNO3, NaNO2, and NaOH. Sketches of the two active heat exchange concepts selected for test are given.

Rapid deuterium exchange-in time for probing conformational change

International Nuclear Information System (INIS)

Dharmasiri, K.; Smith, D.L.

1995-01-01

Isotopic exchange of protein backbone amide hydrogens has been used extensively as a sensitive probe of protein structure. One of the salient features of hydrogen exchange is the vast range of exchange rates in one protein. Isotopic exchange methods have been used to study the structural features including protein folding and unfolding (1), functionally different forms of proteins (2), protein-protein complexation (3), and protein stability parameter. Many backbone amide protons that are surface accessible and are not involved in hydrogen bonding undergo rapid deuterium exchange. In order to study, fast exchanging amide protons, fast exchange-in times are necessary

Ion mobility spectrometry-hydrogen deuterium exchange mass spectrometry of anions: part 1. Peptides to proteins.

Science.gov (United States)

Donohoe, Gregory C; Khakinejad, Mahdiar; Valentine, Stephen J

2015-04-01

Ion mobility spectrometry (IMS) coupled with hydrogen deuterium exchange (HDX)-mass spectrometry (MS) has been used to study the conformations of negatively-charged peptide and protein ions. Results are presented for ion conformers of angiotensin 1, a synthetic peptide (SP), bovine insulin, ubiquitin, and equine cytochrome c. In general, the SP ion conformers demonstrate a greater level of HDX efficiency as a greater proportion of the sites undergo HDX. Additionally, these ions exhibit the fastest rates of exchange. Comparatively, the angiotensin 1 ions exhibit a lower rate of exchange and HDX level presumably because of decreased accessibility of exchange sites by charge sites. The latter are likely confined to the peptide termini. Insulin ions show dramatically reduced HDX levels and exchange rates, which can be attributed to decreased conformational flexibility resulting from the disulfide bonds. For the larger ubiquitin and protein ions, increased HDX is observed for larger ions of higher charge state. For ubiquitin, a conformational transition from compact to more elongated species (from lower to higher charge states) is reflected by an increase in HDX levels. These results can be explained by a combination of interior site protection by compact conformers as well as decreased access by charge sites. The elongated cytochrome c ions provide the largest HDX levels where higher values correlate with charge state. These results are consistent with increased exchange site accessibility by additional charge sites. The data from these enhanced IMS-HDX experiments are described in terms of charge site location, conformer rigidity, and interior site protection.

Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament.

KAUST Repository

Fornander, Louise H; Renodon-Corniè re, Axelle; Kuwabara, Naoyuki; Ito, Kentaro; Tsutsui, Yasuhiro; Shimizu, Toshiyuki; Iwasaki, Hiroshi; Nordé n, Bengt; Takahashi, Masayuki

2013-01-01

The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure

Exchange rate variability, market activity and heterogeneity

OpenAIRE

Rime, Dagfinn; Sucarrat, Genaro

2007-01-01

We study the role played by geographic and bank-size heterogeneity in the relation between exchange rate variability and market activity. We find some support for the hypothesis that increases in short-term global interbank market activity, which can be interpreted as due to variation in information arrival, increase variability. However, our results do not suggest that local short-term activity increases variability. With respect to long-term market activity, which can be interpreted as a me...

Sustained Na+/H+ exchanger activation promotes gliotransmitter release from reactive hippocampal astrocytes following oxygen-glucose deprivation.

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Pelin Cengiz

Full Text Available Hypoxia ischemia (HI-related brain injury is the major cause of long-term morbidity in neonates. One characteristic hallmark of neonatal HI is the development of reactive astrogliosis in the hippocampus. However, the impact of reactive astrogliosis in hippocampal damage after neonatal HI is not fully understood. In the current study, we investigated the role of Na(+/H(+ exchanger isoform 1 (NHE1 protein in mouse reactive hippocampal astrocyte function in an in vitro ischemia model (oxygen/glucose deprivation and reoxygenation, OGD/REOX. 2 h OGD significantly increased NHE1 protein expression and NHE1-mediated H(+ efflux in hippocampal astrocytes. NHE1 activity remained stimulated during 1-5 h REOX and returned to the basal level at 24 h REOX. NHE1 activation in hippocampal astrocytes resulted in intracellular Na(+ and Ca(2+ overload. The latter was mediated by reversal of Na(+/Ca(2+ exchange. Hippocampal astrocytes also exhibited a robust release of gliotransmitters (glutamate and pro-inflammatory cytokines IL-6 and TNFα during 1-24 h REOX. Interestingly, inhibition of NHE1 activity with its potent inhibitor HOE 642 not only reduced Na(+ overload but also gliotransmitter release from hippocampal astrocytes. The noncompetitive excitatory amino acid transporter inhibitor TBOA showed a similar effect on blocking the glutamate release. Taken together, we concluded that NHE1 plays an essential role in maintaining H(+ homeostasis in hippocampal astrocytes. Over-stimulation of NHE1 activity following in vitro ischemia disrupts Na(+ and Ca(2+ homeostasis, which reduces Na(+-dependent glutamate uptake and promotes release of glutamate and cytokines from reactive astrocytes. Therefore, blocking sustained NHE1 activation in reactive astrocytes may provide neuroprotection following HI.

13CHD2–CEST NMR spectroscopy provides an avenue for studies of conformational exchange in high molecular weight proteins

International Nuclear Information System (INIS)

Rennella, Enrico; Huang, Rui; Velyvis, Algirdas; Kay, Lewis E.

2015-01-01

An NMR experiment for quantifying slow (millisecond) time-scale exchange processes involving the interconversion between visible ground state and invisible, conformationally excited state conformers is presented. The approach exploits chemical exchange saturation transfer (CEST) and makes use of 13 CHD 2 methyl group probes that can be readily incorporated into otherwise highly deuterated proteins. The methodology is validated with an application to a G48A Fyn SH3 domain that exchanges between a folded conformation and a sparsely populated and transiently formed unfolded ensemble. Experiments on a number of different protein systems, including a 360 kDa half-proteasome, establish that the sensitivity of this 13 CHD 2 13 C–CEST technique can be upwards of a factor of 5 times higher than for a previously published 13 CH 3 13 C–CEST approach (Bouvignies and Kay in J Biomol NMR 53:303–310, 2012), suggesting that the methodology will be powerful for studies of conformational exchange in high molecular weight proteins

Selective effects of charge on G protein activation by FSH-receptor residues 551-555 and 650-653.

Science.gov (United States)

Grasso, P; Deziel, M R; Reichert, L E

1995-01-01

Two cytosolic regions of the rat testicular FSH receptor (FSHR), residues 533-555 and 645-653, have been identified as G protein-coupling domains. We localized the activity in these domains to their C-terminal sequences, residues 551-555 (KIAKR, net charge +3) and 650-653 (RKSH, net charge +3), and examined the effects of charge on G protein activation by the C-terminal peptides, using synthetic analogs containing additions, through alanine (A) linkages, of arginine (R, +), histidine (H, +) or both. RA-KIAKR (net charge +4) mimicked the effect of FSHR-(551-555) on guanine nucleotide exchange in rat testis membranes, but reduced its ability to inhibit FSH-stimulated estradiol biosynthesis in cultured rat Sertoli cells. Further increasing net charge by the addition of H (HARA-KIAKR, net charge +5) increased guanosine 5'-triphosphate (GTP) binding, but eliminated FSHR-(551-555) effects on FSH-stimulated steroidogenesis. HA-RKSH (net charge +4) significantly inhibited guanine nucleotide exchange in rat testis membranes, but stimulated basal and potentiated FSH-induced estradiol biosynthesis in cultured rat Sertoli cells. Addition of two H residues (HAHA-RKSH, net charge +5) restored GTP binding and further potentiated basal and FSH-stimulated steroidogenesis. These results suggest that positive charges in G protein-coupling domains of the FSHR play a role in modulating G protein activation and postbinding effects of FSH, such as steroidogenesis.

Mixed retention mechanism of proteins in weak anion-exchange chromatography.

Science.gov (United States)

Liu, Peng; Yang, Haiya; Geng, Xindu

2009-10-30

Using four commercial weak anion-exchange chromatography (WAX) columns and 11 kinds of different proteins, we experimentally examined the involvement of hydrophobic interaction chromatography (HIC) mechanism in protein retention on the WAX columns. The HIC mechanism was found to operate in all four WAX columns, and each of these columns had a better resolution in the HIC mode than in the corresponding WAX mode. Detailed analysis of the molecular interactions in a chromatographic system indicated that it is impossible to completely eliminate hydrophobic interactions from a WAX column. Based on these results, it may be possible to employ a single WAX column for protein separation by exploiting mixed modes (WAX and HIC) of retention. The stoichiometric displacement theory and two linear plots were used to show that mechanism of the mixed modes of retention in the system was a combination of two kinds of interactions, i.e., nonselective interactions in the HIC mode and selective interactions in the IEC mode. The obtained U-shaped elution curve of proteins could be distinguished into four different ranges of salt concentration, which also represent four retention regions.

A simple purification and activity assay of the coagulant protein from Moringa oleifera seed.

Science.gov (United States)

Ghebremichael, Kebreab A; Gunaratna, K R; Henriksson, Hongbin; Brumer, Harry; Dalhammar, Gunnel

2005-06-01

Use of extracts from Moringa oleifera (MO) is of great interest for low-cost water treatment. This paper discusses water and salt extraction of a coagulant protein from the seed, purification using ion exchange, its chemical characteristics, coagulation and antimicrobial properties. The coagulant from both extracts is a cationic protein with pI greater than 9.6 and molecular mass less than 6.5 kDa. Mass spectrometric analysis of the purified water extract indicated that it contained at least four homologous proteins, based on MS/MS peptide sequence data. The protein is thermoresistant and remained active after 5h heat treatment at 95 degrees C. The coagulant protein showed both flocculating and antibacterial effects of 1.1--4 log reduction. With samples of high turbidity, the MO extract showed similar coagulation activity as alum. Cecropin A and MO extract were found to have similar flocculation effects for clay and microorganisms. Simple methods for both the purification and assay of MO coagulating proteins are presented, which are necessary for large-scale water treatment applications.

Oxysterol-binding Protein Activation at Endoplasmic Reticulum-Golgi Contact Sites Reorganizes Phosphatidylinositol 4-Phosphate Pools.

Science.gov (United States)

Goto, Asako; Charman, Mark; Ridgway, Neale D

2016-01-15

Oxysterol-binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi/trans-Golgi network. 25-Hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to determine how OSBP controls the availability of PI-4P for this metabolic pathway. Treatment of fibroblasts or Chinese hamster ovary (CHO) cells with 25OH caused a 50-70% reduction in Golgi-associated immunoreactive PI-4P that correlated with Golgi localization of OSBP. In contrast, 25OH caused an OSBP-dependent enrichment in Golgi PI-4P that was detected with a pleckstrin homology domain probe. The cellular mass of phosphatidylinositol monophosphates and Golgi PI-4P measured with an unbiased PI-4P probe (P4M) was unaffected by 25OH and OSBP silencing, indicating that OSBP shifts the distribution of PI-4P upon localization to ER-Golgi contact sites. The PI-4P and sterol binding activities of OSBP were both required for 25OH activation of sphingomyelin synthesis, suggesting that 25OH must be exchanged for PI-4P to be concentrated at contact sites. We propose a model wherein 25OH activation of OSBP promotes the binding and retention of PI-4P at ER-Golgi contact sites. This pool of PI-4P specifically recruits pleckstrin homology domain-containing proteins involved in lipid transfer and metabolism, such as CERT. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Oxygen exchange at gas/oxide interfaces: how the apparent activation energy of the surface exchange coefficient depends on the kinetic regime.

Science.gov (United States)

Fielitz, Peter; Borchardt, Günter

2016-08-10

In the dedicated literature the oxygen surface exchange coefficient KO and the equilibrium oxygen exchange rate [Fraktur R] are considered to be directly proportional to each other regardless of the experimental circumstances. Recent experimental observations, however, contradict the consequences of this assumption. Most surprising is the finding that the apparent activation energy of KO depends dramatically on the kinetic regime in which it has been determined, i.e. surface exchange controlled vs. mixed or diffusion controlled. This work demonstrates how the diffusion boundary condition at the gas/solid interface inevitably entails a correlation between the oxygen surface exchange coefficient KO and the oxygen self-diffusion coefficient DO in the bulk ("on top" of the correlation between KO and [Fraktur R] for the pure surface exchange regime). The model can thus quantitatively explain the range of apparent activation energies measured in the different regimes: in the surface exchange regime the apparent activation energy only contains the contribution of the equilibrium exchange rate, whereas in the mixed or in the diffusion controlled regime the contribution of the oxygen self-diffusivity has also to be taken into account, which may yield significantly higher apparent activation energies and simultaneously quantifies the correlation KO ∝ DO(1/2) observed for a large number of oxides in the mixed or diffusion controlled regime, respectively.

Deciphering the Dynamic Interaction Profile of an Intrinsically Disordered Protein by NMR Exchange Spectroscopy.

Science.gov (United States)

Delaforge, Elise; Kragelj, Jaka; Tengo, Laura; Palencia, Andrés; Milles, Sigrid; Bouvignies, Guillaume; Salvi, Nicola; Blackledge, Martin; Jensen, Malene Ringkjøbing

2018-01-24

Intrinsically disordered proteins (IDPs) display a large number of interaction modes including folding-upon-binding, binding without major structural transitions, or binding through highly dynamic, so-called fuzzy, complexes. The vast majority of experimental information about IDP binding modes have been inferred from crystal structures of proteins in complex with short peptides of IDPs. However, crystal structures provide a mainly static view of the complexes and do not give information about the conformational dynamics experienced by the IDP in the bound state. Knowledge of the dynamics of IDP complexes is of fundamental importance to understand how IDPs engage in highly specific interactions without concomitantly high binding affinity. Here, we combine rotating-frame R 1ρ , Carr-Purcell-Meiboom Gill relaxation dispersion as well as chemical exchange saturation transfer to decipher the dynamic interaction profile of an IDP in complex with its partner. We apply the approach to the dynamic signaling complex formed between the mitogen-activated protein kinase (MAPK) p38α and the intrinsically disordered regulatory domain of the MAPK kinase MKK4. Our study demonstrates that MKK4 employs a subtle combination of interaction modes in order to bind to p38α, leading to a complex displaying significantly different dynamics across the bound regions.

Fluorogen-activating proteins: beyond classical fluorescent proteins

Directory of Open Access Journals (Sweden)

Shengnan Xu

2018-05-01

Full Text Available Fluorescence imaging is a powerful technique for the real-time noninvasive monitoring of protein dynamics. Recently, fluorogen activating proteins (FAPs/fluorogen probes for protein imaging were developed. Unlike the traditional fluorescent proteins (FPs, FAPs do not fluoresce unless bound to their specific small-molecule fluorogens. When using FAPs/fluorogen probes, a washing step is not required for the removal of free probes from the cells, thus allowing rapid and specific detection of proteins in living cells with high signal-to-noise ratio. Furthermore, with different fluorogens, living cell multi-color proteins labeling system was developed. In this review, we describe about the discovery of FAPs, the design strategy of FAP fluorogens, the application of the FAP technology and the advances of FAP technology in protein labeling systems. KEY WORDS: Fluorogen activating proteins, Fluorogens, Genetically encoded sensors, Fluorescence imaging, Molecular imaging

Measurement of backbone hydrogen-deuterium exchange in the type III secretion system needle protein PrgI by solid-state NMR

Science.gov (United States)

Chevelkov, Veniamin; Giller, Karin; Becker, Stefan; Lange, Adam

2017-10-01

In this report we present site-specific measurements of amide hydrogen-deuterium exchange rates in a protein in the solid state phase by MAS NMR. Employing perdeuteration, proton detection and a high external magnetic field we could adopt the highly efficient Relax-EXSY protocol previously developed for liquid state NMR. According to this method, we measured the contribution of hydrogen exchange on apparent 15N longitudinal relaxation rates in samples with differing D2O buffer content. Differences in the apparent T1 times allowed us to derive exchange rates for multiple residues in the type III secretion system needle protein.

Thiol-Disulfide Exchange between Glutaredoxin and Glutathione

DEFF Research Database (Denmark)

Iversen, Rasmus; Andersen, Peter Anders; Jensen, Kristine Steen

2010-01-01

Glutaredoxins are ubiquitous thiol-disulfide oxidoreductases which catalyze the reduction of glutathione-protein mixed disulfides. Belonging to the thioredoxin family, they contain a conserved active site CXXC motif. The N-proximal active site cysteine can form a mixed disulfide with glutathione ...... has been replaced with serine. The exchange reaction between the reduced protein and oxidized glutathione leading to formation of the mixed disulfide could readily be monitored by isothermal titration calorimetry (ITC) due to the enthalpic contributions from the noncovalent interactions...

Isolation and characterization of an RIP (ribosome-inactivating protein)-like protein from tobacco with dual enzymatic activity.

Science.gov (United States)

Sharma, Neelam; Park, Sang-Wook; Vepachedu, Ramarao; Barbieri, Luigi; Ciani, Marialibera; Stirpe, Fiorenzo; Savary, Brett J; Vivanco, Jorge M

2004-01-01

Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a protein termed tobacco RIP (TRIP) was isolated from tobacco (Nicotiana tabacum) leaves and purified using ion exchange and gel filtration chromatography in combination with yeast ribosome depurination assays. TRIP has a molecular mass of 26 kD as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed strong N-glycosidase activity as manifested by the depurination of yeast rRNA. Purified TRIP showed immunoreactivity with antibodies of RIPs from Mirabilis expansa. TRIP released fewer amounts of adenine residues from ribosomal (Artemia sp. and rat ribosomes) and non-ribosomal substrates (herring sperm DNA, rRNA, and tRNA) compared with other RIPs. TRIP inhibited translation in wheat (Triticum aestivum) germ more efficiently than in rabbit reticulocytes, showing an IC50 at 30 ng in the former system. Antimicrobial assays using highly purified TRIP (50 microg mL(-1)) conducted against various fungi and bacterial pathogens showed the strongest inhibitory activity against Trichoderma reesei and Pseudomonas solancearum. A 15-amino acid internal polypeptide sequence of TRIP was identical with the internal sequences of the iron-superoxide dismutase (Fe-SOD) from wild tobacco (Nicotiana plumbaginifolia), Arabidopsis, and potato (Solanum tuberosum). Purified TRIP showed SOD activity, and Escherichia coli Fe-SOD was observed to have RIP activity too. Thus, TRIP may be considered a dual activity enzyme showing RIP-like activity and Fe-SOD characteristics.

Differential multiple quantum relaxation caused by chemical exchange outside the fast exchange limit

International Nuclear Information System (INIS)

Wang Chunyu; Palmer, Arthur G.

2002-01-01

Differential relaxation of multiple quantum coherences is a signature for chemical exchange processes in proteins. Previous analyses of experimental data have used theoretical descriptions applicable only in the limit of fast exchange. Theoretical expressions for differential relaxation rate constants that are accurate outside fast exchange are presented for two-spin-system subject to two-site chemical exchange. The theoretical expressions are validated using experimental results for 15 N- 1 H relaxation in basic pancreatic trypsin inhibitor. The new theoretical expression is valuable for identification and characterization of exchange processes in proteins using differential relaxation of multiple quantum coherences

A Protein Scaffold Coordinates SRC-Mediated JNK Activation in Response to Metabolic Stress.

Science.gov (United States)

Kant, Shashi; Standen, Claire L; Morel, Caroline; Jung, Dae Young; Kim, Jason K; Swat, Wojciech; Flavell, Richard A; Davis, Roger J

2017-09-19

Obesity is a major risk factor for the development of metabolic syndrome and type 2 diabetes. How obesity contributes to metabolic syndrome is unclear. Free fatty acid (FFA) activation of a non-receptor tyrosine kinase (SRC)-dependent cJun NH 2 -terminal kinase (JNK) signaling pathway is implicated in this process. However, the mechanism that mediates SRC-dependent JNK activation is unclear. Here, we identify a role for the scaffold protein JIP1 in SRC-dependent JNK activation. SRC phosphorylation of JIP1 creates phosphotyrosine interaction motifs that bind the SH2 domains of SRC and the guanine nucleotide exchange factor VAV. These interactions are required for SRC-induced activation of VAV and the subsequent engagement of a JIP1-tethered JNK signaling module. The JIP1 scaffold protein, therefore, plays a dual role in FFA signaling by coordinating upstream SRC functions together with downstream effector signaling by the JNK pathway. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

A Protein Scaffold Coordinates SRC-Mediated JNK Activation in Response to Metabolic Stress

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Shashi Kant

2017-09-01

Full Text Available Obesity is a major risk factor for the development of metabolic syndrome and type 2 diabetes. How obesity contributes to metabolic syndrome is unclear. Free fatty acid (FFA activation of a non-receptor tyrosine kinase (SRC-dependent cJun NH2-terminal kinase (JNK signaling pathway is implicated in this process. However, the mechanism that mediates SRC-dependent JNK activation is unclear. Here, we identify a role for the scaffold protein JIP1 in SRC-dependent JNK activation. SRC phosphorylation of JIP1 creates phosphotyrosine interaction motifs that bind the SH2 domains of SRC and the guanine nucleotide exchange factor VAV. These interactions are required for SRC-induced activation of VAV and the subsequent engagement of a JIP1-tethered JNK signaling module. The JIP1 scaffold protein, therefore, plays a dual role in FFA signaling by coordinating upstream SRC functions together with downstream effector signaling by the JNK pathway.

Spatially resolved protein hydrogen exchange measured by subzero-cooled chip-based nanoelectrospray ionization tandem mass spectrometry

DEFF Research Database (Denmark)

Amon, Sabine; Trelle, Morten B; Jensen, Ole N

2012-01-01

. After a given period of deuteration, the exchange reaction is quenched by acidification (pH 2.5) and cooling (0 °C) and the deuterated protein (or a digest thereof) is analyzed by mass spectrometry. The unavoidable loss of deuterium (back-exchange) that occurs under quench conditions is undesired...... as it leads to loss of information. Here we describe the successful application of a chip-based nanoelectrospray ionization mass spectrometry top-down fragmentation approach based on cooling to subzero temperature (-15 °C) which reduces the back-exchange at quench conditions to very low levels. For example...

Strand exchange of telomeric DNA catalyzed by the Werner syndrome protein (WRN) is specifically stimulated by TRF2

Science.gov (United States)

Edwards, Deanna N.; Orren, David K.; Machwe, Amrita

2014-01-01

Werner syndrome (WS), caused by loss of function of the RecQ helicase WRN, is a hereditary disease characterized by premature aging and elevated cancer incidence. WRN has DNA binding, exonuclease, ATPase, helicase and strand annealing activities, suggesting possible roles in recombination-related processes. Evidence indicates that WRN deficiency causes telomeric abnormalities that likely underlie early onset of aging phenotypes in WS. Furthermore, TRF2, a protein essential for telomere protection, interacts with WRN and influences its basic helicase and exonuclease activities. However, these studies provided little insight into WRN's specific function at telomeres. Here, we explored the possibility that WRN and TRF2 cooperate during telomeric recombination processes. Our results indicate that TRF2, through its interactions with both WRN and telomeric DNA, stimulates WRN-mediated strand exchange specifically between telomeric substrates; TRF2's basic domain is particularly important for this stimulation. Although TRF1 binds telomeric DNA with similar affinity, it has minimal effects on WRN-mediated strand exchange of telomeric DNA. Moreover, TRF2 is displaced from telomeric DNA by WRN, independent of its ATPase and helicase activities. Together, these results suggest that TRF2 and WRN act coordinately during telomeric recombination processes, consistent with certain telomeric abnormalities associated with alteration of WRN function. PMID:24880691

Protein Internal Dynamics Associated With Pre-System Glass Transition Temperature Endothermic Events: Investigation of Insulin and Human Growth Hormone by Solid State Hydrogen/Deuterium Exchange.

Science.gov (United States)

Fang, Rui; Grobelny, Pawel J; Bogner, Robin H; Pikal, Michael J

2016-11-01

Lyophilized proteins are generally stored below their glass transition temperature (T g ) to maintain long-term stability. Some proteins in the (pure) solid state showed a distinct endotherm at a temperature well below the glass transition, designated as a pre-T g endotherm. The pre-T g endothermic event has been linked with a transition in protein internal mobility. The aim of this study was to investigate the internal dynamics of 2 proteins, insulin and human growth hormone (hGH), both of which exhibit the pre-T g endothermic event with onsets at 50°C-60°C. Solid state hydrogen/deuterium (H/D) exchange of both proteins was characterized by Fourier transform infrared spectroscopy over a temperature range from 30°C to 80°C. A distinct sigmoidal transition in the extent of H/D exchange had a midpoint of 56.1 ± 1.2°C for insulin and 61.7 ± 0.9°C for hGH, suggesting a transition to greater mobility in the protein molecules at these temperatures. The data support the hypothesis that the pre-T g event is related to a transition in internal protein mobility associated with the protein dynamical temperature. Exceeding the protein dynamical temperature is expected to activate protein internal motion and therefore may have stability consequences. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Hydrogen exchange kinetics in a membrane protein determined by 15N NMR spectroscopy: Use of the INEPT [insensitive nucleus enhancement by polarization transfer] experiment to follow individual amides in detergent-solubilized M13 coat protein

International Nuclear Information System (INIS)

Henry, G.D.; Sykes, B.D.

1990-01-01

The coat protein of the filamentous coliphage M13 is a 50-residue polypeptide which spans the inner membrane of the Escherichia coli host upon infection. Amide hydrogen exchange kinetics have been used to probe the structure and dynamics of M13 coat protein which has been solubilized in sodium dodecyl sulfate (SDS) micelles. In a previous 1 H nuclear magnetic resonance (NMR) study, multiple exponential analysis of the unresolved amide proton envelope revealed the existence of two slow kinetic sets containing a total of about 30 protons. The slower set (15-20 amides) originates from the hydrophobic membrane-spanning region and exchanges at least 10 5 -fold slower than the unstructured, non-H-bonded model polypeptide poly(DL-alanine). Herein the authors use 15 N NMR spectroscopy of biosynthetically labeled coat protein to follow individual, assigned, slowly exchanging amides in or near the hydrophobic segment. The INEPT (insensitive nucleus enhancement by polarization transfer) experiments can be used to transfer magnetization to the 15 N nucleus from a coupled proton; when 15 N-labeled protonated protein is dissolved in 2 H 2 O, the INEPT signal disappears with time as the amide protons are replaced by solvent deuterons. Amide hydrogen exchange is catalyzed by both H + and OH - ions. The time-dependent exchange-out experiment is suitable for slow exchange rates (k ex ). The INEPT experiment was also adapted to measure some of the more rapidly exchanging amides in the coat protein using either saturation transfer from water or exchange effects on the polarization transfer step itself. The results of all of these experiments are consistent with previous models of the coat protein in which a stable segment extends from the hydrophobic membrane-spanning region through to the C-terminus, whereas the N-terminal region is undergoing more extensive dynamic fluctuations

BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein regulates neurite development via PI3K-AKT and ERK signaling pathways.

Science.gov (United States)

Zhou, C; Li, C; Li, D; Wang, Y; Shao, W; You, Y; Peng, J; Zhang, X; Lu, L; Shen, X

2013-12-19

The elongation of neuron is highly dependent on membrane trafficking. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein 1 (BIG1) functions in the membrane trafficking between the Golgi apparatus and the plasma membrane. BFA, an uncompetitive inhibitor of BIG1 can inhibit neurite outgrowth and polarity development. In this study, we aimed to define the possible role of BIG1 in neurite development and to further investigate the potential mechanism. By immunostaining, we found that BIG1 was extensively colocalized with synaptophysin, a marker for synaptic vesicles in soma and partly in neurites. The amount of both protein and mRNA of BIG1 were up-regulated during rat brain development. BIG1 depletion significantly decreased the neurite length and inhibited the phosphorylation of phosphatidylinositide 3-kinase (PI3K) and protein kinase B (AKT). Inhibition of BIG1 guanine nucleotide-exchange factor (GEF) activity by BFA or overexpression of the dominant-negative BIG1 reduced PI3K and AKT phosphorylation, indicating regulatory effects of BIG1 on PI3K-AKT signaling pathway is dependent on its GEF activity. BIG1 siRNA or BFA treatment also significantly reduced extracellular signal-regulated kinase (ERK) phosphorylation. Overexpression of wild-type BIG1 significantly increased ERK phosphorylation, but the dominant-negative BIG1 had no effect on ERK phosphorylation, indicating the involvement of BIG1 in ERK signaling regulation may not be dependent on its GEF activity. Our result identified a novel function of BIG1 in neurite development. The newly recognized function integrates the function of BIG1 in membrane trafficking with the activation of PI3K-AKT and ERK signaling pathways which are critical in neurite development. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Natural clinoptilolite exchanged with iron: characterization and catalytic activity in nitrogen monoxide reduction

Directory of Open Access Journals (Sweden)

Daria Tito-Ferro

2016-12-01

Full Text Available The aim of this work was to characterize the natural clinoptilolite from Tasajeras deposit, Cuba, modified by hydrothermal ion-exchange with solutions of iron (II sulfate and iron (III nitrate in acid medium. Besides this, its catalytic activity to reduce nitrogen monoxide with carbon monoxide/propene in the presence of oxygen was evaluated. The characterization was performed by Mössbauer and UV-Vis diffuse reflectance spectroscopies and adsorption measurements. The obtained results lead to conclude that in exchanged samples, incorporated divalent and trivalent irons are found in octahedral coordination. Both irons should be mainly in cationic extra-framework positions inside clinoptilolite channels as charge compensating cations, and also as iron oxy-hydroxides resulting from limited hydrolysis of these cations. The iron (III exchanged samples has a larger amount of iron oxy-hydroxides agglomerates. The iron (II exchanged samples have additionally iron (II sulfate adsorbed. The catalytic activity in the nitrogen monoxide reduction is higher in the exchanged zeolites than starting. Among all samples, those exchanged of iron (II has the higher catalytic activity. This lead to outline that, main catalytically active centers are associated with divalent iron.

Probing the Binding Interfaces of Protein Complexes Using Gas-Phase H/D Exchange Mass Spectrometry

DEFF Research Database (Denmark)

Mistarz, Ulrik H; Brown, Jeffery M; Haselmann, Kim F

2016-01-01

Fast gas-phase hydrogen/deuterium exchange mediated by ND3 gas and measured by mass spectrometry (gas-phase HDX-MS) is a largely unharnessed, fast, and sensitive method for probing primary- and higher-order polypeptide structure. Labeling of heteroatom-bound non-amide hydrogens in a sub-milliseco......Fast gas-phase hydrogen/deuterium exchange mediated by ND3 gas and measured by mass spectrometry (gas-phase HDX-MS) is a largely unharnessed, fast, and sensitive method for probing primary- and higher-order polypeptide structure. Labeling of heteroatom-bound non-amide hydrogens in a sub......-millisecond time span after electrospray ionization by ND3 gas can provide structural insights into protein conformers present in solution. Here, we have explored the use of gas-phase HDX-MS for probing the higher-order structure and binding interfaces of protein complexes originating from native solution...

ABH antigens as recognition sites for the activation of red blood cell anion exchange by the lectin ulex europaeus agglutinin I.

Science.gov (United States)

Engelmann, B

1993-11-01

The blood group antigen H (blood group O) and fucose-specific lectin Ulex europaeus agglutinin I (UEA1) (10 micrograms/ml) was found to increase the rate constant of Cl- efflux into 100 mM Na+ oxalate media by about 40% in erythrocytes taken from antigen H donors. In 100 mM K+ oxalate, 150 mM Na+ pyruvate and in 150 mM Na+ acetate media the lectin elevated the rate constant of Cl- efflux by 20-50%. The acceleration of Cl- efflux by UEA1 was completely blocked by 10 microM 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) indicating that the effect of the lectin is mediated by the anion exchanger of human erythrocytes (band 3 protein). In antigen A1 erythrocytes no significant stimulation of anion exchange by UEA1 was seen. The activation of Cl- efflux was completely prevented by addition of 1 mM fucose to the medium. These results suggest that the effect of UEA1 is mediated through interaction with the fucose residues of H antigens. Increasing extracellular Ca++ from 0.5 to 5 mM in Na+ pyruvate or Na+ acetate media slightly reduced the acceleration of anion exchange by the lectin. On the other hand, replacing part of extracellular chloride by bicarbonate did not considerably alter the (previously reported) stimulatory effect of UEA1 on red blood cell Ca++ uptake. This suggests that the acceleration of anion exchange and of Ca++ uptake by UEA1, respectively, are mediated by different mechanisms. It is concluded that UEA1 activates anion exchange of human erythrocytes most probably by a direct interaction with H antigens present on extracellular domains of the band 3 protein.

Laser-based optical activity detection of amino acids and proteins

Energy Technology Data Exchange (ETDEWEB)

Reitsma, B.H.

1987-01-01

The optical activity detector (OAD) for HPLC is a selective detector for optically active substances including amino acids and proteins. This study illustrates the use of the OAD in three related areas. Section I illustrates the separation of four free amino acids using cation-exchange chromatography. Detection by coupling the OAD to a refractive index detector (RI) for proline and threonine and the OAD to an ultraviolet absorbance detector (UV) for tyrosine and phenylalanine allows the calculation of enantiomeric (D/L) ratios of these amino acids without physical separation. Specific rotations of these four amino acids are also reported. Section II illustrates the separation of 16 dansyl-L-amino acids by RP-HPLC with detection by OA/UV. Section III illustrates the RP-HPLC separation of conformers of soybean trypsin inhibitor. Detection by OA/UV provides insights from the chromatogram unavailable for UV absorbance detection alone. In addition, identification of impurities is simplified with OA/UV. Specific rotations of the separated protein fractions show no significant change accompanying change in conformation.

Active Gating, Molecular Pumping, and Turnover Determination in Biomimetic Lipidic Cubic Mesophases with Reconstituted Membrane Proteins.

Science.gov (United States)

Speziale, Chiara; Zabara, Alexandru Florian; Drummond, Calum John; Mezzenga, Raffaele

2017-11-28

Understanding the mechanisms controlling molecular transport in bioinspired materials is a central topic in many branches of nanotechnology. In this work, we show that biomolecules of fundamental importance in biological processes, such as glucose, can be transported in an active, controlled, and selective manner across macroscopic lipidic cubic mesophases, by correctly reconstituting within them their corresponding membrane protein transporters, such as Staphylococcus epidermidis (GlcP Se ). Importantly, by duly exploiting the symporter properties of GlcP Se of coupled glucose/H + transport, the diffusion of glucose can further be tuned by independent physiological stimuli, such as parallel or antiparallel pH gradients, offering an important model to study molecular exchange processes in cellular machinery. We finally show that by measuring the transport properties of the lipidic mesophases with and without the GlcP Se membrane protein reconstituted within, it becomes possible to determine its intrinsic conductance. We generalize these findings to other membrane proteins from the antiporters family, such as the bacterial ClC exchanger from Escherichia coli (EcClC), providing a robust method for evaluating the turnover rate of the membrane proteins in general.

A plasma coagulation assay for an activated protein C-independent anticoagulant activity of protein S

NARCIS (Netherlands)

van Wijnen, M.; van 't Veer, C.; Meijers, J. C.; Bertina, R. M.; Bouma, B. N.

1998-01-01

To study the physiological importance of the activated protein C (APC)-independent anticoagulant activity of protein S, we developed an assay specific for this activity. The ability of protein S to prolong the clotting time in an APC-independent way was expressed as the ratio of the clotting time in

Activation energies for iodine-exchange systems containing organic iodine compounds

Energy Technology Data Exchange (ETDEWEB)

Ikeda, N. (Takyo Univ. of Education (Japan). Faculty of Science) Takahashi, Yasuko

1976-01-01

In studies on the nonequilibrium isotopic exchange method for determining iodine in organic iodine compounds, activation energies have been measured to find systems having appropriate rate of exchange reactions. Activation energies are discussed by considering the effect of the structure of organic iodine compounds, the concentrations of reactants and solvent, etc. In homogeneous systems, activation energy is found to become larger in the order of CH/sub 3/Iactivation energy is less in 100% acetone than in 90% acetone solution. In heterogeneous systems, e.g. org. I(aq.)--I/sub 2/(CCL/sub 4/ or C/sub 2/H/sub 4/Cl/sub 2/), activation energy increases in the order of 3,5-diiodotyrosine<3-iodotyrosine<5-iodouracil. The catalytic effect of I/sub 2/ is large, and the iodine ratio between I/sub 2/ and organic iodine is a predominant factor in determining the rate of the exchange reaction.

Resveratrol Inhibits Porcine Intestinal Glucose and Alanine Transport: Potential Roles of Na+/K+-ATPase Activity, Protein Kinase A, AMP-Activated Protein Kinase and the Association of Selected Nutrient Transport Proteins with Detergent Resistant Membranes

Directory of Open Access Journals (Sweden)

Stefanie Klinger

2018-03-01

Full Text Available Background: Beneficial effects of Resveratrol (RSV have been demonstrated, including effects on transporters and channels. However, little is known about how RSV influences intestinal transport. The aim of this study was to further characterize the effects of RSV on intestinal transport and the respective mechanisms. Methods: Porcine jejunum and ileum were incubated with RSV (300 µM, 30 min in Ussing chambers (functional studies and tissue bathes (detection of protein expression, phosphorylation, association with detergent resistant membranes (DRMs. Results: RSV reduced alanine and glucose-induced short circuit currents (ΔIsc and influenced forskolin-induced ΔIsc. The phosphorylation of sodium–glucose-linked transporter 1 (SGLT1, AMP-activated protein kinase (AMPK, protein kinase A substrates (PKA-S and liver kinase B1 (LKB1 increased but a causative relation to the inhibitory effects could not directly be established. The DRM association of SGLT1, peptide transporter 1 (PEPT1 and (phosphorylated Na+/H+-exchanger 3 (NHE3 did not change. Conclusion: RSV influences the intestinal transport of glucose, alanine and chloride and is likely to affect other transport processes. As the effects of protein kinase activation vary between the intestinal localizations, it would appear that increasing cyclic adenosine monophosphate (cAMP levels are part of the mechanism. Nonetheless, the physiological responses depend on cell type-specific structures.

Protein hydrogen exchange measured at single-residue resolution by electron transfer dissociation mass spectrometry

DEFF Research Database (Denmark)

Rand, Kasper D; Zehl, Martin; Jensen, Ole Nørregaard

2009-01-01

Because of unparalleled sensitivity and tolerance to protein size, mass spectrometry (MS) has become a popular method for measuring the solution hydrogen (1H/2H) exchange (HX) of biologically relevant protein states. While incorporated deuterium can be localized to different regions by pepsin....... The deuterium labeling pattern of beta2-microglobulin is retained in the gaseous fragment ions by employing mild declustering conditions for electrospray ionization. A recently developed model peptide is used to arrive at such ion source declustering conditions that prevent the occurrence of intramolecular gas...

Hydrogen Exchange Differences between Chemoreceptor Signaling Complexes Localize to Functionally Important Subdomains

Science.gov (United States)

2015-01-01

The goal of understanding mechanisms of transmembrane signaling, one of many key life processes mediated by membrane proteins, has motivated numerous studies of bacterial chemotaxis receptors. Ligand binding to the receptor causes a piston motion of an α helix in the periplasmic and transmembrane domains, but it is unclear how the signal is then propagated through the cytoplasmic domain to control the activity of the associated kinase CheA. Recent proposals suggest that signaling in the cytoplasmic domain involves opposing changes in dynamics in different subdomains. However, it has been difficult to measure dynamics within the functional system, consisting of extended arrays of receptor complexes with two other proteins, CheA and CheW. We have combined hydrogen exchange mass spectrometry with vesicle template assembly of functional complexes of the receptor cytoplasmic domain to reveal that there are significant signaling-associated changes in exchange, and these changes localize to key regions of the receptor involved in the excitation and adaptation responses. The methylation subdomain exhibits complex changes that include slower hydrogen exchange in complexes in a kinase-activating state, which may be partially consistent with proposals that this subdomain is stabilized in this state. The signaling subdomain exhibits significant protection from hydrogen exchange in complexes in a kinase-activating state, suggesting a tighter and/or larger interaction interface with CheA and CheW in this state. These first measurements of the stability of protein subdomains within functional signaling complexes demonstrate the promise of this approach for measuring functionally important protein dynamics within the various physiologically relevant states of multiprotein complexes. PMID:25420045

A non-catalytic N-terminal domain negatively influences the nucleotide exchange activity of translation elongation factor 1Bα.

Science.gov (United States)

Trosiuk, Tetiana V; Shalak, Vyacheslav F; Szczepanowski, Roman H; Negrutskii, Boris S; El'skaya, Anna V

2016-02-01

Eukaryotic translation elongation factor 1Bα (eEF1Bα) is a functional homolog of the bacterial factor EF-Ts, and is a component of the macromolecular eEF1B complex. eEF1Bα functions as a catalyst of guanine nucleotide exchange on translation elongation factor 1A (eEF1A). The C-terminal domain of eEF1Bα is necessary and sufficient for its catalytic activity, whereas the N-terminal domain interacts with eukaryotic translation elongation factor 1Bγ (eEF1Bγ) to form a tight complex. However, eEF1Bγ has been shown to enhance the catalytic activity of eEF1Bα attributed to the C-terminal domain of eEF1Bα. This suggests that the N-terminal domain of eEF1Bα may in some way influence the guanine nucleotide exchange process. We have shown that full-length recombinant eEF1Bα and its truncated forms are non-globular proteins with elongated shapes. Truncation of the N-terminal domain of eEF1Bα, which is dispensable for catalytic activity, resulted in acceleration of the rate of guanine nucleotide exchange on eEF1A compared to full-length eEF1Bα. A similar effect on the catalytic activity of eEF1Bα was observed after its interaction with eEF1Bγ. We suggest that the non-catalytic N-terminal domain of eEF1Bα may interfere with eEF1A binding to the C-terminal catalytic domain, resulting in a decrease in the overall rate of the guanine nucleotide exchange reaction. Formation of a tight complex between the eEF1Bγ and eEF1Bα N-terminal domains abolishes this inhibitory effect. © 2015 FEBS.

Probing slowly exchanging protein systems via {sup 13}C{sup {alpha}}-CEST: monitoring folding of the Im7 protein

Energy Technology Data Exchange (ETDEWEB)

Hansen, Alexandar L.; Bouvignies, Guillaume; Kay, Lewis E., E-mail: kay@pound.med.utoronto.ca [University of Toronto, Departments of Molecular Genetics, Biochemistry and Chemistry (Canada)

2013-03-15

A {sup 13}C{sup {alpha}} chemical exchange saturation transfer based experiment is presented for the study of protein systems undergoing slow interconversion between an 'observable' ground state and one or more 'invisible' excited states. Here a labeling strategy whereby [2-{sup 13}C]-glucose is the sole carbon source is exploited, producing proteins with {sup 13}C at the C{sup {alpha}} position, while the majority of residues remain unlabeled at CO or C{sup {beta}}. The new experiment is demonstrated with an application to the folding reaction of the Im7 protein that involves an on-pathway excited state. The obtained excited state {sup 13}C{sup {alpha}} chemical shifts are cross validated by comparison to values extracted from analysis of CPMG relaxation dispersion profiles, establishing the utility of the methodology.

The interaction of protein S with the phospholipid surface is essential for the activated protein C-independent activity of protein S

NARCIS (Netherlands)

van Wijnen, M.; Stam, J. G.; van't Veer, C.; Meijers, J. C.; Reitsma, P. H.; Bertina, R. M.; Bouma, B. N.

1996-01-01

Protein S is a vitamin-K dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Recent data showed a direct anticoagulant role of protein S independent of APC, as demonstrated by the inhibition of prothrombinase and tenase activity both in

Rapid Conformational Analysis of Protein Drugs in Formulation by Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS)

DEFF Research Database (Denmark)

Esmail Nazari, Zeinab; van de Weert, Marco; Bou-Assaf, George

2016-01-01

Hydrogen Deuterium Exchange coupled to Mass Spectrometry (HDX-MS) has become an established method for analysis of protein higher-order structure. Here, we use HDX-MS methodology based on manual Solid-Phase Extraction (SPE) to allow fast and simplified conformational analysis of proteins under...... pharmaceutically relevant formulation conditions. Of significant practical utility, the methodology allows global HDX-MS analyses to be performed without refrigeration or external cooling of the setup. In Mode 1, we used DMSO-containing solvents for SPE, allowing the HDX-MS analysis to be performed at acceptable...... in formulation, using an internal HDX reference peptide (P7I) to control for any sample-to-sample variations in back exchange. Advantages of the methodology include low sample use, optimized excipient removal using multiple solvents, and fast data acquisition. Our results indicate that the SPE-HDX-MS system can...

The role of polymer nanolayer architecture on the separation performance of anion-exchange membrane adsorbers: I. Protein separations.

Science.gov (United States)

Bhut, Bharat V; Weaver, Justin; Carter, Andrew R; Wickramasinghe, S Ranil; Husson, Scott M

2011-11-01

This contribution describes the preparation of strong anion-exchange membranes with higher protein binding capacities than the best commercial resins. Quaternary amine (Q-type) anion-exchange membranes were prepared by grafting polyelectrolyte nanolayers from the surfaces of macroporous membrane supports. A focus of this study was to better understand the role of polymer nanolayer architecture on protein binding. Membranes were prepared with different polymer chain graft densities using a newly developed surface-initiated polymerization protocol designed to provide uniform and variable chain spacing. Bovine serum albumin and immunoglobulin G were used to measure binding capacities of proteins with different size. Dynamic binding capacities of IgG were measured to evaluate the impact of polymer chain density on the accessibility of large size protein to binding sites within the polyelectrolyte nanolayer under flow conditions. The dynamic binding capacity of IgG increased nearly linearly with increasing polymer chain density, which suggests that the spacing between polymer chains is sufficient for IgG to access binding sites all along the grafted polymer chains. Furthermore, the high dynamic binding capacity of IgG (>130 mg/mL) was independent of linear flow velocity, which suggests that the mass transfer of IgG molecules to the binding sites occurs primarily via convection. Overall, this research provides clear evidence that the dynamic binding capacities of large biologics can be higher for well-designed macroporous membrane adsorbers than commercial membrane or resin ion-exchange products. Specifically, using controlled polymerization leads to anion-exchange membrane adsorbers with high binding capacities that are independent of flow rate, enabling high throughput. Results of this work should help to accelerate the broader implementation of membrane adsorbers in bioprocess purification steps. Copyright © 2011 Wiley Periodicals, Inc.

Herpes simplex virus internalization into epithelial cells requires Na+/H+ exchangers and p21-activated kinases but neither clathrin- nor caveolin-mediated endocytosis.

Science.gov (United States)

Devadas, Deepika; Koithan, Thalea; Diestel, Randi; Prank, Ute; Sodeik, Beate; Döhner, Katinka

2014-11-01

Herpes simplex virus 1 (HSV-1) is an alphaherpesvirus that has been reported to infect some epithelial cell types by fusion at the plasma membrane but others by endocytosis. To determine the molecular mechanisms of productive HSV-1 cell entry, we perturbed key endocytosis host factors using specific inhibitors, RNA interference (RNAi), or overexpression of dominant negative proteins and investigated their effects on HSV-1 infection in the permissive epithelial cell lines Vero, HeLa, HEp-2, and PtK2. HSV-1 internalization required neither endosomal acidification nor clathrin- or caveolin-mediated endocytosis. In contrast, HSV-1 gene expression and internalization were significantly reduced after treatment with 5-(N-ethyl-N-isopropyl)amiloride (EIPA). EIPA blocks the activity of Na(+)/H(+) exchangers, which are plasma membrane proteins implicated in all forms of macropinocytosis. HSV-1 internalization furthermore required the function of p21-activated kinases that contribute to macropinosome formation. However, in contrast to some forms of macropinocytosis, HSV-1 did not enlist the activities of protein kinase C (PKC), tyrosine kinases, C-terminal binding protein 1, or dynamin to activate its internalization. These data suggest that HSV-1 depends on Na(+)/H(+) exchangers and p21-activated kinases either for macropinocytosis or for local actin rearrangements required for fusion at the plasma membrane or subsequent passage through the actin cortex underneath the plasma membrane. After initial replication in epithelial cells, herpes simplex viruses (HSVs) establish latent infections in neurons innervating these regions. Upon primary infection and reactivation from latency, HSVs cause many human skin and neurological diseases, particularly in immunocompromised hosts, despite the availability of effective antiviral drugs. Many viruses use macropinocytosis for virus internalization, and many host factors mediating this entry route have been identified, although the

Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression.

Science.gov (United States)

Schaper, F; Gendo, C; Eck, M; Schmitz, J; Grimm, C; Anhuf, D; Kerr, I M; Heinrich, P C

1998-11-01

Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.

Ligand exchange chromatography of free amino acids and proteins on porous microparticulate zirconium oxide

International Nuclear Information System (INIS)

Blackwell, J.A.; Carr, P.W.

1992-01-01

The Lewis acid sites present on the underlying zirconium oxide particles are responsible for the unusual elution sequence for amino acids on copper loaded, phosphated zirconium oxide supports reported in an earlier study. To more thoroughly examine the effect of these strong Lewis acid sites in this paper. The authors have studied ligand exchange chromatography on copper loaded zirconium oxide particles. It is shown here that carboxylate functional groups on amino acid solutes strongly interact with surface Lewis acid sites. Addition of competing hard Lewis bases to the eluent attenuates these specific interactions. The result is a chromatographic system with high selectivity which is also suitable for ligand exchange chromatography of proteins

The influence of zinc(II) on thioredoxin/glutathione disulfide exchange: QM/MM studies to explore how zinc(II) accelerates exchange in higher dielectric environments.

Science.gov (United States)

Kurian, Roby; Bruce, Mitchell R M; Bruce, Alice E; Amar, François G

2015-08-01

QM/MM studies were performed to explore the energetics of exchange reactions of glutathione disulfide (GSSG) and the active site of thioredoxin [Cys32-Gly33-Pro34-Cys35] with and without zinc(II), in vacuum and solvated models. The activation energy for exchange, in the absence of zinc, is 29.7 kcal mol(-1) for the solvated model. This is 3.3 kcal mol(-1) higher than the activation energy for exchange in the gas phase, due to ground state stabilization of the active site Cys-32 thiolate in a polar environment. In the presence of zinc, the activation energy for exchange is 4.9 kcal mol(-1) lower than in the absence of zinc (solvated models). The decrease in activation energy is attributed to stabilization of the charge-separated transition state, which has a 4-centered, cyclic arrangement of Zn-S-S-S with an estimated dipole moment of 4.2 D. A difference of 4.9 kcal mol(-1) in activation energy would translate to an increase in rate by a factor of about 4000 for zinc-assisted thiol-disulfide exchange. The calculations are consistent with previously reported experimental results, which indicate that metal-thiolate, disulfide exchange rates increase as a function of solvent dielectric. This trend is opposite to that observed for the influence of the dielectric environment on the rate of thiol-disulfide exchange in the absence of metal. The results suggest a dynamic role for zinc in thiol-disulfide exchange reactions, involving accessible cysteine sites on proteins, which may contribute to redox regulation and mechanistic pathways during oxidative stress.

Hydrogen/deuterium exchange in mass spectrometry.

Science.gov (United States)

Kostyukevich, Yury; Acter, Thamina; Zherebker, Alexander; Ahmed, Arif; Kim, Sunghwan; Nikolaev, Eugene

2018-03-30

The isotopic exchange approach is in use since the first observation of such reactions in 1933 by Lewis. This approach allows the investigation of the pathways of chemical and biochemical reactions, determination of structure, composition, and conformation of molecules. Mass spectrometry has now become one of the most important analytical tools for the monitoring of the isotopic exchange reactions. Investigation of conformational dynamics of proteins, quantitative measurements, obtaining chemical, and structural information about individual compounds of the complex natural mixtures are mainly based on the use of isotope exchange in combination with high resolution mass spectrometry. The most important reaction is the Hydrogen/Deuterium exchange, which is mainly performed in the solution. Recently we have developed the approach allowing performing of the Hydrogen/Deuterium reaction on-line directly in the ionization source under atmospheric pressure. Such approach simplifies the sample preparation and can accelerate the exchange reaction so that certain hydrogens that are considered as non-labile will also participate in the exchange. The use of in-ionization source H/D exchange in modern mass spectrometry for structural elucidation of molecules serves as the basic theme in this review. We will focus on the mechanisms of the isotopic exchange reactions and on the application of in-ESI, in-APCI, and in-APPI source Hydrogen/Deuterium exchange for the investigation of petroleum, natural organic matter, oligosaccharides, and proteins including protein-protein complexes. The simple scenario for adaptation of H/D exchange reactions into mass spectrometric method is also highlighted along with a couple of examples collected from previous studies. © 2018 Wiley Periodicals, Inc.

Activation of Extracellular Signal-Regulated Kinase but Not of p38 Mitogen-Activated Protein Kinase Pathways in Lymphocytes Requires Allosteric Activation of SOS

Science.gov (United States)

Jun, Jesse E.; Yang, Ming; Chen, Hang; Chakraborty, Arup K.

2013-01-01

Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cγ (PLCγ)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation. PMID:23589333

Structural Dynamics Control Allosteric Activation of Cytohesin Family Arf GTPase Exchange Factors

Energy Technology Data Exchange (ETDEWEB)

Malaby, Andrew W.; Das, Sanchaita; Chakravarthy, Srinivas; Irving, Thomas C.; Bilsel, Osman; Lambright, David G.

2018-01-01

Membrane dynamic processes including vesicle biogenesis depend on Arf guanosine triphosphatase (GTPase) activation by guanine nucleotide exchange factors (GEFs) containing a catalytic Sec7 domain and a membrane-targeting module such as a pleckstrin homology (PH) domain. The catalytic output of cytohesin family Arf GEFs is controlled by autoinhibitory interactions that impede accessibility of the exchange site in the Sec7 domain. These restraints can be relieved through activator Arf-GTP binding to an allosteric site comprising the PH domain and proximal autoinhibitory elements (Sec7-PH linker and C-terminal helix). Small-angle X-ray scattering and negative-stain electron microscopy were used to investigate the structural organization and conformational dynamics of cytohesin-3 (Grp1) in autoinhibited and active states. The results support a model in which hinge dynamics in the autoinhibited state expose the activator site for Arf-GTP binding, while subsequent C-terminal helix unlatching and repositioning unleash conformational entropy in the Sec7-PH linker to drive exposure of the exchange site.

Top-Down Hydrogen-Deuterium Exchange Analysis of Protein Structures Using Ultraviolet Photodissociation.

Science.gov (United States)

Brodie, Nicholas I; Huguet, Romain; Zhang, Terry; Viner, Rosa; Zabrouskov, Vlad; Pan, Jingxi; Petrotchenko, Evgeniy V; Borchers, Christoph H

2018-03-06

Top-down hydrogen-deuterium exchange (HDX) analysis using electron capture or transfer dissociation Fourier transform mass spectrometry (FTMS) is a powerful method for the analysis of secondary structure of proteins in solution. The resolution of the method is a function of the degree of fragmentation of backbone bonds in the proteins. While fragmentation is usually extensive near the N- and C-termini, electron capture (ECD) or electron transfer dissociation (ETD) fragmentation methods sometimes lack good coverage of certain regions of the protein, most often in the middle of the sequence. Ultraviolet photodissociation (UVPD) is a recently developed fast-fragmentation technique, which provides extensive backbone fragmentation that can be complementary in sequence coverage to the aforementioned electron-based fragmentation techniques. Here, we explore the application of electrospray ionization (ESI)-UVPD FTMS on an Orbitrap Fusion Lumos Tribrid mass spectrometer to top-down HDX analysis of proteins. We have incorporated UVPD-specific fragment-ion types and fragment-ion mixtures into our isotopic envelope fitting software (HDX Match) for the top-down HDX analysis. We have shown that UVPD data is complementary to ETD, thus improving the overall resolution when used as a combined approach.

Deletion mutants of the Escherichia coli K-12 mannitol permease: dissection of transport-phosphorylation, phospho-exchange, and mannitol-binding activities.

Science.gov (United States)

Grisafi, P L; Scholle, A; Sugiyama, J; Briggs, C; Jacobson, G R; Lengeler, J W

1989-05-01

We have constructed a series of deletion mutations of the cloned Escherichia coli K-12 mtlA gene, which encodes the mannitol-specific enzyme II of the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system. This membrane-bound permease consists of 637 amino acid residues and is responsible for the concomitant transport and phosphorylation of D-mannitol in E. coli. Deletions into the 3' end of mtlA were constructed by exonuclease III digestion. Restriction mapping of the resultant plasmids identified several classes of deletions that lacked approximately 5% to more than 75% of the gene. Immunoblotting experiments revealed that many of these plasmids expressed proteins within the size range predicted by the restriction analyses, and all of these proteins were membrane localized, which demonstrated that none of the C-terminal half of the permease is required for membrane insertion. Functional analyses of the deletion proteins, expressed in an E. coli strain deleted for the chromosomal copy of mtlA, showed that all but one of the strains containing confirmed deletions were inactive in transport and PEP-dependent phosphorylation of mannitol, but deletions removing up to at least 117 amino acid residues from the C terminus of the permease were still active in catalyzing phospho exchange between mannitol 1-phosphate and mannitol. A deletion protein that lacked 240 residues from the C terminus of the permease was inactive in phospho exchange but still bound mannitol with high affinity. These experiments localize sites important for transport and PEP-dependent phosphorylation to the extreme C terminus of the mannitol permease, sites important for phospho exchange to between residues 377 and 519, and sites necessary for mannitol binding to the N-terminal 60% of the molecule. The results are discussed with respect to the fact that the mannitol permease consists of structurally independent N- and C-terminal domains.

Comparative Molecular Dynamics Simulations of Mitogen-Activated Protein Kinase-Activated Protein Kinase 5

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Inger Lindin

2014-03-01

Full Text Available The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD simulations of: (1 MK5 alone; (2 MK5 in complex with an inhibitor; and (3 MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding.

Characterization of mu s-ms dynamics of proteins using a combined analysis of N-15 NMR relaxation and chemical shift: Conformational exchange in plastocyanin induced by histidine protonations

DEFF Research Database (Denmark)

Hass, M. A. S.; Thuesen, Marianne Hallberg; Christensen, Hans Erik Mølager

2004-01-01

of the exchanging species can be determined independently of the relaxation rates. The applicability of the approach is demonstrated by a detailed analysis of the conformational exchange processes previously observed in the reduced form of the blue copper protein, plastocyanin from the cyanobacteria Anabaena......An approach is presented that allows a detailed, quantitative characterization of conformational exchange processes in proteins on the mus-ms time scale. The approach relies on a combined analysis of NMR relaxation rates and chemical shift changes and requires that the chemical shift...... quantitatively by the correlation between the R-ex terms and the corresponding chemical shift differences of the exchanging species. By this approach, the R-ex terms of N-15 nuclei belonging to contiguous regions in the protein could be assigned to the same exchange process. Furthermore, the analysis...

Semiautomatic exchanger of samples for carry out neutron activation analysis

International Nuclear Information System (INIS)

Aguilar H, F.; Quintana C, G.; Torres R, C. E.; Mejia J, J. O.

2015-09-01

In this paper the design methodology and implementation of a semiautomatic exchanger of samples for the Analysis Laboratory by Neutron Activation of the Reactor department is presented. Taking into account the antecedents, the necessities of improvement are described, as well as the equipment that previously contained the Laboratory. The project of the semiautomatic exchanger of samples was developed at Instituto Nacional de Investigaciones Nucleares, with its own technology to increase independence from commercial equipment. Each element of the semiautomatic exchanger of samples is described both in the design phase as construction. The achieved results are positive and encouraging for the fulfillment of the proposed objective that is to increase the capacity of the Laboratory. (Author)

Protein kinase activity associated with the corticosteroid binder IB

International Nuclear Information System (INIS)

Vujicic, M.; Djordjevic-Markovic, R.; Radic, O.; Krstic, M.; Kanazir, D.

1997-01-01

The physiological effects elicited by glucocorticoids are mediated via glucocorticoid receptors (GR). Analysis of specific glucocorticoid binding to radioactively labelled [ 3 H] triamcinolone acetonide in rat liver cytosol and analysis by ion exchange chromatography have revealed the presence of two distinct molecular species. The major form, designated as binder II appears to correspond to the well characterized glucocorticoid receptor by virtue of its size, charge, steroid binding characteristics and ability to bind to DNA.The second form, designated as corticosteroid binder IB, is a minor binding component in the liver. The binder IB differs from the binder II receptor by virtue of its lower molecular weight and its elution in the pre gradient of DEAE-Sephadex A-50 column which retains the un activated binder II receptor complexes. We examined the kinase activity of partially purified corticosteroid binder IB. Using (γ 3 2 P) ATP we detected kinase activity associated with the IB fraction from the rat liver. This kinase phosphorylate mixed histones and and dose not phosphorylate IB protein in vitro. The kinase activity is completely inhibited by the addition of Mg 2 + ions and is partially inhibited by the addition of Ca 2 +ions. (author)

Ion-Exchange Chromatography: Basic Principles and Application.

Science.gov (United States)

Cummins, Philip M; Rochfort, Keith D; O'Connor, Brendan F

2017-01-01

Ion-Exchange Chromatography (IEC) allows for the separation of ionizable molecules on the basis of differences in charge properties. Its large sample-handling capacity, broad applicability (particularly to proteins and enzymes), moderate cost, powerful resolving ability, and ease of scale-up and automation have led to it becoming one of the most versatile and widely used of all liquid chromatography (LC) techniques. In this chapter, we review the basic principles of IEC, as well as the broader criteria for selecting IEC conditions. By way of further illustration, we outline basic laboratory protocols to partially purify a soluble serine peptidase from bovine whole brain tissue, covering crude tissue extract preparation through to partial purification of the target enzyme using anion-exchange chromatography. Protocols for assaying total protein and enzyme activity in both pre- and post-IEC fractions are also described.

Hydrogen Exchange Mass Spectrometry of Functional Membrane-bound Chemotaxis Receptor Complexes

Science.gov (United States)

Koshy, Seena S.; Eyles, Stephen J.; Weis, Robert M.; Thompson, Lynmarie K.

2014-01-01

The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended membrane-bound arrays. Previous studies have shown that attractant binding induces a small (~2 Å) piston displacement of one helix of the periplasmic and transmembrane domains towards the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 Å away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston motion could propagate through a dynamic domain to control CheA kinase activity. To address this, we have developed a method for measuring dynamics of the receptor cytoplasmic fragment (CF) in functional complexes with CheA and CheW. Hydrogen exchange mass spectrometry (HDX-MS) measurements of global exchange of CF demonstrate that CF exhibits significantly slower exchange in functional complexes than in solution. Since the exchange rates in functional complexes are comparable to that of other proteins of similar structure, the CF appears to be a well-structured protein within these complexes, which is compatible with its role in propagating a signal that appears to be a tiny conformational change in the periplasmic and transmembrane domains of the receptor. We also demonstrate the feasibility of this protocol for local exchange measurements, by incorporating a pepsin digest step to produce peptides with 87% sequence coverage and only 20% back exchange. This method extends HDX-MS to membrane-bound functional complexes without detergents that may perturb the stability or structure of the system. PMID:24274333

Neurodevelopmental disease-associated de novo mutations and rare sequence variants affect TRIO GDP/GTP exchange factor activity.

Science.gov (United States)

Katrancha, Sara M; Wu, Yi; Zhu, Minsheng; Eipper, Betty A; Koleske, Anthony J; Mains, Richard E

2017-12-01

Bipolar disorder, schizophrenia, autism and intellectual disability are complex neurodevelopmental disorders, debilitating millions of people. Therapeutic progress is limited by poor understanding of underlying molecular pathways. Using a targeted search, we identified an enrichment of de novo mutations in the gene encoding the 330-kDa triple functional domain (TRIO) protein associated with neurodevelopmental disorders. By generating multiple TRIO antibodies, we show that the smaller TRIO9 isoform is the major brain protein product, and its levels decrease after birth. TRIO9 contains two guanine nucleotide exchange factor (GEF) domains with distinct specificities: GEF1 activates both Rac1 and RhoG; GEF2 activates RhoA. To understand the impact of disease-associated de novo mutations and other rare sequence variants on TRIO function, we utilized two FRET-based biosensors: a Rac1 biosensor to study mutations in TRIO (T)GEF1, and a RhoA biosensor to study mutations in TGEF2. We discovered that one autism-associated de novo mutation in TGEF1 (K1431M), at the TGEF1/Rac1 interface, markedly decreased its overall activity toward Rac1. A schizophrenia-associated rare sequence variant in TGEF1 (F1538Intron) was substantially less active, normalized to protein level and expressed poorly. Overall, mutations in TGEF1 decreased GEF1 activity toward Rac1. One bipolar disorder-associated rare variant (M2145T) in TGEF2 impaired inhibition by the TGEF2 pleckstrin-homology domain, resulting in dramatically increased TGEF2 activity. Overall, genetic damage to both TGEF domains altered TRIO catalytic activity, decreasing TGEF1 activity and increasing TGEF2 activity. Importantly, both GEF changes are expected to decrease neurite outgrowth, perhaps consistent with their association with neurodevelopmental disorders. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin

Science.gov (United States)

Lin, Changsheng; Ear, Jason; Midde, Krishna; Lopez-Sanchez, Inmaculada; Aznar, Nicolas; Garcia-Marcos, Mikel; Kufareva, Irina; Abagyan, Ruben; Ghosh, Pradipta

2014-01-01

A long-standing issue in the field of signal transduction is to understand the cross-talk between receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major and distinct signaling hubs that control eukaryotic cell behavior. Although stimulation of many RTKs leads to activation of trimeric G proteins, the molecular mechanisms behind this phenomenon remain elusive. We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins. Using a combination of homology modeling, protein–protein interaction, and kinase assays, we demonstrate that a stretch of ∼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands. Using protein–protein interaction assays, we demonstrated that both SH2 and GEF domains of GIV are required for the formation of a ligand-activated ternary complex between GIV, Gαi, and growth factor receptors and for activation of Gαi after growth factor stimulation. Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV—Akt enhancement, actin remodeling, and cell migration. The mechanistic and structural insights gained here shed light on the long-standing questions surrounding RTK/G protein cross-talk, set a novel paradigm, and characterize a unique pharmacological target for uncoupling GIV-dependent signaling downstream of multiple oncogenic RTKs. PMID:25187647

Exploration of the dynamic properties of protein complexes predicted from spatially constrained protein-protein interaction networks.

Directory of Open Access Journals (Sweden)

Eric A Yen

2014-05-01

Full Text Available Protein complexes are not static, but rather highly dynamic with subunits that undergo 1-dimensional diffusion with respect to each other. Interactions within protein complexes are modulated through regulatory inputs that alter interactions and introduce new components and deplete existing components through exchange. While it is clear that the structure and function of any given protein complex is coupled to its dynamical properties, it remains a challenge to predict the possible conformations that complexes can adopt. Protein-fragment Complementation Assays detect physical interactions between protein pairs constrained to ≤8 nm from each other in living cells. This method has been used to build networks composed of 1000s of pair-wise interactions. Significantly, these networks contain a wealth of dynamic information, as the assay is fully reversible and the proteins are expressed in their natural context. In this study, we describe a method that extracts this valuable information in the form of predicted conformations, allowing the user to explore the conformational landscape, to search for structures that correlate with an activity state, and estimate the abundance of conformations in the living cell. The generator is based on a Markov Chain Monte Carlo simulation that uses the interaction dataset as input and is constrained by the physical resolution of the assay. We applied this method to an 18-member protein complex composed of the seven core proteins of the budding yeast Arp2/3 complex and 11 associated regulators and effector proteins. We generated 20,480 output structures and identified conformational states using principle component analysis. We interrogated the conformation landscape and found evidence of symmetry breaking, a mixture of likely active and inactive conformational states and dynamic exchange of the core protein Arc15 between core and regulatory components. Our method provides a novel tool for prediction and

Anxiety and depression with neurogenesis defects in exchange protein directly activated by cAMP 2-deficient mice are ameliorated by a selective serotonin reuptake inhibitor, Prozac

Science.gov (United States)

Zhou, L; Ma, S L; Yeung, P K K; Wong, Y H; Tsim, K W K; So, K F; Lam, L C W; Chung, S K

2016-01-01

Intracellular cAMP and serotonin are important modulators of anxiety and depression. Fluoxetine, a selective serotonin reuptake inhibitor (SSRI) also known as Prozac, is widely used against depression, potentially by activating cAMP response element-binding protein (CREB) and increasing brain-derived neurotrophic factor (BDNF) through protein kinase A (PKA). However, the role of Epac1 and Epac2 (Rap guanine nucleotide exchange factors, RAPGEF3 and RAPGEF4, respectively) as potential downstream targets of SSRI/cAMP in mood regulations is not yet clear. Here, we investigated the phenotypes of Epac1 (Epac1−/−) or Epac2 (Epac2−/−) knockout mice by comparing them with their wild-type counterparts. Surprisingly, Epac2−/− mice exhibited a wide range of mood disorders, including anxiety and depression with learning and memory deficits in contextual and cued fear-conditioning tests without affecting Epac1 expression or PKA activity. Interestingly, rs17746510, one of the three single-nucleotide polymorphisms (SNPs) in RAPGEF4 associated with cognitive decline in Chinese Alzheimer's disease (AD) patients, was significantly correlated with apathy and mood disturbance, whereas no significant association was observed between RAPGEF3 SNPs and the risk of AD or neuropsychiatric inventory scores. To further determine the detailed role of Epac2 in SSRI/serotonin/cAMP-involved mood disorders, we treated Epac2−/− mice with a SSRI, Prozac. The alteration in open field behavior and impaired hippocampal cell proliferation in Epac2−/− mice were alleviated by Prozac. Taken together, Epac2 gene polymorphism is a putative risk factor for mood disorders in AD patients in part by affecting the hippocampal neurogenesis. PMID:27598965

Electrochemical reduction of disulfide-containing proteins for hydrogen/deuterium exchange monitored by mass spectrometry

DEFF Research Database (Denmark)

Mysling, Simon; Salbo, Rune; Ploug, Michael

2014-01-01

Characterization of disulfide bond-containing proteins by hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) requires reduction of the disulfide bonds under acidic and cold conditions, where the amide hydrogen exchange reaction is quenched (pH 2.5, 0 °C). The reduction typically...... of TCEP. In the present study, we explore the feasibility of using electrochemical reduction as a substitute for TCEP in HDX-MS analyses. Our results demonstrate that efficient disulfide bond reduction is readily achieved by implementing an electrochemical cell into the HDX-MS workflow. We also identify...... some challenges in using electrochemical reduction in HDX-MS analyses and provide possible conditions to attenuate these limitations. For example, high salt concentrations hamper disulfide bond reduction, necessitating additional dilution of the sample with aqueous acidic solution at quench conditions....

Determinants of protein elution rates from preparative ion-exchange adsorbents.

Science.gov (United States)

Angelo, James M; Lenhoff, Abraham M

2016-04-01

The rate processes involved in elution in preparative chromatography can affect both peak resolution and hence selectivity as well as practical factors such as facility fit. These processes depend on the physical structure of the adsorbent particles, the amount of bound solute, the solution conditions for operation or some combination of these factors. Ion-exchange adsorbents modified with covalently attached or grafted polymer layers have become widely used in preparative chromatography. Their often easily accessible microstructures offer substantial binding capacities for biomolecules, but elution has sometimes been observed to be undesirably slow. In order to determine which physicochemical phenomena control elution behavior, commercially available cellulosic, dextran-grafted and unmodified agarose materials were characterized here by their elution profiles at various conditions, including different degrees of loading. Elution data were analyzed under the assumption of purely diffusion-limited control, including the role of pore structure properties such as porosity and tortuosity. In general, effective elution rates decreased with the reduction of accessible pore volume, but differences among different proteins indicated the roles of additional factors. Additional measurements and analysis, including the use of confocal laser scanning microscopy to observe elution within single chromatographic particles, indicated the importance of protein association within the particle during elution. The use of protein stabilizing agents was explored in systems presenting atypical elution behavior, and l-arginine and disaccharide excipients were shown to alleviate the effects for one protein, lysozyme, in the presence of sodium chloride. Incorporation of these excipients into eluent buffer gave rise to faster elution and significantly lower pool volumes in elution from polymer-modified adsorbents. Copyright © 2016 Elsevier B.V. All rights reserved.

The guanine nucleotide exchange factor RIC8 regulates conidial germination through Gα proteins in Neurospora crassa.

Directory of Open Access Journals (Sweden)

Carla J Eaton

Full Text Available Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth.

Amine-functionalized, silver-exchanged zeolite NaY: Preparation, characterization and antibacterial activity

Energy Technology Data Exchange (ETDEWEB)

Hanim, Siti Aishah Mohd; Malek, Nik Ahmad Nizam Nik, E-mail: niknizam@fbb.utm.my; Ibrahim, Zaharah

2016-01-01

Graphical abstract: - Highlights: • Functionalization of Ag-exchanged zeolite NaY with 3-aminopropyltriethoxysilane APTES (ZSA) as antibacterial agent. • Antibacterial assay of ZSA was performed against Escherichia coli ATCC11229 and Staphylococcus aureus ATCC6538. • Functionalization of Ag-exchanged zeolite NaY with APTES significantly increased the antibacterial agent. • Different mechanisms of bacterial death were suggested for each bacteria type by the functionalized Ag-exchanged zeolite NaY. - Abstract: Amine-functionalized, silver-exchanged zeolite NaY (ZSA) were prepared with three different concentrations of 3-aminopropyltriethoxysilane (APTES) (0.01, 0.20 and 0.40 M) and four different concentrations of silver ions (25%, 50%, 100% and 200% from zeolite cation exchange capacity (CEC)). The samples were characterized by Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), energy-dispersive X-ray (EDX), surface area analysis, thermogravimetric analysis (TGA) and zeta potential (ZP) analysis. The FTIR results indicated that the zeolite was functionalized by APTES and that the intensity of the peaks corresponding to APTES increased as the concentration of APTES used was increased. The antibacterial activities of the silver-exchanged zeolite NaY (ZS) and ZSA were studied against Escherichia coli ATCC11229 and Staphylococcus aureus ATCC6538 using the disc diffusion technique (DDT) and minimum inhibitory concentration (MIC). The antibacterial activity of ZSA increased with the increase in APTES on ZS, and E. coli was more susceptible towards the sample compared to S. aureus. The FESEM micrographs of the bacteria after contact with the ZSA suggested different mechanisms of bacterial death for these two bacteria due to exposure to the studied sample. The functionalization of ZS with APTES improved the antibacterial activity of the silver-zeolite, depending on the concentration of silver

The case for foreign exchange intervention: the government as an active reserve manager

OpenAIRE

Christopher J. Neely

2005-01-01

This paper argues that major governments should actively manage their foreign exchange portfolios to maximize the risk-adjusted return to the taxpayer by exploiting long-term, fundamental based predictability in floating exchange rates. Such transactions—equivalent to foreign exchange intervention—would improve welfare by transferring risk from private agents to the risk-tolerant government. Interventions explicitly designed to profit the reserve management authority would be more likely to b...

Hydrogen exchange rate of tyrosine hydroxyl groups in proteins as studied by the deuterium isotope effect on C(zeta) chemical shifts.

Science.gov (United States)

Takeda, Mitsuhiro; Jee, Jungoo; Ono, Akira Mei; Terauchi, Tsutomu; Kainosho, Masatsune

2009-12-30

We describe a new NMR method for monitoring the individual hydrogen exchange rates of the hydroxyl groups of tyrosine (Tyr) residues in proteins. The method utilizes (2S,3R)-[beta(2),epsilon(1,2)-(2)H(3);0,alpha,beta,zeta-(13)C(4);(15)N]-Tyr, zeta-SAIL Tyr, to detect and assign the (13)C(zeta) signals of Tyr rings efficiently, either by indirect (1)H-detection through 7-8 Hz (1)H(delta)-(13)C(zeta) spin couplings or by direct (13)C(zeta) observation. A comparison of the (13)C(zeta) chemical shifts of three Tyr residues of an 18.2 kDa protein, EPPIb, dissolved in H(2)O and D(2)O, revealed that all three (13)C(zeta) signals in D(2)O appeared at approximately 0.13 ppm ( approximately 20 Hz at 150.9 MHz) higher than those in H(2)O. In a H(2)O/D(2)O (1:1) mixture, however, one of the three signals for (13)C(zeta) appeared as a single peak at the averaged chemical shifts, and the other two appeared as double peaks at exactly the same chemical shifts in H(2)O and D(2)O, in 50 mM phosphate buffer (pH 6.6) at 40 degrees C. These three peaks were assigned to Tyr-36, Tyr-120, and Tyr-30, from the lower to higher chemical shifts, respectively. The results indicate that the hydroxyl proton of Tyr-120 exchanges faster than a few milliseconds, whereas those of Tyr-30 and Tyr-36 exchange more slowly. The exchange rate of the Tyr-30 hydroxyl proton, k(ex), under these conditions was determined by (13)C NMR exchange spectroscopy (EXSY) to be 9.2 +/- 1.1 s(-1). The Tyr-36 hydroxyl proton, however, exchanges too slowly to be determined by EXSY. These profound differences among the hydroxyl proton exchange rates are closely related to their relative solvent accessibility and the hydrogen bonds associated with the Tyr hydroxyl groups in proteins.

[Correlation between gaseous exchange rate, body temperature, and mitochondrial protein content in the liver of mice].

Science.gov (United States)

Muradian, Kh K; Utko, N O; Mozzhukhina, T H; Pishel', I M; Litoshenko, O Ia; Bezrukov, V V; Fraĭfel'd, V E

2002-01-01

Correlative and regressive relations between the gaseous exchange, thermoregulation and mitochondrial protein content were analyzed by two- and three-dimensional statistics in mice. It has been shown that the pair wise linear methods of analysis did not reveal any significant correlation between the parameters under exploration. However, it became evident at three-dimensional and non-linear plotting for which the coefficients of multivariable correlation reached and even exceeded 0.7-0.8. The calculations based on partial differentiation of the multivariable regression equations allow to conclude that at certain values of VO2, VCO2 and body temperature negative relations between the systems of gaseous exchange and thermoregulation become dominating.

Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

DEFF Research Database (Denmark)

Ngo, HT; Pham, Long; Kim, JW

2013-01-01

Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray...... inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA...... increased HCV IRES-mediated translation and MAPKAPK3-dependent HCV IRES activity was further increased by core protein. These data suggest that HCV core may modulate MAPKAPK3 to facilitate its own propagation....

An NMR-based quenched hydrogen exchange investigation of model amyloid fibrils formed by cold shock protein A.

Science.gov (United States)

Alexandrescu, A T

2001-01-01

Acid-denatured cold shock protein A (CspA) self-assembles into polymers with properties typical of amyloid fibrils. In the present work, a quenched hydrogen exchange experiment was used to probe the interactions of CspA fibrils with solvent. Exchange was initiated by incubating suspensions of fibrils in D2O, and quenched by flash freezing. Following lyophilization, exchange-quenched samples were dissolved in 90% DMSO/10% D2O, giving DMSO-denatured monomers. Intrinsic exchange rates for denatured CspA in 90% DMSO/10% D2O (pH* 4.5) were sufficiently slow (approximately 1 x 10(-3) min-1) to enable quantification of NMR signal intensity decays due to H/D exchange in the fibrils. Hydrogen exchange rate constants for CspA fibrils were found to vary less than 3-fold from a mean value of 5 x 10(-5) min-1. The uniformity of rate constants suggests that exchange is in the EX1 limit, and that the mechanism of exchange involves a cooperative dissociation of CspA monomers from fibrils, concomitant with unfolding. Previous studies indicated that the highest protection factors in native CspA are approximately 10(3), and that protection factors for the acid-denatured monomer precursors of CspA fibrils are close to unity. Because exchange in is in the EX1 regime, it is only possible to place a lower limit of at least 10(5) on protection factors in CspA fibrils. The observation that all amide protons are protected from exchange indicates that the entire CspA polypeptide chain is structured in the fibrils.

Isolation of two biologically active cell surface proteins from Brucella abortus by chromatofocusing

International Nuclear Information System (INIS)

Tabatabai, L.B.; Deyoe, B.L.

1983-01-01

Brucella abortus contains a group of immunogenic cell surface proteins which have potential value as a vaccine or as a diagnostic reagent for the prevention and diagnosis of bovine brucellosis. Under nondenaturing conditions, these proteins range in molecular weight from 10,000-124,000, as determined by high performance liquid chromatography (HPLC) on TSK 3000sw. By analytical isoelectrofocusing, 6 major protein bands could be distinguished with pI's ranging from 4.0 to 6.0 and 3 additional major proteins with pI's of 7.5, 9.5, and 10. By chromatofocusing on Polybuffer Exchanger 94 with a pH gradient from 6-4, two of the six proteins from pI 4-6 were separated, a pI 4.9 and a pI 4.7 protein; a third fraction contained the high pI proteins. The former two proteins were homogeneous by analytical isoelectrofocusing, and a molecular weight of 54,000 daltons was found for both protein species by HPLC on TSK 3000sw. The pI 4-6 and not the pI 9.5 and 10 proteins, could be radiolabeled when intact cells were radioiodinated with diazotized ( 125 I)-iodosulfanilic acid. Biological activity of the proteins as assessed in lemmings indicated that immunization with the pI 4.7 and 4.9 proteins afforded better protection against experimental brucellosis than immunization with the high pI proteins. These results support our view that a single surface protein may be sufficient for the prevention of experimental brucellosis

Isolation of two biologically active cell surface proteins from Brucella abortus by chromatofocusing

Energy Technology Data Exchange (ETDEWEB)

Tabatabai, L.B.; Deyoe, B.L.

1983-01-01

Brucella abortus contains a group of immunogenic cell surface proteins which have potential value as a vaccine or as a diagnostic reagent for the prevention and diagnosis of bovine brucellosis. Under nondenaturing conditions, these proteins range in molecular weight from 10,000-124,000, as determined by high performance liquid chromatography (HPLC) on TSK 3000sw. By analytical isoelectrofocusing, 6 major protein bands could be distinguished with pI's ranging from 4.0 to 6.0 and 3 additional major proteins with pI's of 7.5, 9.5, and 10. By chromatofocusing on Polybuffer Exchanger 94 with a pH gradient from 6-4, two of the six proteins from pI 4-6 were separated, a pI 4.9 and a pI 4.7 protein; a third fraction contained the high pI proteins. The former two proteins were homogeneous by analytical isoelectrofocusing, and a molecular weight of 54,000 daltons was found for both protein species by HPLC on TSK 3000sw. The pI 4-6 and not the pI 9.5 and 10 proteins, could be radiolabeled when intact cells were radioiodinated with diazotized (/sup 125/I)-iodosulfanilic acid. Biological activity of the proteins as assessed in lemmings indicated that immunization with the pI 4.7 and 4.9 proteins afforded better protection against experimental brucellosis than immunization with the high pI proteins. These results support our view that a single surface protein may be sufficient for the prevention of experimental brucellosis.

Oxysterol-binding Protein Activation at Endoplasmic Reticulum-Golgi Contact Sites Reorganizes Phosphatidylinositol 4-Phosphate Pools*

OpenAIRE

Goto, Asako; Charman, Mark; Ridgway, Neale D.

2015-01-01

Oxysterol-binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi/trans-Golgi network. 25-Hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to de...

Effect of complete protein 4.1R deficiency on ion transport properties of murine erythrocytes

International Nuclear Information System (INIS)

Rivera, Alicia; De Franceschi, Lucia; Peters, Luanne L.; Gascard, Philippe; Mohandas, Narla; Brugnara, Carlo

2006-01-01

Moderate hemolytic anemia, abnormal erythrocyte morphology (spherocytosis), and decreased membrane stability are observed in mice with complete deficiency of all erythroid protein 4.1 protein isoforms (4.1-/-; Shi TS et al., J. Clin. Invest. 103:331,1999). We have examined the effects of erythroid protein 4.1 (4.1R) deficiency on erythrocyte cation transport and volume regulation. 4.1-/- mice exhibited erythrocyte dehydration that was associated with reduced cellular K and increased Na content. Increased Na permeability was observed in these mice, mostly mediated by Na/H exchange with normal Na-K pump and Na-K-2Cl cotransport activities. The Na/H exchange of 4.1-/- erythrocytes was markedly activated by exposure to hypertonic conditions (18.2+- 3.2 in 4.1 -/- vs.9.8 +- 1.3 mmol/1013 cell x h in control mice), with an abnormal dependence on osmolarity, (K0.5=417 +- 42 in 4.1 -/- vs. 460 +- 35 mOsmin control mice) suggestive of an up-regulated functional state. While the affinity for internal protons was not altered (K0.5= 489.7 +- 0.7 vs.537.0 +- 0.56 nM in control mice), the Vmax of the H-induced Na/H exchange activity was markedly elevated in 4.1-/- erythrocytes Vmax 91.47 Moderate hemolytic anemia, abnormal erythrocyte morphology (spherocytosis), and decreased membrane stability are observed in mice with complete deficiency of all erythroid protein 4.1 protein isoforms (4.1-/-; Shi TSet al., J. Clin. Invest. 103:331,1999). We have examined the effects of erythroid protein 4.1 (4.1R) deficiency on erythrocyte cation transport and volume regulation. 4.1-/- mice exhibited erythrocyte dehydration that was associated with reduced cellular K and increased Na content. Increased Na permeability was observed in these mice, mostly mediated by Na/H exchange with normal Na-K pump and Na-K-2Cl cotransport activities. The Na/H exchange of 4.1-/- erythrocytes was markedly activated by exposure to hypertonic conditions (18.2 +- 3.2 in 4.1 -/- vs. 9.8 +- 1.3mmol/1013 cell x h in

Mitogen-activated protein kinases mediate Mycobacterium ...

Indian Academy of Sciences (India)

2012-01-19

Jan 19, 2012 ... CD44, an adhesion molecule, has been reported to be a binding site for ... receptors in mediating mitogen-activated protein kinase activation. ... surface expression and tumour necrosis factor-alpha levels, ... Abbreviations used: Abs, antibodies; ANOVA, analysis of variance; AP-1, activator protein -1; BCG, ...

A photoactivatable probe for the Na+/H+ exchanger cross-links a 66-kDa renal brush border membrane protein

International Nuclear Information System (INIS)

Ross, W.; Bertrand, W.; Morrison, A.

1990-01-01

Earlier studies on LLC-PK1 cells have demonstrated two pharmacologically distinct Na+/H+ exchangers in renal epithelia. In addition, the cDNA clone for the human Na+/H+ antiporter which is growth factor activatable has been isolated and expressed. We report here the synthesis of an amiloride analogue that can be photoactivated and labeled with 125I. This analogue covalently cross-links a 66-kDa protein of bovine renal brush border membranes. A rabbit polyclonal antibody that was directed against a 20-amino acid peptide of the cytoplasmic domain of its human Na+/H+ antiporter also gives a positive Western against 66-kDa protein of bovine brush border membranes. Thus, the photoactive probe may be helpful in the isolation and purification of the brush border Na+/H+ exchanger

Active heat exchange system development for latent heat thermal energy storage

Science.gov (United States)

Alario, J.; Kosson, R.; Haslett, R.

1980-01-01

Various active heat exchange concepts were identified from among three generic categories: scrapers, agitators/vibrators and slurries. The more practical ones were given a more detailed technical evaluation and an economic comparison with a passive tube-shell design for a reference application (300 MW sub t storage for 6 hours). Two concepts were selected for hardware development: (1) a direct contact heat exchanger in which molten salt droplets are injected into a cooler counterflowing stream of liquid metal carrier fluid, and (2) a rotating drum scraper in which molten salt is sprayed onto the circumference of a rotating drum, which contains the fluid salt is sprayed onto the circumference of a rotating drum, which contains the fluid heat sink in an internal annulus near the surface. A fixed scraper blade removes the solidified salt from the surface which was nickel plated to decrease adhesion forces. In addition to improving performance by providing a nearly constant transfer rate during discharge, these active heat exchanger concepts were estimated to cost at least 25% less than the passive tube-shell design.

Proteomics strategy for identifying candidate bioactive proteins in complex mixtures: application to the platelet releasate.

LENUS (Irish Health Repository)

O'Connor, Roisin

2010-01-01

Proteomic approaches have proven powerful at identifying large numbers of proteins, but there are fewer reports of functional characterization of proteins in biological tissues. Here, we describe an experimental approach that fractionates proteins released from human platelets, linking bioassay activity to identity. We used consecutive orthogonal separation platforms to ensure sensitive detection: (a) ion-exchange of intact proteins, (b) SDS-PAGE separation of ion-exchange fractions and (c) HPLC separation of tryptic digests coupled to electrospray tandem mass spectrometry. Migration of THP-1 monocytes in response to complete or fractionated platelet releasate was assessed and located to just one of the forty-nine ion-exchange fractions. Over 300 proteins were identified in the releasate, with a wide range of annotated biophysical and biochemical properties, in particular platelet activation, adhesion, and wound healing. The presence of PEDF and involucrin, two proteins not previously reported in platelet releasate, was confirmed by western blotting. Proteins identified within the fraction with monocyte promigratory activity and not in other inactive fractions included vimentin, PEDF, and TIMP-1. We conclude that this analytical platform is effective for the characterization of complex bioactive samples.

The effect of monetary policy of Central Bank on activities of Tehran Stock Exchange

Directory of Open Access Journals (Sweden)

Hossein Vazifehdust

2013-08-01

Full Text Available This paper examines the relationship between monetary policy and activity of the Tehran Stock Exchange. The statistical population of the research consists of all companies listed in Tehran Stock Exchange and central bank monetary policy variables including time series generated by the central bank seasonally. For the purpose of data analysis, econometric autoregressive system models, and two-stage ADF regression with unit roots test, co-integration and reliability were used to determine level of effect and type of effect of the four components of monetary policy on exchange activity. The results of this study show that there is a strong relation between share price index and monetary policy variables and between monetary policy variables and trading volume. However, the relation between monetary policy variables and cash yield index was not so strong, but monetary policy variables’ effect on stock exchange activity was acceptable considering strong relation between the two first variables. It is suggested that if this work is done using non-linear models, it will yield better results.

Protein Kinase A (PKA) Type I Interacts with P-Rex1, a Rac Guanine Nucleotide Exchange Factor

Science.gov (United States)

Chávez-Vargas, Lydia; Adame-García, Sendi Rafael; Cervantes-Villagrana, Rodolfo Daniel; Castillo-Kauil, Alejandro; Bruystens, Jessica G. H.; Fukuhara, Shigetomo; Taylor, Susan S.; Mochizuki, Naoki; Reyes-Cruz, Guadalupe; Vázquez-Prado, José

2016-01-01

Morphology of migrating cells is regulated by Rho GTPases and fine-tuned by protein interactions and phosphorylation. PKA affects cell migration potentially through spatiotemporal interactions with regulators of Rho GTPases. Here we show that the endogenous regulatory (R) subunit of type I PKA interacts with P-Rex1, a Rac guanine nucleotide exchange factor that integrates chemotactic signals. Type I PKA holoenzyme interacts with P-Rex1 PDZ domains via the CNB B domain of RIα, which when expressed by itself facilitates endothelial cell migration. P-Rex1 activation localizes PKA to the cell periphery, whereas stimulation of PKA phosphorylates P-Rex1 and prevents its activation in cells responding to SDF-1 (stromal cell-derived factor 1). The P-Rex1 DEP1 domain is phosphorylated at Ser-436, which inhibits the DH-PH catalytic cassette by direct interaction. In addition, the P-Rex1 C terminus is indirectly targeted by PKA, promoting inhibitory interactions independently of the DEP1-PDZ2 region. A P-Rex1 S436A mutant construct shows increased RacGEF activity and prevents the inhibitory effect of forskolin on sphingosine 1-phosphate-dependent endothelial cell migration. Altogether, these results support the idea that P-Rex1 contributes to the spatiotemporal localization of type I PKA, which tightly regulates this guanine exchange factor by a multistep mechanism, initiated by interaction with the PDZ domains of P-Rex1 followed by direct phosphorylation at the first DEP domain and putatively indirect regulation of the C terminus, thus promoting inhibitory intramolecular interactions. This reciprocal regulation between PKA and P-Rex1 might represent a key node of integration by which chemotactic signaling is fine-tuned by PKA. PMID:26797121

Two Chimeric Regulators of G-protein Signaling (RGS) Proteins Differentially Modulate Soybean Heterotrimeric G-protein Cycle*

Science.gov (United States)

Roy Choudhury, Swarup; Westfall, Corey S.; Laborde, John P.; Bisht, Naveen C.; Jez, Joseph M.; Pandey, Sona

2012-01-01

Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gβ, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1–4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1–4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks. PMID:22474294

Activity systems modeling as a theoretical lens for social exchange studies

Directory of Open Access Journals (Sweden)

Ernest Jones

2016-01-01

Full Text Available The social exchange perspective seeks to acknowledge, understand and predict the dynamics of social interactions. Empirical research involving social exchange constructs have grown to be highly technical including confirmatory factor analysis to assess construct distinctiveness and structural equation modeling to assess construct causality. Each study seemingly strives to assess how underlying social exchange theoretic constructs interrelate. Yet despite this methodological depth and resultant explanatory and predictive power, a significant number of studies report findings that, once synthesized, suggest an underlying persistent threat of conceptual or construct validity brought about by a search for epistemological parsimony. Further, it is argued that a methodological approach that embraces inherent complexity such as activity systems modeling facilitates the search for simplified models while not ignoring contextual factors.

Hydrogen/deuterium exchange mass spectrometry with top-down electron capture dissociation for characterizing structural transitions of a 17 kDa protein.

Science.gov (United States)

Pan, Jingxi; Han, Jun; Borchers, Christoph H; Konermann, Lars

2009-09-09

Amide H/D exchange (HDX) mass spectrometry (MS) is widely used for protein structural studies. Traditionally, this technique involves protein labeling in D(2)O, followed by acid quenching, proteolytic digestion, and analysis of peptide deuteration levels by HPLC/MS. There is great interest in the development of alternative HDX approaches involving the top-down fragmentation of electrosprayed protein ions, instead of relying on enzymatic cleavage and solution-phase separations. A number of recent studies have demonstrated that electron capture dissociation (ECD) results in fragmentation of gaseous protein ions with little or no H/D scrambling. However, the successful application of this approach for in-depth protein conformational studies has not yet been demonstrated. The current work uses horse myoglobin as a model system for assessing the suitability of HDX-MS with top-down ECD for experiments of this kind. It is found that ECD can pinpoint the locations of protected amides with an average resolution of less than two residues for this 17 kDa protein. Native holo-myoglobin (hMb) shows considerable protection from exchange in all of its helices, whereas loops are extensively deuterated. Fraying is observable at some helix termini. Removal of the prosthetic heme group from hMb produces apo-myoglobin (aMb). Both hMb and aMb share virtually the same HDX protection pattern in helices A-E, whereas helix F is unfolded in aMb. In addition, destabilization is evident for some residues close to the beginning of helix G, the end of helix H, and the C-terminus of the protein. The structural changes reported herein are largely consistent with earlier NMR data for sperm whale myoglobin, although small differences between the two systems are evident. Our findings demonstrate that the level of structural information obtainable with top-down ECD for small to medium-sized proteins considerably surpasses that of traditional HDX-MS experiments, while at the same time greatly reducing

Characterization of a PEGylated protein therapeutic by ion exchange chromatography with on-line detection by native ESI MS and MS/MS.

Science.gov (United States)

Muneeruddin, K; Bobst, C E; Frenkel, R; Houde, D; Turyan, I; Sosic, Z; Kaltashov, I A

2017-01-16

Detailed profiling of both enzymatic (e.g., glycosylation) and non-enzymatic (e.g., oxidation and deamidation) post-translational modifications (PTMs) is frequently required for the quality assessment of protein-based drugs. Challenging as it is, this task is further complicated for the so-called second-generation biopharmaceuticals, which also contain "designer PTMs" introduced to either enhance their pharmacokinetic profiles (e.g., PEGylated proteins) or endow them with therapeutic activity (e.g., protein-drug conjugates). Such modifications of protein covalent structure can dramatically increase structural heterogeneity, making the very notion of "molecular mass" meaningless, as ions representing different glycoforms of a PEGylated protein may have nearly identical distributions of ionic current as a function of m/z, making their contributions to the mass spectrum impossible to distinguish. In this work we demonstrate that a combination of ion exchange chromatography (IXC) with on-line detection by electrospray ionization mass spectrometry (ESI MS) and methods of ion manipulation in the gas phase (limited charge reduction and collision-induced dissociation) allows meaningful structural information to be obtained on a structurally heterogeneous sample of PEGylated interferon β-1a. IXC profiling of the protein sample gives rise to a convoluted chromatogram with several partially resolved peaks which can represent both deamidation and different glycosylation patterns within the protein, as well as varying extent of PEGylation. Thus, profiling the protein with on-line IXC/ESI/MS/MS allows it to be characterized by providing information on three different types of PTMs (designer, enzymatic and non-enzymatic) within a single protein therapeutic.

Electrochemical ion-exchange for medium active liquid waste treatment

International Nuclear Information System (INIS)

Bridger, N.J.; Turner, A.D.

1987-01-01

Electrochemical ion-exchange has already been demonstrated to be a robust, effective process for the treatment of active liquid wastes -with high decontamination and volume reduction factors, and only a low energy requirement. The primary aim of this new programme is to scale up this process - initially to 0.1m 3 /h, and ultimately to 1 3 m/h. A new 0.4m 2 electrode module has been designed and constructed, together with 3m 3 feed tanks for the first phase of this work. Further development work is also being carried out on alternative electrode designs and fabrication methods, as well as new exchange media (including inorganic absorbers and organic chelating resins) in order to optimize selectivity performance. (author)

Chitin and stress induced protein kinase activation

DEFF Research Database (Denmark)

Kenchappa, Chandra Shekar; Azevedo da Silva, Raquel; Bressendorff, Simon

2017-01-01

The assays described here are pertinent to protein kinase studies in any plant. They include an immunoblot phosphorylation/activation assay and an in-gel activity assay for MAP kinases (MPKs) using the general protein kinase substrate myelin basic protein. They also include a novel in-gel peptide...... substrate assay for Snf1-related kinase family 2 members (SnRK2s). This kinase family-specific assay overcomes some limitations of in-gel assays and permits the identification of different types of kinase activities in total protein extracts....

Application of resistivity measurements to the control of protein elutions using cellulose ion-exchangers; Application de la mesure de resistivite au controle d'une elution de proteines a partir de celluloses echangeuses d'ions

Energy Technology Data Exchange (ETDEWEB)

Duplaa, A M; Brandolin, G [Commissariat a l' Energie Atomique, Grenoble (France). Centre d' Etudes Nucleaires

1969-07-01

The eluate obtained by chromatography of a mixture of proteins on columns of cellulose ion-exchangers (DEAE-cellulose and CM-cellulose) sometimes have very low proteins concentrations. The resistivity measurement gives more information than the UV control which is often inadequate. The modifications undergone by elution buffers are recorded and the best conditions for the extraction of an enzymatic protein are determined. The tests are performed without proteins on the columns; they consist in a double control of resistivity and ph of elution buffers after they pass on the exchangers columns. (author) [French] Apres chromatographie d'un melange de proteine; sur colonnes d'echangeurs d'ions tels que DEAE-cellulose et CM-cellulose, les eluats obtenus ont quelquefois des concentrations en proteines tres faibles. Au controle en UV souvent insuffisant, on a ajoute la mesure de resistivite apportant des donnees complementaires. Des essais, realises en l'absence de proteines et consistant a effectuer un double controle de resistivite et de pH des tampons d'elution apres leur passage sur colonnes d'echangeurs, ont permis d'enregistrer les modifications subies par ces tampons et de determiner leurs meilleures conditions d'utilisation pour l'extraction d'une proteine enzymatique. (auteur)

Solvent Exchange Rates of Side-chain Amide Protons in Proteins

International Nuclear Information System (INIS)

Rajagopal, Ponni; Jones, Bryan E.; Klevit, Rachel E.

1998-01-01

Solvent exchange rates and temperature coefficients for Asn/Gln side-chain amide protons have been measured in Escherichia coli HPr. The protons of the eight side-chain amide groups (two Asn and six Gln) exhibit varying exchange rates which are slower than some of the fast exchanging backbone amide protons. Differences in exchange rates of the E and Z protons of the same side-chain amide group are obtained by measuring exchange rates at pH values > 8. An NOE between a side-chain amide proton and a bound water molecule was also observed

Chaperone activity of human small heat shock protein-GST fusion proteins.

Science.gov (United States)

Arbach, Hannah; Butler, Caley; McMenimen, Kathryn A

2017-07-01

Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST

Differential Mobility Spectrometry-Hydrogen Deuterium Exchange (DMS-HDX) as a Probe of Protein Conformation in Solution.

Science.gov (United States)

Zhu, Shaolong; Campbell, J Larry; Chernushevich, Igor; Le Blanc, J C Yves; Wilson, Derek J

2016-06-01

Differential mobility spectrometry (DMS) is an ion mobility technique that has been adopted chiefly as a pre-filter for small- to medium-sized analytes (DMS-field asymmetric waveform ion mobility spectroscopy (FAIMS)-the application of DMS to intact biomacromolecules remains largely unexplored. In this work, we employ DMS combined with gas-phase hydrogen deuterium exchange (DMS-HDX) to probe the gas-phase conformations generated from proteins that were initially folded, partially-folded, and unfolded in solution. Our findings indicate that proteins with distinct structural features in solution exhibit unique deuterium uptake profiles as function of their optimal transmission through the DMS. Ultimately we propose that DMS-HDX can, if properly implemented, provide rapid measurements of liquid-phase protein structural stability that could be of use in biopharmaceuticals development. Graphical Abstract ᅟ.

Crystallization and preliminary crystallographic analysis of the human calcineurin homologous protein CHP2 bound to the cytoplasmic region of the Na+/H+ exchanger NHE1

International Nuclear Information System (INIS)

Ben Ammar, Youssef; Takeda, Soichi; Sugawara, Mitsuaki; Miyano, Masashi; Mori, Hidezo; Wakabayashi, Shigeo

2005-01-01

Crystallization of the human CHP2–NHE1 binding domain complex. Calcineurin homologous protein (CHP) is a Ca 2+ -binding protein that directly interacts with and regulates the activity of all plasma-membrane Na + /H + -exchanger (NHE) family members. In contrast to the ubiquitous isoform CHP1, CHP2 is highly expressed in cancer cells. To understand the regulatory mechanism of NHE1 by CHP2, the complex CHP2–NHE1 (amino acids 503–545) has been crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as precipitant. The crystals diffract to 2.7 Å and belong to a tetragonal space group, with unit-cell parameters a = b = 49.96, c = 103.20 Å

Theory and applications of a novel ion exchange chromatographic technology using controlled pH gradients for separating proteins on anionic and cationic stationary phases.

Science.gov (United States)

Tsonev, Latchezar I; Hirsh, Allen G

2008-07-25

pISep is a major new advance in low ionic strength ion exchange chromatography. It enables the formation of externally controlled pH gradients over the very broad pH range from 2 to 12. The gradients can be generated on either cationic or anionic exchangers over arbitrary pH ranges wherein the stationary phases remain totally charged. Associated pISep software makes possible the calculation of either linear, nonlinear or combined, multi-step, multi-slope pH gradients. These highly reproducible pH gradients, while separating proteins and glycoproteins in the order of their electrophoretic pIs, provide superior chromatographic resolution compared to salt. This paper also presents a statistical mechanical model for protein binding to ion exchange stationary phases enhancing the electrostatic interaction theory for the general dependence of retention factor k, on both salt and pH simultaneously. It is shown that the retention factors computed from short time isocratic salt elution data of a model protein can be used to accurately predict its salt elution concentration in varying slope salt elution gradients formed at varying isocratic pH as well as the pH at which it will be eluted from an anionic exchange column by a pISep pH gradient in the absence of salt.

Venom Protein C activators as diagnostic agents for defects of protein C System.

Science.gov (United States)

Ramzan, Faiqah; Asmat, Andleeb

2018-06-18

Background Protein C is a vitamin K dependent plasma zymogen. It prevents clotting by inhibiting clotting by inactivating factor V and factor VIII. Protein C activation pathway involves three steps: (i) Activation of protein C; (ii) Inhibition of coagulation through inactivating factor V and VIII by activated protein C and (iii) Inhibition of activated protein C by plasma protease inhibitors specific for this enzyme. Proteinases converts the zymogen Protein C (PC) of vertebrates into activated PC, which has been detected in several snake venoms. Most PC activators have been purified from venom of snake species belonging to the genera of the Agkistrodon complex. Unlike the physiological thrombin-catalyzed PC activation reaction which requires thrombomodulin as a cofactor, most snake venom activators directly convert the zymogen PC into the catalytically active form which can easily be determined by means of coagulation or chromogenic substrate techniques. Conclusion The fast-acting PC activator Protac® from Agkistrodon contortrix (southern copperhead snake) venom has been found to have broad application in diagnostic practice for the determination of disorders in the PC pathway. Recently, screening assays for the PC pathway have been introduced, based on the observation that the PC pathway is probably the most important physiological barrier against thrombosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Platelet factor 4 impairs the anticoagulant activity of activated protein C.

LENUS (Irish Health Repository)

Preston, Roger J S

2012-02-01

Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q\\/R679Q and FVa-R306Q\\/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg(306) by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation.

Platelet factor 4 impairs the anticoagulant activity of activated protein C.

LENUS (Irish Health Repository)

Preston, Roger J S

2009-02-27

Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q\\/R679Q and FVa-R306Q\\/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg(306) by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation.

Membrane association of the Arabidopsis ARF exchange factor GNOM involves interaction of conserved domains

DEFF Research Database (Denmark)

Anders, Nadine; Nielsen, Michael M.; Keicher, Jutta

2008-01-01

vesicle formation by activating ARF GTPases on specific membranes in animals, plants, and fungi. However, apart from the catalytic exchange activity of the SEC7 domain, the functional significance of other conserved domains is virtually unknown. Here, we show that a distinct N-terminal domain of GNOM......The GNOM protein plays a fundamental role in Arabidopsis thaliana development by regulating endosome-to-plasma membrane trafficking required for polar localization of the auxin efflux carrier PIN1. GNOM is a family member of large ARF guanine nucleotide exchange factors (ARF-GEFs), which regulate...... mediates dimerization and in addition interacts heterotypically with two other conserved domains in vivo. In contrast with N-terminal dimerization, the heterotypic interaction is essential for GNOM function, as mutations abolishing this interaction inactivate the GNOM protein and compromise its membrane...

Lipid exchange by ultracentrifugation

DEFF Research Database (Denmark)

Drachmann, Nikolaj Düring; Olesen, Claus

2014-01-01

, and the complex interplay between the lipids and the P-type ATPases are still not well understood. We here describe a robust method to exchange the majority of the lipids surrounding the ATPase after solubilisation and/or purification with a target lipid of interest. The method is based on an ultracentrifugation...... step, where the protein sample is spun through a dense buffer containing large excess of the target lipid, which results in an approximately 80-85 % lipid exchange. The method is a very gently technique that maintains protein folding during the process, hence allowing further characterization...

Probing Conformational Dynamics of Tau Protein by Hydrogen/Deuterium Exchange Mass Spectrometry

Science.gov (United States)

Huang, Richard Y.-C.; Iacob, Roxana E.; Sankaranarayanan, Sethu; Yang, Ling; Ahlijanian, Michael; Tao, Li; Tymiak, Adrienne A.; Chen, Guodong

2018-01-01

Fibrillization of the microtubule-associated protein tau has been recognized as one of the signature pathologies of the nervous system in Alzheimer's disease, progressive supranuclear palsy, and other tauopathies. The conformational transition of tau in the fibrillization process, tau monomer to soluble aggregates to fibrils in particular, remains unclear. Here we report on the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) in combination with other biochemical approaches, including Thioflavin S fluorescence measurements, enzyme-linked immunosorbent assay (ELISA), and Western blotting to understand the heparin-induced tau's fibrillization. HDX-MS studies including anti-tau antibody epitope mapping experiments provided molecular level details of the full-length tau's conformational dynamics and its regional solvent accessibility upon soluble aggregates formation. The results demonstrate that R3 region in the full-length tau's microtubule binding repeat region (MTBR) is stabilized in the aggregation process, leaving both N and C terminal regions to be solvent exposed in the soluble aggregates and fibrils. The findings also illustrate the practical utility of orthogonal analytical methodologies for the characterization of protein higher order structure. [Figure not available: see fulltext.

Empirical Correction for Differences in Chemical Exchange Rates in Hydrogen Exchange-Mass Spectrometry Measurements.

Science.gov (United States)

Toth, Ronald T; Mills, Brittney J; Joshi, Sangeeta B; Esfandiary, Reza; Bishop, Steven M; Middaugh, C Russell; Volkin, David B; Weis, David D

2017-09-05

A barrier to the use of hydrogen exchange-mass spectrometry (HX-MS) in many contexts, especially analytical characterization of various protein therapeutic candidates, is that differences in temperature, pH, ionic strength, buffering agent, or other additives can alter chemical exchange rates, making HX data gathered under differing solution conditions difficult to compare. Here, we present data demonstrating that HX chemical exchange rates can be substantially altered not only by the well-established variables of temperature and pH but also by additives including arginine, guanidine, methionine, and thiocyanate. To compensate for these additive effects, we have developed an empirical method to correct the hydrogen-exchange data for these differences. First, differences in chemical exchange rates are measured by use of an unstructured reporter peptide, YPI. An empirical chemical exchange correction factor, determined by use of the HX data from the reporter peptide, is then applied to the HX measurements obtained from a protein of interest under different solution conditions. We demonstrate that the correction is experimentally sound through simulation and in a proof-of-concept experiment using unstructured peptides under slow-exchange conditions (pD 4.5 at ambient temperature). To illustrate its utility, we applied the correction to HX-MS excipient screening data collected for a pharmaceutically relevant IgG4 mAb being characterized to determine the effects of different formulations on backbone dynamics.

Outer Membrane Protein 25 of Brucella Activates Mitogen-Activated Protein Kinase Signal Pathway in Human Trophoblast Cells

Directory of Open Access Journals (Sweden)

Jing Zhang

2017-12-01

Full Text Available Outer membrane protein 25 (OMP25, a virulence factor from Brucella, plays an important role in maintaining the structural stability of Brucella. Mitogen-activated protein kinase (MAPK signal pathway widely exists in eukaryotic cells. In this study, human trophoblast cell line HPT-8 and BALB/c mice were infected with Brucella abortus 2308 strain (S2308 and 2308ΔOmp25 mutant strain. The expression of cytokines and activation of MAPK signal pathway were detected. We found that the expressions of tumor necrosis factor-α, interleukin-1, and interleukin-10 (IL-10 were increased in HPT-8 cells infected with S2308 and 2308ΔOmp25 mutant. S2308 also activated p38 phosphorylation protein, extracellular-regulated protein kinases (ERK, and Jun-N-terminal kinase (JNK from MAPK signal pathway. 2308ΔOmp25 could not activate p38, ERK, and JNK branches. Immunohistochemistry experiments showed that S2308 was able to activate phosphorylation of p38 and ERK in BABL/c mice. However, 2308ΔOmp25 could weakly activate phosphorylation of p38 and ERK. These results suggest that Omp25 played an important role in the process of Brucella activation of the MAPK signal pathway.

Dietary protein considerations to support active aging.

Science.gov (United States)

Wall, Benjamin T; Cermak, Naomi M; van Loon, Luc J C

2014-11-01

Given our rapidly aging world-wide population, the loss of skeletal muscle mass with healthy aging (sarcopenia) represents an important societal and public health concern. Maintaining or adopting an active lifestyle alleviates age-related muscle loss to a certain extent. Over time, even small losses of muscle tissue can hinder the ability to maintain an active lifestyle and, as such, contribute to the development of frailty and metabolic disease. Considerable research focus has addressed the application of dietary protein supplementation to support exercise-induced gains in muscle mass in younger individuals. In contrast, the role of dietary protein in supporting the maintenance (or gain) of skeletal muscle mass in active older persons has received less attention. Older individuals display a blunted muscle protein synthetic response to dietary protein ingestion. However, this reduced anabolic response can largely be overcome when physical activity is performed in close temporal proximity to protein consumption. Moreover, recent evidence has helped elucidate the optimal type and amount of dietary protein that should be ingested by the older adult throughout the day in order to maximize the skeletal muscle adaptive response to physical activity. Evidence demonstrates that when these principles are adhered to, muscle maintenance or hypertrophy over prolonged periods can be further augmented in active older persons. The present review outlines the current understanding of the role that dietary protein occupies in the lifestyle of active older adults as a means to increase skeletal muscle mass, strength and function, and thus support healthier aging.

Effective Application of Bicelles for Conformational Analysis of G Protein-Coupled Receptors by Hydrogen/Deuterium Exchange Mass Spectrometry

Science.gov (United States)

Duc, Nguyen Minh; Du, Yang; Thorsen, Thor S.; Lee, Su Youn; Zhang, Cheng; Kato, Hideaki; Kobilka, Brian K.; Chung, Ka Young

2015-05-01

G protein-coupled receptors (GPCRs) have important roles in physiology and pathology, and 40% of drugs currently on the market target GPCRs for the treatment of various diseases. Because of their therapeutic importance, the structural mechanism of GPCR signaling is of great interest in the field of drug discovery. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for analyzing ligand binding sites, the protein-protein interaction interface, and conformational changes of proteins. However, its application to GPCRs has been limited for various reasons, including the hydrophobic nature of GPCRs and the use of detergents in their preparation. In the present study, we tested the application of bicelles as a means of solubilizing GPCRs for HDX-MS studies. GPCRs (e.g., β2-adrenergic receptor [β2AR], μ-opioid receptor, and protease-activated receptor 1) solubilized in bicelles produced better sequence coverage (greater than 90%) than GPCRs solubilized in n-dodecyl-β-D-maltopyranoside (DDM), suggesting that bicelles are a more effective method of solubilization for HDX-MS studies. The HDX-MS profile of β2AR in bicelles showed that transmembrane domains (TMs) undergo lower deuterium uptake than intracellular or extracellular regions, which is consistent with the fact that the TMs are highly ordered and embedded in bicelles. The overall HDX-MS profiles of β2AR solubilized in bicelles and in DDM were similar except for intracellular loop 3. Interestingly, we detected EX1 kinetics, an important phenomenon in protein dynamics, at the C-terminus of TM6 in β2AR. In conclusion, we suggest the application of bicelles as a useful method for solubilizing GPCRs for conformational analysis by HDX-MS.

Virtual Protein Purification: A Simple Exercise to Introduce pH as A Parameter That Effects Ion Exchange Chromatography

Science.gov (United States)

Clark, Daniel D.; Edwards, Daniel J.

2018-01-01

This article describes a simple exercise using a free, easy-to-use, established online program. The exercise helps to reinforce protein purification concepts and introduces undergraduates to pH as a parameter that affects anion-exchange chromatography. The exercise was tested with biochemistry majors at California State University-Chico. Given the…

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

Hydrogen/deuterium exchange of multiply-protonated cytochrome c ions

International Nuclear Information System (INIS)

Wood, T.D.; Guan, Ziqiang; O'Connor, P.B.

1995-01-01

Low resolution measurements show gaseous multiply-protonated cytochrome c ions undergo hydrogen/deuterium (H/D) exchange with pseudo first-order kinetics at three distinct exchange levels, suggesting the co-existence of gaseous protein conformations. Although exchange levels first increase with increasing charge values, they decrease at the highest charge values, consistent with solution-phase behavior of cytochrome c, where the native structure unfolds with decreasing pH until folding into a compact A-state at lowest pH. High resolution measurements indicate the presence of at least six H/D exchange levels. Infrared (IR) laser heating and fast collisions via quadrupolar excitation (QE) increase H/D exchange levels (unfolding) while charge-stripping ions to lower charge values can increase or decrease H/D exchange levels (unfolding or folding). Wolynes has suggested studying proteins in vacuo could play an important role in delineating the contributions various forces play in the protein folding process, provided appropriate comparisons can be made between gas-phase and solution-phase structures

Ric-8A, a Gα protein guanine nucleotide exchange factor potentiates taste receptor signaling

Directory of Open Access Journals (Sweden)

Claire J Fenech

2009-10-01

Full Text Available Taste receptors for sweet, bitter and umami tastants are G-protein coupled receptors (GPCRs. While much effort has been devoted to understanding G-protein-receptor interactions and identifying the components of the signalling cascade downstream of these receptors, at the level of the G-protein the modulation of receptor signal transduction remains relatively unexplored. In this regard a taste-specific regulator of G-protein signaling (RGS, RGS21, has recently been identified. To study whether guanine nucleotide exchange factors (GEFs are involved in the transduction of the signal downstream of the taste GPCRs we investigated the expression of Ric-8A and Ric-8B in mouse taste cells and their interaction with G-protein subunits found in taste buds. Mammalian Ric-8 proteins were initially identified as potent GEFs for a range of Gα subunits and Ric-8B has recently been shown to amplify olfactory signal transduction. We find that both Ric-8A and Ric-8B are expressed in a large portion of taste bud cells and that most of these cells contain IP3R-3 a marker for sweet, umami and bitter taste receptor cells. Ric-8A interacts with Gα-gustducin and Gαi2 through which it amplifies the signal transduction of hTas2R16, a receptor for bitter compounds. Overall, these findings are consistent with a role for Ric-8 in mammalian taste signal transduction.

Quantitation of chemical exchange rates using pulsed-field-gradient diffusion measurements

International Nuclear Information System (INIS)

Andrec, Michael; Prestegard, James H.

1997-01-01

A new approach to the quantitation of chemical exchange rates is presented, and its utility is illustrated with application to the exchange of protein amide protons with bulk water. The approach consists of a selective-inversion exchange HMQC experiment in which a short spin echo diffusion filter has been inserted into the exchange period. In this way, the kinetics of exchange are encoded directly in an apparent diffusion coefficient which is a function of the position of the diffusion filter in the pulse sequence. A detailed theoretical analysis of this experiment indicates that, in addition to the measurement of simple exchange rates, the experiment is capable of measuring the effect of mediated exchange, e.g. the transfer of magnetization from bulk water to an amide site mediated by an internal bound water molecule or a labile protein side-chain proton in fast exchange with bulk water. Experimental results for rapid water/amide exchange in acyl carrier protein are shown to be quantitatively consistent with the exchange rates measured using a selective-inversion exchange experiment

Cellular reprogramming through mitogen-activated protein kinases

Directory of Open Access Journals (Sweden)

Justin eLee

2015-10-01

Full Text Available Mitogen-activated protein kinase (MAPK cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554 in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression – including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding and degradation steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

Ras activation by SOS

DEFF Research Database (Denmark)

Iversen, Lars; Tu, Hsiung-Lin; Lin, Wan-Chen

2014-01-01

Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual ...

Preparation of a novel dual-function strong cation exchange/hydrophobic interaction chromatography stationary phase for protein separation.

Science.gov (United States)

Zhao, Kailou; Yang, Li; Wang, Xuejiao; Bai, Quan; Yang, Fan; Wang, Fei

2012-08-30

We have explored a novel dual-function stationary phase which combines both strong cation exchange (SCX) and hydrophobic interaction chromatography (HIC) characteristics. The novel dual-function stationary phase is based on porous and spherical silica gel functionalized with ligand containing sulfonic and benzyl groups capable of electrostatic and hydrophobic interaction functionalities, which displays HIC character in a high salt concentration, and IEC character in a low salt concentration in mobile phase employed. As a result, it can be employed to separate proteins with SCX and HIC modes, respectively. The resolution and selectivity of the dual-function stationary phase were evaluated under both HIC and SCX modes with standard proteins and can be comparable to that of conventional IEC and HIC columns. More than 96% of mass and bioactivity recoveries of proteins can be achieved in both HIC and SCX modes, respectively. The results indicated that the novel dual-function column could replace two individual SCX and HIC columns for protein separation. Mixed retention mechanism of proteins on this dual-function column based on stoichiometric displacement theory (SDT) in LC was investigated to find the optimal balance of the magnitude of electrostatic and hydrophobic interactions between protein and the ligand on the silica surface in order to obtain high resolution and selectivity for protein separation. In addition, the effects of the hydrophobicity of the ligand of the dual-function packings and pH of the mobile phase used on protein separation were also investigated in detail. The results show that the ligand with suitable hydrophobicity to match the electrostatic interaction is very important to prepare the dual-function stationary phase, and a better resolution and selectivity can be obtained at pH 6.5 in SCX mode. Therefore, the dual-function column can replace two individual SCX and HIC columns for protein separation and be used to set up two-dimensional liquid

Purification and characterization of a novel anti-HSV-2 protein with antiproliferative and peroxidase activities from Stellaria media

Institute of Scientific and Technical Information of China (English)

Yu Shan; Yuhong Zheng; Fuqin Guan; Jianjian Zhou; Haiguang Zhao; Bing Xia; Xu Feng

2013-01-01

A novel antiviral protein,designated as Stellarmedin A,was purified from Stellaria media (L.) Vill.(Caryophyllaceae) by using ammonium sulfate precipitation,cation-exchange chromatography system.Gel electrophoresis analysis showed that Stellarmedin A is a highly basic glycoprotein with a molecular weight of 35.1 kDa and an isoelectric point of ～8.7.The Nterminal 14-amino acid sequence,MGNTGVLTGERNDR,is similar to those of other plant peroxidases.This protein inhibited herpes simplex virus type 2 (HSV-2) replication in vitro with an ICso of 13.18 μg/ml and a therapeutic index exceeding 75.9.It was demonstrated that Stellarmedin A affects the initial stage of HSV-2 infection and is able to inhibit the proliferation of promyelocytic leukemia HL-60 and colon carcinoma LoVo cells with an ICso of 9.09 and 12.32 μM,respectively.Moreover,Stellarmedin A has a peroxidase activity of 36.6 μmol/min/mg protein,when gualacol was used as substrate.To our knowledge,this is the first report about an anti-HSV-2 protein with antiproliferative and peroxidase activities from S.media.

Dioscorin, the major tuber storage protein of yam (Dioscorea batatas decne) with carbonic anhydrase and trypsin inhibitor activities.

Science.gov (United States)

Hou, W C; Liu, J S; Chen, H J; Chen, T E; Chang, C F; Lin, Y H

1999-05-01

Dioscorin, the tuber storage protein of yam (Dioscorea batatas Decne), was purified successively by ammonium sulfate fractionation, DE-52 ion exchange chromatography, and Sephadex G-75 column. Two protein bands (82 and 28 kDa) were found under nonreducing conditions after SDS-PAGE; but only one band (32 kDa) was detected under reducing conditions. The first 21 amino acids in the N-terminal region of the 28 kDa form were VEDEFSYIEGNPNGPENWGNL, which was highly homologous to deductive sequence of dioscorin from cDNA of another yam species (Dioscoreacayenensis Lam) reported by Conlan et al. (Plant Mol. Biol. 1995, 28, 369-380). Hewett-Emmett and Tashian (Mol. Phylogenet. Evol. 1996, 5, 50 -77) mentioned that, according to DNA alignments, dioscorin from yam (D. cayenensis) was alpha-carbonic anhydrase (alpha-CA) related. In this report, we found that the purified dioscorin showed both CA dehydration activity using sodium bicarbonate as a substrate and CA activity staining after SDS-PAGE. A polyclonal antibody, which was raised against trypsin inhibitor (TI), a storage protein of sweet potato (Ipomoea batatas [L.] Lam var. Tainong 57), cross-reacted with dioscorin, which also showed TI activity determined by both activity staining after SDS-PAGE and trypsin inhibition determination.

Dissociation of activated protein C functions by elimination of protein S cofactor enhancement.

LENUS (Irish Health Repository)

Harmon, Shona

2008-11-07

Activated protein C (APC) plays a critical anticoagulant role in vivo by inactivating procoagulant factor Va and factor VIIIa and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor can initiate protease-activated receptor-1 (PAR-1)-mediated cytoprotective signaling. Protein S constitutes a critical cofactor for the anticoagulant function of APC but is not known to be involved in regulating APC-mediated protective PAR-1 signaling. In this study we utilized a site-directed mutagenesis strategy to characterize a putative protein S binding region within the APC Gla domain. Three single amino acid substitutions within the APC Gla domain (D35T, D36A, and A39V) were found to mildly impair protein S-dependent anticoagulant activity (<2-fold) but retained entirely normal cytoprotective activity. However, a single amino acid substitution (L38D) ablated the ability of protein S to function as a cofactor for this APC variant. Consequently, in assays of protein S-dependent factor Va proteolysis using purified proteins or in the plasma milieu, APC-L38D variant exhibited minimal residual anticoagulant activity compared with wild type APC. Despite the location of Leu-38 in the Gla domain, APC-L38D interacted normally with endothelial cell protein C receptor and retained its ability to trigger PAR-1 mediated cytoprotective signaling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to separate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.

Casting granular ion exchange resins with medium-active waste in cement

International Nuclear Information System (INIS)

Beijer, O.

1980-01-01

Medium active waste from nuclear power stations in Sweden is trapped in granular ion exchange resins. The resin is mixed with cement paste and cast in a concrete shell which is cubic and has an edge dimension of 1.2 m. In some cases the ion exchange cement mortar has cracked. The report presents laboratory sutdies of the properties of the ion exchange resin and the mortar. Also the leaching of the moulds has been investigated. It was shown that a mixture with a water cement ratio higher than about 0.5 swells considerably during the first weeks after casting. The diffusion constant for cesium 137 has been determined at 3.10 -4 cm 2 /24-hour period in conjunction with exposure of the mould and mortar to sea water. The Swedish language report has 400 pages with 90 figures and 30 tables. (author)

Integration of G protein α (Gα) signaling by the regulator of G protein signaling 14 (RGS14).

Science.gov (United States)

Brown, Nicole E; Goswami, Devrishi; Branch, Mary Rose; Ramineni, Suneela; Ortlund, Eric A; Griffin, Patrick R; Hepler, John R

2015-04-03

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gαi/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gαi1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gαi1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gαo-AlF4(-). Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gαi1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gαo-AlF4(-) and an AlF4(-)-insensitive mutant (G42R) of Gαi1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gαi1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gαo-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Na+/H+ exchanger, NHE1, differentially regulates mitogen-activated protein kinase subfamilies after osmotic shrinkage in Ehrlich Lettre Ascites cells

DEFF Research Database (Denmark)

Petersen, Stine Helene Falsig; Rasmussen, Maria; Darborg, Barbara Vasek

2007-01-01

Osmotic stress modulates mitogen activated protein kinase (MAPK) activities, leading to altered gene transcription and cell death/survival balance, however, the mechanisms involved are incompletely elucidated. Here, we show, using a combination of biochemical and molecular biology approaches...... by human (h) NHE1 expression in cells lacking endogenous NHE1 activity. The effect of NHE1 on ERK1/2 was pH(i)-independent and upstream of MEK1/2. Shrinkage-activation of JNK1/2 was attenuated by EIPA, augmented by hNHE1 expression, and abolished in the presence of HCO(3)(-). Basal JNK activity...

Application of inorganic ion exchangers for low and medium activity radioactive effluent decontamination

International Nuclear Information System (INIS)

Dozol, J.F.; Eymard, S.; Gambade, R.; La Rosa, G.

1986-01-01

This study proposes an alternative pretreatment or treatment for low and medium activity liquid wastes, allowing to improve the quality of containment and decrease the cost of storage. Inorganic ion exchangers are used to remove alpha emitters and long lived fission products and concentrate them in a small volume; these exchangers can be converted into a stable matrix by thermal treatment. This treatment, at least for some liquid wastes, don't exclude a complementary decontamination by chemical precipitation. Sludges, arising from precipitation, exempt from alpha emitters and long lived fission products can be stored in a shallow land burial. This study includes two parts: - Measurements of distribution coefficients for the main nuclides in order to choose, for each liquid wastes, the most suitable ion exchanger. - Estimation of performances of selected inorganic ion exchangers, from tests of percolation of genuine effluents

Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

Science.gov (United States)

Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

2005-01-01

The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research.

Crystallization and preliminary crystallographic analysis of the human calcineurin homologous protein CHP2 bound to the cytoplasmic region of the Na{sup +}/H{sup +} exchanger NHE1

Energy Technology Data Exchange (ETDEWEB)

Ben Ammar, Youssef [Department of Molecular Physiology, National Cardiovascular Center Research Institute, Fujishiro-dai 5-7-1, Suita, Osaka 565-8565 (Japan); Takeda, Soichi [Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Fujishiro-dai 5-7-1, Suita, Osaka 565-8565 (Japan); Sugawara, Mitsuaki; Miyano, Masashi [Structural Biophysics Laboratory, RIKEN Harima Institute at SPring-8, Kouto, Mikazuki, Sayo, Hyogo 679-5148 (Japan); Mori, Hidezo [Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Fujishiro-dai 5-7-1, Suita, Osaka 565-8565 (Japan); Wakabayashi, Shigeo, E-mail: wak@ri.ncvc.go.jp [Department of Molecular Physiology, National Cardiovascular Center Research Institute, Fujishiro-dai 5-7-1, Suita, Osaka 565-8565 (Japan)

2005-10-01

Crystallization of the human CHP2–NHE1 binding domain complex. Calcineurin homologous protein (CHP) is a Ca{sup 2+}-binding protein that directly interacts with and regulates the activity of all plasma-membrane Na{sup +}/H{sup +}-exchanger (NHE) family members. In contrast to the ubiquitous isoform CHP1, CHP2 is highly expressed in cancer cells. To understand the regulatory mechanism of NHE1 by CHP2, the complex CHP2–NHE1 (amino acids 503–545) has been crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as precipitant. The crystals diffract to 2.7 Å and belong to a tetragonal space group, with unit-cell parameters a = b = 49.96, c = 103.20 Å.

In silico study of protein to protein interaction analysis of AMP-activated protein kinase and mitochondrial activity in three different farm animal species

Science.gov (United States)

Prastowo, S.; Widyas, N.

2018-03-01

AMP-activated protein kinase (AMPK) is cellular energy censor which works based on ATP and AMP concentration. This protein interacts with mitochondria in determine its activity to generate energy for cell metabolism purposes. For that, this paper aims to compare the protein to protein interaction of AMPK and mitochondrial activity genes in the metabolism of known animal farm (domesticated) that are cattle (Bos taurus), pig (Sus scrofa) and chicken (Gallus gallus). In silico study was done using STRING V.10 as prominent protein interaction database, followed with biological function comparison in KEGG PATHWAY database. Set of genes (12 in total) were used as input analysis that are PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3, PPARGC1, ACC, CPT1B, NRF2 and SOD. The first 7 genes belong to gene in AMPK family, while the last 5 belong to mitochondrial activity genes. The protein interaction result shows 11, 8 and 5 metabolism pathways in Bos taurus, Sus scrofa and Gallus gallus, respectively. The top pathway in Bos taurus is AMPK signaling pathway (10 genes), Sus scrofa is Adipocytokine signaling pathway (8 genes) and Gallus gallus is FoxO signaling pathway (5 genes). Moreover, the common pathways found in those 3 species are Adipocytokine signaling pathway, Insulin signaling pathway and FoxO signaling pathway. Genes clustered in Adipocytokine and Insulin signaling pathway are PRKAA2, PPARGC1A, PRKAB1 and PRKAG2. While, in FoxO signaling pathway are PRKAA2, PRKAB1, PRKAG2. According to that, we found PRKAA2, PRKAB1 and PRKAG2 are the common genes. Based on the bioinformatics analysis, we can demonstrate that protein to protein interaction shows distinct different of metabolism in different species. However, further validation is needed to give a clear explanation.

Gonadotropin stimulates oocyte translation by increasing magnesium activity through intracellular potassium-magnesium exchange

International Nuclear Information System (INIS)

Horowitz, S.B.; Tluczek, L.J.

1989-01-01

We previously showed that gonadotropin increases the K + activity in Xenopus oocytes and that this is a signal for increased translation. However, K + need not act to control synthesis directly but may act through an unidentified downstream effector. Using microinjection to vary the salt content of oocytes and concomitantly measuring [ 3 H]leucine incorporation, we found that small changes in Mg 2+ greatly affect translation rates. (Ca 2+ had little influence.) By measuring intracellular ion activities, we found that oocyte cations existed in a buffer-like (ion-exchange) equilibrium in which K + and Mg 2+ are the preponderant monovalent and divalent cations. Hence, increasing cellular K + activity might increase translation by causing Mg 2+ activity to rise. If so, the increased translation rates produced by hormone treatment or K + injection would be prevented by EDTA, a Mg 2+ chelating agent. This prediction was tested and confirmed. We conclude that, when gonadotropin increases K + activity, the cell's internal ion-exchange equilibrium is altered thereby increasing Mg 2+ activity and this up-regulates translation

Effects of phorbol ester on mitogen-activated protein kinase kinase activity in wild-type and phorbol ester-resistant EL4 thymoma cells.

Science.gov (United States)

Gause, K C; Homma, M K; Licciardi, K A; Seger, R; Ahn, N G; Peterson, M J; Krebs, E G; Meier, K E

1993-08-05

Phorbol ester-sensitive and -resistant EL4 thymoma cell lines differ in their ability to activate mitogen-activated protein kinase (MAPK) in response to phorbol ester. Treatment of wild-type EL4 cells with phorbol ester results in the rapid activations of MAPK and pp90rsk kinase, a substrate for MAPK, while neither kinase is activated in response to phorbol ester in variant EL4 cells. This study examines the activation of MAPK kinase (MAPKK), an activator of MAPK, in wild-type and variant EL4 cells. Phosphorylation of a 40-kDa substrate, identified as MAPK, was observed following in vitro phosphorylation reactions using cytosolic extracts or Mono Q column fractions prepared from phorbol ester-treated wild-type EL4 cells. MAPKK activity coeluted with a portion of the inactive MAPK upon Mono Q anion-exchange chromatography, permitting detection of the MAPKK activity in fractions containing both kinases. This MAPKK activity was present in phorbol ester-treated wild-type cells, but not in phorbol ester-treated variant cells or in untreated wild-type or variant cells. The MAPKK from wild-type cells was able to activate MAPK prepared from either wild-type or variant cells. MAPKK activity could be stimulated in both wildtype and variant EL4 cells in response to treatment of cells with okadaic acid. These results indicate that the failure of variant EL4 cells to activate MAP kinase in response to phorbol ester is due to a failure to activate MAPKK. Therefore, the step that confers phorbol ester resistance to variant EL4 cells lies between the activation of protein kinase C and the activation of MAPKK.

Differential regulation of the Rac1 GTPase-activating protein (GAP) BCR during oxygen/glucose deprivation in hippocampal and cortical neurons.

Science.gov (United States)

Smith, Katharine R; Rajgor, Dipen; Hanley, Jonathan G

2017-12-08

Brain ischemia causes oxygen and glucose deprivation (OGD) in neurons, triggering a cascade of events leading to synaptic accumulation of glutamate. Excessive activation of glutamate receptors causes excitotoxicity and delayed cell death in vulnerable neurons. Following global cerebral ischemia, hippocampal CA1 pyramidal neurons are more vulnerable to injury than their cortical counterparts, but the mechanisms that underlie this difference are unclear. Signaling via Rho-family small GTPases, their upstream guanine nucleotide exchange factors, and GTPase-activating proteins (GAPs) is differentially dysregulated in response to OGD/ischemia in hippocampal and cortical neurons. Increased Rac1 activity caused by OGD/ischemia contributes to neuronal death in hippocampal neurons via diverse effects on NADPH oxidase activity and dendritic spine morphology. The Rac1 guanine nucleotide exchange factor Tiam1 mediates an OGD-induced increase in Rac1 activity in hippocampal neurons; however, the identity of an antagonistic GAP remains elusive. Here we show that the Rac1 GAP breakpoint cluster region (BCR) associates with NMDA receptors (NMDARs) along with Tiam1 and that this protein complex is more abundant in hippocampal compared with cortical neurons. Although total BCR is similar in the two neuronal types, BCR is more active in hippocampal compared with cortical neurons. OGD causes an NMDAR- and Ca 2+ -permeable AMPAR-dependent deactivation of BCR in hippocampal but not cortical neurons. BCR knockdown occludes OGD-induced Rac1 activation in hippocampal neurons. Furthermore, disrupting the Tiam1-NMDAR interaction with a fragment of Tiam1 blocks OGD-induced Tiam1 activation but has no effect on the deactivation of BCR. This work identifies BCR as a critical player in Rac1 regulation during OGD in hippocampal neurons. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

PRTR ion exchange vault column sampling

International Nuclear Information System (INIS)

Cornwell, B.C.

1995-01-01

This report documents ion exchange column sampling and Non Destructive Assay (NDA) results from activities in 1994, for the Plutonium Recycle Test Reactor (PRTR) ion exchange vault. The objective was to obtain sufficient information to prepare disposal documentation for the ion exchange columns found in the PRTR Ion exchange vault. This activity also allowed for the monitoring of the liquid level in the lower vault. The sampling activity contained five separate activities: (1) Sampling an ion exchange column and analyzing the ion exchange media for purpose of waste disposal; (2) Gamma and neutron NDA testing on ion exchange columns located in the upper vault; (3) Lower vault liquid level measurement; (4) Radiological survey of the upper vault; and (5) Secure the vault pending waste disposal

Efficacy of independence sampling in replica exchange simulations of ordered and disordered proteins.

Science.gov (United States)

Lee, Kuo Hao; Chen, Jianhan

2017-11-15

Recasting temperature replica exchange (T-RE) as a special case of Gibbs sampling has led to a simple and efficient scheme for enhanced mixing (Chodera and Shirts, J. Chem. Phys., 2011, 135, 194110). To critically examine if T-RE with independence sampling (T-REis) improves conformational sampling, we performed T-RE and T-REis simulations of ordered and disordered proteins using coarse-grained and atomistic models. The results demonstrate that T-REis effectively increase the replica mobility in temperatures space with minimal computational overhead, especially for folded proteins. However, enhanced mixing does not translate well into improved conformational sampling. The convergences of thermodynamic properties interested are similar, with slight improvements for T-REis of ordered systems. The study re-affirms the efficiency of T-RE does not appear to be limited by temperature diffusion, but by the inherent rates of spontaneous large-scale conformational re-arrangements. Due to its simplicity and efficacy of enhanced mixing, T-REis is expected to be more effective when incorporated with various Hamiltonian-RE protocols. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Neuronal Functions of Activators of G Protein Signaling

Directory of Open Access Journals (Sweden)

Man K. Tse

2012-05-01

Full Text Available G protein-coupled receptors (GPCRs are one of the most important gateways for signal transduction across the plasma membrane. Over the past decade, several classes of alternative regulators of G protein signaling have been identified and reported to activate the G proteins independent of the GPCRs. One group of such regulators is the activator of G protein signaling (AGS family which comprises of AGS1-10. They have entirely different activation mechanisms for G proteins as compared to the classic model of GPCR-mediated signaling and confer upon cells new avenues of signal transduction. As GPCRs are widely expressed in our nervous system, it is believed that the AGS family plays a major role in modulating the G protein signaling in neurons. In this article, we will review the current knowledge on AGS proteins in relation to their potential roles in neuronal regulations.

Petunia nectar proteins have ribonuclease activity.

Science.gov (United States)

Hillwig, Melissa S; Liu, Xiaoteng; Liu, Guangyu; Thornburg, Robert W; Macintosh, Gustavo C

2010-06-01

Plants requiring an insect pollinator often produce nectar as a reward for the pollinator's visitations. This rich secretion needs mechanisms to inhibit microbial growth. In Nicotiana spp. nectar, anti-microbial activity is due to the production of hydrogen peroxide. In a close relative, Petunia hybrida, limited production of hydrogen peroxide was found; yet petunia nectar still has anti-bacterial properties, suggesting that a different mechanism may exist for this inhibition. The nectar proteins of petunia plants were compared with those of ornamental tobacco and significant differences were found in protein profiles and function between these two closely related species. Among those proteins, RNase activities unique to petunia nectar were identified. The genes corresponding to four RNase T2 proteins from Petunia hybrida that show unique expression patterns in different plant tissues were cloned. Two of these enzymes, RNase Phy3 and RNase Phy4 are unique among the T2 family and contain characteristics similar to both S- and S-like RNases. Analysis of amino acid patterns suggest that these proteins are an intermediate between S- and S-like RNases, and support the hypothesis that S-RNases evolved from defence RNases expressed in floral parts. This is the first report of RNase activities in nectar.

The Emerging Role of Guanine Exchange Factors in ALS and other neurodegenerative diseases

Directory of Open Access Journals (Sweden)

Cristian eDroppelmann

2014-09-01

Full Text Available Small GTPases participate in a broad range of cellular processes such as proliferation, differentiation and migration. The exchange of GDP for GTP resulting in the activation of these GTPases is catalyzed by a group of enzymes called guanine nucleotide exchange factors (GEFs, of which two classes: Dbl-related exchange factors and the more recently described Dock family exchange factors. Increasingly, deregulation of normal GEF activity or function has been associated with a broad range of disease states, including neurodegeneration and neurodevelopmental disorders. In this review, we examine this evidence with special emphasis on the novel role of Rho guanine nucleotide exchange factor (RGNEF/p190RhoGEF in the pathogenesis of amyotrophic lateral sclerosis (ALS. RGNEF is the first neurodegeneration-linked GEF that regulates not only RhoA GTPase activation but also functions as an RNA binding protein that directly acts with low molecular weight neurofilament (NEFL mRNA 3’UTR to regulate its stability. This dual role for RGNEF, coupled with the increasing understanding of the key role for GEFs in modulating the GTPase function in cell survival suggests a prominent role for GEFs in mediating a critical balance between cytotoxicity and neuroprotection which, when disturbed, contributes to neuronal loss.

Estimation of activity in primary coolant heat exchanger of Apsara reactor after 50 years of reactor operation

International Nuclear Information System (INIS)

Prasad, S.K.; Anilkumar, S.; Vajpayee, L.K.; Belhe, M.S.; Yadav, R.K.B.; Deolekar, S.S.

2012-01-01

The primary coolant heat exchanger of Apsara Reactor was in operation for 53 years and as a part of partial decommissioning of Apsara Primary Coolant Heat Exchanger (PHEx) was decommissioned and disposed off as active waste. The long lived component deposited in the SS tubes inside the heat exchanger was assessed by taking the scrape samples and in situ gamma spectrometry technique employing NaI(Tl) detector. The data obtained by experimental measurements were validated by Monte Carlo simulation method. From the present studies, it was shown that 137 Cs and 144 Ce as the major isotopes deposited on the SS tube of heat exchanger. In this paper the authors describes the details of the methodology adopted for the assessment of radioactivity content and the results obtained. This give a reliable method to estimate the activity disposed for waste management accounting purpose in a long and heavy reactor component. The upper bound of total activity in PHEx 39.0μCi. (author)

Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum

DEFF Research Database (Denmark)

Holst, B; Tachibana, C; Winther, Jakob R.

1997-01-01

Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a', each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS....... Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a' domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC......-deleted strains overexpressing the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences for C(L/I)HC increased the growth rate significantly, however, further highlighting the importance of the oxidizing function for optimal growth....

Similarities and differences in the nucleic acid chaperone activity of HIV-2 and HIV-1 nucleocapsid proteins in vitro.

Science.gov (United States)

Pachulska-Wieczorek, Katarzyna; Stefaniak, Agnieszka K; Purzycka, Katarzyna J

2014-07-03

The nucleocapsid domain of Gag and mature nucleocapsid protein (NC) act as nucleic acid chaperones and facilitate folding of nucleic acids at critical steps of retroviral replication cycle. The basic N-terminus of HIV-1 NC protein was shown most important for the chaperone activity. The HIV-2 NC (NCp8) and HIV-1 NC (NCp7) proteins possess two highly conserved zinc fingers, flanked by basic residues. However, the NCp8 N-terminal domain is significantly shorter and contains less positively charged residues. This study characterizes previously unknown, nucleic acid chaperone activity of the HIV-2 NC protein. We have comparatively investigated the in vitro nucleic acid chaperone properties of the HIV-2 and HIV-1 NC proteins. Using substrates derived from the HIV-1 and HIV-2 genomes, we determined the ability of both proteins to chaperone nucleic acid aggregation, annealing and strand exchange in duplex structures. Both NC proteins displayed comparable, high annealing activity of HIV-1 TAR DNA and its complementary nucleic acid. Interesting differences between the two NC proteins were discovered when longer HIV substrates, particularly those derived from the HIV-2 genome, were used in chaperone assays. In contrast to NCp7, NCp8 weakly facilitates annealing of HIV-2 TAR RNA to its complementary TAR (-) DNA. NCp8 is also unable to efficiently stimulate tRNALys3 annealing to its respective HIV-2 PBS motif. Using truncated NCp8 peptide, we demonstrated that despite the fact that the N-terminus of NCp8 differs from that of NCp7, this domain is essential for NCp8 activity. Our data demonstrate that the HIV-2 NC protein displays reduced nucleic acid chaperone activity compared to that of HIV-1 NC. We found that NCp8 activity is limited by substrate length and stability to a greater degree than that of NCp7. This is especially interesting in light of the fact that the HIV-2 5'UTR is more structured than that of HIV-1. The reduced chaperone activity observed with NCp8 may

Protein S binding to human endothelial cells is required for expression of cofactor activity for activated protein C

NARCIS (Netherlands)

Hackeng, T. M.; Hessing, M.; van 't Veer, C.; Meijer-Huizinga, F.; Meijers, J. C.; de Groot, P. G.; van Mourik, J. A.; Bouma, B. N.

1993-01-01

An important feedback mechanism in blood coagulation is supplied by the protein C/protein S anticoagulant pathway. In this study we demonstrate that the binding of human protein S to cultured human umbilical vein endothelial cells (HUVECs) is required for the expression of cofactor activity of

Protein structural dynamics at the gas/water interface examined by hydrogen exchange mass spectrometry.

Science.gov (United States)

Xiao, Yiming; Konermann, Lars

2015-08-01

Gas/water interfaces (such as air bubbles or foam) are detrimental to the stability of proteins, often causing aggregation. This represents a potential problem for industrial processes, for example, the production and handling of protein drugs. Proteins possess surfactant-like properties, resulting in a high affinity for gas/water interfaces. The tendency of previously buried nonpolar residues to maximize contact with the gas phase can cause significant structural distortion. Most earlier studies in this area employed spectroscopic tools that could only provide limited information. Here we use hydrogen/deuterium exchange (HDX) mass spectrometry (MS) for probing the conformational dynamics of the model protein myoglobin (Mb) in the presence of N(2) bubbles. HDX/MS relies on the principle that unfolded and/or highly dynamic regions undergo faster deuteration than tightly folded segments. In bubble-free solution Mb displays EX2 behavior, reflecting the occurrence of short-lived excursions to partially unfolded conformers. A dramatically different behavior is seen in the presence of N(2) bubbles; EX2 dynamics still take place, but in addition the protein shows EX1 behavior. The latter results from interconversion of the native state with conformers that are globally unfolded and long-lived. These unfolded species likely correspond to Mb that is adsorbed to the surface of gas bubbles. N(2) sparging also induces aggregation. To explain the observed behavior we propose a simple model, that is, "semi-unfolded" ↔ "native" ↔ "globally unfolded" → "aggregated". This model quantitatively reproduces the experimentally observed kinetics. To the best of our knowledge, the current study marks the first exploration of surface denaturation phenomena by HDX/MS. © 2015 The Protein Society.

Regulation of heterotrimeric G-protein signaling by NDPK/NME proteins and caveolins: an update.

Science.gov (United States)

Abu-Taha, Issam H; Heijman, Jordi; Feng, Yuxi; Vettel, Christiane; Dobrev, Dobromir; Wieland, Thomas

2018-02-01

Heterotrimeric G proteins are pivotal mediators of cellular signal transduction in eukaryotic cells and abnormal G-protein signaling plays an important role in numerous diseases. During the last two decades it has become evident that the activation status of heterotrimeric G proteins is both highly localized and strongly regulated by a number of factors, including a receptor-independent activation pathway of heterotrimeric G proteins that does not involve the classical GDP/GTP exchange and relies on nucleoside diphosphate kinases (NDPKs). NDPKs are NTP/NDP transphosphorylases encoded by the nme/nm23 genes that are involved in a variety of cellular events such as proliferation, migration, and apoptosis. They therefore contribute, for example, to tumor metastasis, angiogenesis, retinopathy, and heart failure. Interestingly, NDPKs are translocated and/or upregulated in human heart failure. Here we describe recent advances in the current understanding of NDPK functions and how they have an impact on local regulation of G-protein signaling.

Contractions activate hormone-sensitive lipase in rat muscle by protein kinase C and mitogen-activated protein kinase

DEFF Research Database (Denmark)

Donsmark, Morten; Langfort, Jozef; Holm, Cecilia

2003-01-01

and contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction......Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by both adrenaline......-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50% by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None...

Using the multi-objective optimization replica exchange Monte Carlo enhanced sampling method for protein-small molecule docking.

Science.gov (United States)

Wang, Hongrui; Liu, Hongwei; Cai, Leixin; Wang, Caixia; Lv, Qiang

2017-07-10

In this study, we extended the replica exchange Monte Carlo (REMC) sampling method to protein-small molecule docking conformational prediction using RosettaLigand. In contrast to the traditional Monte Carlo (MC) and REMC sampling methods, these methods use multi-objective optimization Pareto front information to facilitate the selection of replicas for exchange. The Pareto front information generated to select lower energy conformations as representative conformation structure replicas can facilitate the convergence of the available conformational space, including available near-native structures. Furthermore, our approach directly provides min-min scenario Pareto optimal solutions, as well as a hybrid of the min-min and max-min scenario Pareto optimal solutions with lower energy conformations for use as structure templates in the REMC sampling method. These methods were validated based on a thorough analysis of a benchmark data set containing 16 benchmark test cases. An in-depth comparison between MC, REMC, multi-objective optimization-REMC (MO-REMC), and hybrid MO-REMC (HMO-REMC) sampling methods was performed to illustrate the differences between the four conformational search strategies. Our findings demonstrate that the MO-REMC and HMO-REMC conformational sampling methods are powerful approaches for obtaining protein-small molecule docking conformational predictions based on the binding energy of complexes in RosettaLigand.

In nucleoli, the steady state of nucleolar proteins is leptomycin B-sensitive.

Science.gov (United States)

Muro, Eleonora; Hoang, Thang Q; Jobart-Malfait, Aude; Hernandez-Verdun, Danièle

2008-05-01

The nucleolus is a dynamic structure. It has been demonstrated that nucleolar proteins rapidly associate with and dissociate from nucleolar components in continuous exchanges with the nucleoplasm using GFP (green fluorescent protein)-tagged proteins. However, how the exchanges within one nucleolus and between nucleoli within the nuclear volume occurred is still poorly understood. The movement of PAGFP (photoactivatable GFP)-tagged proteins that become visible after photoactivation can be followed. In the present study, we establish the protocol allowing quantification of the traffic of PAGFP-tagged nucleolar proteins in nuclei containing two nucleoli. The traffic in the activated area, at the periphery of the activated area and to the neighbouring nucleolus is measured. Protein B23 is rapidly replaced in the activated area, and at the periphery of the activated area the steady state suggests intranucleolar recycling of B23; this recycling is LMB (leptomycin B)-sensitive. The pool of activated B23 is equally distributed in the volume of the two nucleoli within 2 min. The three-dimensional distribution of the proteins Nop52 and fibrillarin is less rapid than that of B23 but is also LMB-sensitive. In contrast, traffic of fibrillarin from the nucleoli to the CB (Cajal body) was not modified by LMB. We propose that the steady state of nucleolar proteins in nucleoli depends on the affinity of the proteins for their partners and on intranucleolar recycling. This steady state can be impaired by LMB but not the uptake in the neighbouring nucleolus or the CB.

Mechanistic insights into dioxygen activation, oxygen atom exchange and substrate epoxidation by AsqJ dioxygenase from quantum mechanical/molecular mechanical calculations.

Science.gov (United States)

Song, Xudan; Lu, Jiarui; Lai, Wenzhen

2017-08-02

Herein, we use in-protein quantum mechanical/molecular mechanical (QM/MM) calculations to elucidate the mechanism of dioxygen activation, oxygen atom exchange and substrate epoxidation processes by AsqJ, an Fe II /α-ketoglutarate-dependent dioxygenase (α-KGD) using a 2-His-1-Asp facial triad. Our results demonstrated that the whole reaction proceeds through a quintet surface. The dioxygen activation by AsqJ leads to a quintet penta-coordinated Fe IV -oxo species, which has a square pyramidal geometry with the oxo group trans to His134. This penta-coordinated Fe IV -oxo species is not the reactive one in the substrate epoxidation reaction since its oxo group is pointing away from the target C[double bond, length as m-dash]C bond. Instead, it can undergo the oxo group isomerization followed by water binding or the water binding followed by oxygen atom exchange to form the reactive hexa-coordinated Fe IV -oxo species with the oxo group trans to His211. The calculated parameters of Mössbauer spectra for this hexa-coordinated Fe IV -oxo intermediate are in excellent agreement with the experimental values, suggesting that it is most likely the experimentally trapped species. The calculated energetics indicated that the rate-limiting step is the substrate C[double bond, length as m-dash]C bond activation. This work improves our understanding of the dioxygen activation by α-KGD and provides important structural information about the reactive Fe IV -oxo species.

Calmodulin-lanthanide ion exchange kinetics

International Nuclear Information System (INIS)

Buccigross, J.; O'Donnell, C.; Nelson, D.

1985-01-01

A flow dialysis apparatus suitable for the study of high affinity metal binding proteins has been utilized to study calmodulin-metal exchange kinetics. Calmodulin labeled with Eu-155 and Gd-153 was dialyzed against buffer containing various competing metal ions. The rate of metal exchange was monitored by a gamma-ray scintillation detector. The kinetics of exchange are first order, and the rates fall into two categories: Ca (II) and CD (II) in one, and the lanthanides Eu (III), Gd (III), and La (III) in the other

Optimization of elution salt concentration in stepwise elution of protein chromatography using linear gradient elution data. Reducing residual protein A by cation-exchange chromatography in monoclonal antibody purification.

Science.gov (United States)

Ishihara, Takashi; Kadoya, Toshihiko; Endo, Naomi; Yamamoto, Shuichi

2006-05-05

Our simple method for optimization of the elution salt concentration in stepwise elution was applied to the actual protein separation system, which involves several difficulties such as detection of the target. As a model separation system, reducing residual protein A by cation-exchange chromatography in human monoclonal antibody (hMab) purification was chosen. We carried out linear gradient elution experiments and obtained the data for the peak salt concentration of hMab and residual protein A, respectively. An enzyme-linked immunosorbent assay was applied to the measurement of the residual protein A. From these data, we calculated the distribution coefficient of the hMab and the residual protein A as a function of salt concentration. The optimal salt concentration of stepwise elution to reduce the residual protein A from the hMab was determined based on the relationship between the distribution coefficient and the salt concentration. Using the optimized condition, we successfully performed the separation, resulting in high recovery of hMab and the elimination of residual protein A.

Estimating Exchange Market Pressure and the Degree of Exchange Market Intervention for Finland during the Floating Exchange Rate Regime

OpenAIRE

Pösö, Mika; Spolander, Mikko

1997-01-01

In this paper, we use a fairly simple monetary macro model to calculate the quarterly measures of exchange market pressure and the degree of the Bank of Finland's intervention during the time the markka was floated. Exchange market pressure measures the size of the exchange rate change that would have occurred if the central bank had unexpectedly refrained from intervening in the foreign exchange market. Intervention activity of the central bank is measured as the proportion of exchange marke...

Purinergic receptors stimulate Na+/Ca2+ exchange in pancreatic duct cells: possible role of proteins handling and transporting Ca2+

DEFF Research Database (Denmark)

Hansen, Mette R; Krabbe, Simon; Ankorina-Stark, Ieva

2009-01-01

). Since NCX can also be connected with epithelial Ca(2+) transport, we also investigated expression of some Ca(2+)-handling/transporting proteins. Expression analysis revealed that pancreatic ducts of rat and human duct cell line CFPAC-1 (also PANC-1 and Capan-1) express the Na(+)/Ca(2+) exchanger (splice...

Activation of the Unfolded Protein Response Contributes toward the Antitumor Activity of Vorinostat

Directory of Open Access Journals (Sweden)

Soumen Kahali

2010-01-01

Full Text Available Histone deacetylase (HDAC inhibitors represent an emerging class of anticancer agents progressing through clinical trials. Although their primary target is thought to involve acetylation of core histones, several nonhistone substrates have been identified, including heat shock protein (HSP 90, which may contribute towards their antitumor activity. Glucose-regulated protein 78 (GRP78 is a member of the HSP family of molecular chaperones and plays a central role in regulating the unfolded protein response (UPR. Emerging data suggest that GRP78 is critical in cellular adaptation and survival associated with oncogenesis and may serve as a cancer-specific therapeutic target. On the basis of shared homology with HSP family proteins, we sought to determine whether GRP78 could serve as a molecular target of the HDAC inhibitor vorinostat. Vorinostat treatment led to GRP78 acetylation, dissociation, and subsequent activation of its client protein double-stranded RNA-activated protein-like endoplasmic reticulum kinase (PERK. Investigations in a panel of cancer cell lines identified that UPR activation after vorinostat exposure is specific to certain lines. Mass spectrometry performed on immunoprecipitated GRP78 identified lysine-585 as a specific vorinostat-induced acetylation site of GRP78. Downstream activation of the UPR was confirmed, including eukaryotic initiating factor 2α phosphorylation and increase in ATF4 and C/EBP homologous protein expression. To determine the biologic relevance of UPR activation after vorinostat, RNA interference of PERK was performed, demonstrating significantly decreased sensitivity to vorinostat-induced cytotoxicity. Collectively, these findings indicate that GRP78 is a biologic target of vorinostat, and activation of the UPR through PERK phosphorylation contributes toward its antitumor activity.

Synergistic inhibition of the intrinsic factor X activation by protein S and C4b-binding protein

NARCIS (Netherlands)

Koppelman, S.J.

1995-01-01

The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for

Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms

DEFF Research Database (Denmark)

Massip, L; Garand, C; Labbé, A

2010-01-01

show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1......), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease...... activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCdelta and PKCbetaII in the membrane fraction of cells. In contrast...

Electron self-exchange in hemoglobins revealed by deutero-hemin substitution.

Science.gov (United States)

Athwal, Navjot Singh; Alagurajan, Jagannathan; Sturms, Ryan; Fulton, D Bruce; Andreotti, Amy H; Hargrove, Mark S

2015-09-01

Hemoglobins (phytoglobins) from rice plants (nsHb1) and from the cyanobacterium Synechocystis (PCC 6803) (SynHb) can reduce hydroxylamine with two electrons to form ammonium. The reaction requires intermolecular electron transfer between protein molecules, and rapid electron self-exchange might play a role in distinguishing these hemoglobins from others with slower reaction rates, such as myoglobin. A relatively rapid electron self-exchange rate constant has been measured for SynHb by NMR, but the rate constant for myoglobin is equivocal and a value for nsHb1 has not yet been measured. Here we report electron self-exchange rate constants for nsHb1 and Mb as a test of their role in hydroxylamine reduction. These proteins are not suitable for analysis by NMR ZZ exchange, so a method was developed that uses cross-reactions between each hemoglobin and its deutero-hemin substituted counterpart. The resulting electron transfer is between identical proteins with low driving forces and thus closely approximates true electron self-exchange. The reactions can be monitored spectrally due to the distinct spectra of the prosthetic groups, and from this electron self-exchange rate constants of 880 (SynHb), 2900 (nsHb1), and 0.05M(-1) s(-1) (Mb) have been measured for each hemoglobin. Calculations of cross-reactions using these values accurately predict hydroxylamine reduction rates for each protein, suggesting that electron self-exchange plays an important role in the reaction. Copyright © 2015 Elsevier Inc. All rights reserved.

An Econometric Analysis of the Impact of Real Effective Exchange Rate on Economic Activities in Nepal

OpenAIRE

NARA BAHADUR THAPA

2002-01-01

This paper tests the relationship between REER and GDP on the Nepalese economy. In the literature, two channels of transmission exist for the real exchange rate to affect economic activities; these are the aggregate demand channel and the aggregate supply channel. The traditional view has it that the real exchange rate operates through the aggregate demand channel. This means that the depreciation of the real exchange rate enhances the international competitiveness of domestic goods, boosts n...

Isolation and structure-function characterization of a signaling-active rhodopsin-G protein complex.

Science.gov (United States)

Gao, Yang; Westfield, Gerwin; Erickson, Jon W; Cerione, Richard A; Skiniotis, Georgios; Ramachandran, Sekar

2017-08-25

The visual photo-transduction cascade is a prototypical G protein-coupled receptor (GPCR) signaling system, in which light-activated rhodopsin (Rho*) is the GPCR catalyzing the exchange of GDP for GTP on the heterotrimeric G protein transducin (G T ). This results in the dissociation of G T into its component α T -GTP and β 1 γ 1 subunit complex. Structural information for the Rho*-G T complex will be essential for understanding the molecular mechanism of visual photo-transduction. Moreover, it will shed light on how GPCRs selectively couple to and activate their G protein signaling partners. Here, we report on the preparation of a stable detergent-solubilized complex between Rho* and a heterotrimer (G T *) comprising a Gα T /Gα i1 chimera (α T *) and β 1 γ 1 The complex was formed on native rod outer segment membranes upon light activation, solubilized in lauryl maltose neopentyl glycol, and purified with a combination of affinity and size-exclusion chromatography. We found that the complex is fully functional and that the stoichiometry of Rho* to Gα T * is 1:1. The molecular weight of the complex was calculated from small-angle X-ray scattering data and was in good agreement with a model consisting of one Rho* and one G T *. The complex was visualized by negative-stain electron microscopy, which revealed an architecture similar to that of the β 2 -adrenergic receptor-G S complex, including a flexible α T * helical domain. The stability and high yield of the purified complex should allow for further efforts toward obtaining a high-resolution structure of this important signaling complex. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

ARSENIC REMOVAL FROM DRINKING WATER BY ION EXCHANGE AND ACTIVATED ALUMINA PLANTS

Science.gov (United States)

This report documents a long term performance study of two ion exchange (IE) and two activated alumina (AA) treatment plants to remove arsenic from drinking water. Performance information was collected on these systems that are located in the northeast for one full year. The stud...

The human protein disulfide isomerase gene family

Directory of Open Access Journals (Sweden)

Galligan James J

2012-07-01

Full Text Available Abstract Enzyme-mediated disulfide bond formation is a highly conserved process affecting over one-third of all eukaryotic proteins. The enzymes primarily responsible for facilitating thiol-disulfide exchange are members of an expanding family of proteins known as protein disulfide isomerases (PDIs. These proteins are part of a larger superfamily of proteins known as the thioredoxin protein family (TRX. As members of the PDI family of proteins, all proteins contain a TRX-like structural domain and are predominantly expressed in the endoplasmic reticulum. Subcellular localization and the presence of a TRX domain, however, comprise the short list of distinguishing features required for gene family classification. To date, the PDI gene family contains 21 members, varying in domain composition, molecular weight, tissue expression, and cellular processing. Given their vital role in protein-folding, loss of PDI activity has been associated with the pathogenesis of numerous disease states, most commonly related to the unfolded protein response (UPR. Over the past decade, UPR has become a very attractive therapeutic target for multiple pathologies including Alzheimer disease, Parkinson disease, alcoholic and non-alcoholic liver disease, and type-2 diabetes. Understanding the mechanisms of protein-folding, specifically thiol-disulfide exchange, may lead to development of a novel class of therapeutics that would help alleviate a wide range of diseases by targeting the UPR.

Mycobacterium tuberculosis nucleoid-associated DNA-binding protein H-NS binds with high-affinity to the Holliday junction and inhibits strand exchange promoted by RecA protein.

Science.gov (United States)

Sharadamma, N; Harshavardhana, Y; Singh, Pawan; Muniyappa, K

2010-06-01

A number of studies have shown that the structure and composition of bacterial nucleoid influences many a processes related to DNA metabolism. The nucleoid-associated proteins modulate not only the DNA conformation but also regulate the DNA metabolic processes such as replication, recombination, repair and transcription. Understanding of how these processes occur in the context of Mycobacterium tuberculosis nucleoid is of considerable medical importance because the nucleoid structure may be constantly remodeled in response to environmental signals and/or growth conditions. Many studies have concluded that Escherichia coli H-NS binds to DNA in a sequence-independent manner, with a preference for A-/T-rich tracts in curved DNA; however, recent studies have identified the existence of medium- and low-affinity binding sites in the vicinity of the curved DNA. Here, we show that the M. tuberculosis H-NS protein binds in a more structure-specific manner to DNA replication and repair intermediates, but displays lower affinity for double-stranded DNA with relatively higher GC content. Notably, M. tuberculosis H-NS was able to bind Holliday junction (HJ), the central recombination intermediate, with substantially higher affinity and inhibited the three-strand exchange promoted by its cognate RecA. Likewise, E. coli H-NS was able to bind the HJ and suppress DNA strand exchange promoted by E. coli RecA, although much less efficiently compared to M. tuberculosis H-NS. Our results provide new insights into a previously unrecognized function of H-NS protein, with implications for blocking the genome integration of horizontally transferred genes by homologous and/or homeologous recombination.

Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication? [version 1; referees: 2 approved

Directory of Open Access Journals (Sweden)

Simon Imhof

2016-04-01

Full Text Available Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes.  Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer.  Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature.

Role of adaptor proteins and clathrin in the trafficking of human kidney anion exchanger 1 (kAE1) to the cell surface.

Science.gov (United States)

Junking, Mutita; Sawasdee, Nunghathai; Duangtum, Natapol; Cheunsuchon, Boonyarit; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-thai

2014-07-01

Kidney anion exchanger 1 (kAE1) plays an important role in acid-base homeostasis by mediating chloride/bicarbornate (Cl-/HCO3-) exchange at the basolateral membrane of α-intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of SLC4A1 encoding kAE1 results in kidney disease - distal renal tubular acidosis (dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans-Golgi network (TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral-related sorting proteins, including the mu1 subunit of adaptor protein (AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non-polarized kidney cells. By using RNA interference, co-immunoprecipitation, yellow fluorescent protein-based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin (but not AP-1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral-related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid-secreting α-intercalated cells. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Minimizing Back Exchange in the Hydrogen Exchange-Mass Spectrometry Experiment

Science.gov (United States)

Walters, Benjamin T.; Ricciuti, Alec; Mayne, Leland; Englander, S. Walter

2012-12-01

The addition of mass spectrometry (MS) analysis to the hydrogen exchange (HX) proteolytic fragmentation experiment extends powerful HX methodology to the study of large biologically important proteins. A persistent problem is the degradation of HX information due to back exchange of deuterium label during the fragmentation-separation process needed to prepare samples for MS measurement. This paper reports a systematic analysis of the factors that influence back exchange (solution pH, ionic strength, desolvation temperature, LC column interaction, flow rates, system volume). The many peptides exhibit a range of back exchange due to intrinsic amino acid HX rate differences. Accordingly, large back exchange leads to large variability in D-recovery from one residue to another as well as one peptide to another that cannot be corrected for by reference to any single peptide-level measurement. The usual effort to limit back exchange by limiting LC time provides little gain. Shortening the LC elution gradient by 3-fold only reduced back exchange by ~2 %, while sacrificing S/N and peptide count. An unexpected dependence of back exchange on ionic strength as well as pH suggests a strategy in which solution conditions are changed during sample preparation. Higher salt should be used in the first stage of sample preparation (proteolysis and trapping) and lower salt (<20 mM) and pH in the second stage before electrospray injection. Adjustment of these and other factors together with recent advances in peptide fragment detection yields hundreds of peptide fragments with D-label recovery of 90 % ± 5 %.

Minimizing back exchange in the hydrogen exchange-mass spectrometry experiment.

Science.gov (United States)

Walters, Benjamin T; Ricciuti, Alec; Mayne, Leland; Englander, S Walter

2012-12-01

The addition of mass spectrometry (MS) analysis to the hydrogen exchange (HX) proteolytic fragmentation experiment extends powerful HX methodology to the study of large biologically important proteins. A persistent problem is the degradation of HX information due to back exchange of deuterium label during the fragmentation-separation process needed to prepare samples for MS measurement. This paper reports a systematic analysis of the factors that influence back exchange (solution pH, ionic strength, desolvation temperature, LC column interaction, flow rates, system volume). The many peptides exhibit a range of back exchange due to intrinsic amino acid HX rate differences. Accordingly, large back exchange leads to large variability in D-recovery from one residue to another as well as one peptide to another that cannot be corrected for by reference to any single peptide-level measurement. The usual effort to limit back exchange by limiting LC time provides little gain. Shortening the LC elution gradient by 3-fold only reduced back exchange by ~2%, while sacrificing S/N and peptide count. An unexpected dependence of back exchange on ionic strength as well as pH suggests a strategy in which solution conditions are changed during sample preparation. Higher salt should be used in the first stage of sample preparation (proteolysis and trapping) and lower salt (<20 mM) and pH in the second stage before electrospray injection. Adjustment of these and other factors together with recent advances in peptide fragment detection yields hundreds of peptide fragments with D-label recovery of 90% ± 5%.

Protein crystallography and drug discovery: recollections of knowledge exchange between academia and industry.

Science.gov (United States)

Blundell, Tom L

2017-07-01

The development of structure-guided drug discovery is a story of knowledge exchange where new ideas originate from all parts of the research ecosystem. Dorothy Crowfoot Hodgkin obtained insulin from Boots Pure Drug Company in the 1930s and insulin crystallization was optimized in the company Novo in the 1950s, allowing the structure to be determined at Oxford University. The structure of renin was developed in academia, on this occasion in London, in response to a need to develop antihypertensives in pharma. The idea of a dimeric aspartic protease came from an international academic team and was discovered in HIV; it eventually led to new HIV antivirals being developed in industry. Structure-guided fragment-based discovery was developed in large pharma and biotechs, but has been exploited in academia for the development of new inhibitors targeting protein-protein interactions and also antimicrobials to combat mycobacterial infections such as tuberculosis. These observations provide a strong argument against the so-called 'linear model', where ideas flow only in one direction from academic institutions to industry. Structure-guided drug discovery is a story of applications of protein crystallography and knowledge exhange between academia and industry that has led to new drug approvals for cancer and other common medical conditions by the Food and Drug Administration in the USA, as well as hope for the treatment of rare genetic diseases and infectious diseases that are a particular challenge in the developing world.

Gamma irradiation effect on soy protein modification, protein - phenolic interaction and antioxidant activity in soybean

International Nuclear Information System (INIS)

Kumari, Sweta; Dahuja, Anil; Vinutha, T.; Singh, Bhupinder

2014-01-01

Soy protein is one of the most important sources of protein to feed the world population in the future. Consumption of soybean quality protein and their texture is dependent on the protein modification. In the present study, four soybean genotypes PL5039 (black), EC 472143 (black), Pusa 9814 (yellow) and SL525 (yellow), differing in their seed coat colour were gamma irradiated at 0.5,1.0, 2.0 and 5.0 kGy and the extent of protein modification and parameters affecting it viz. free phenolics, bound phenolics, lip oxygenase and antioxidant activity were analysed. Modifications of soybean proteins were investigated by chemical analysis and electrophoresis. The irradiation dose of 1.0 kGy showed decreased turbidity, protein oxidation, surface hydrophobicity but increased solubility and sulfhydryl and disulfide contents in all the genotypes. Further, SDS PAGE profile of treated soybean seeds revealed remarkable difference in electrophoretic bands as compared to the untreated seeds. Lipoxygense activity in all the genotypes decreased with increased exposure of gamma irradiation, which produced peroxide products that changes the structural characteristics of soy protein. Free phenolics, bound phenolics and total antioxidant activity measured in terms of FRAP in all the genotypes increased significantly at a dose of 2.0 kGy and it declined at a dose of 5.0 kGy. Antioxidant potential measured in terms of 1,1-diphenyl-2- picrylhydrazyl (DPPH) scavenging activity showed an increasing trend with dose, indicating that radiation processing as a method of food preservation has a positive nutritional implication. Hence, it is suggested that, mild gamma irradiation upto 2.0 kGy may reduce the protein oxidation, enhance the antioxidant activity and improve the soybean protein quality compared to higher dose 5.0 kGy, which reduced the protein quality. (author)

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.

Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies.

Science.gov (United States)

Yamaguchi, Hiroshi; Miyazaki, Masaya

2014-02-20

Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

Refolding Techniques for Recovering Biologically Active Recombinant Proteins from Inclusion Bodies

Directory of Open Access Journals (Sweden)

Hiroshi Yamaguchi

2014-02-01

Full Text Available Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

Conferring specificity in redox pathways by enzymatic thiol/disulfide exchange reactions.

Science.gov (United States)

Netto, Luis Eduardo S; de Oliveira, Marcos Antonio; Tairum, Carlos A; da Silva Neto, José Freire

2016-01-01

Thiol-disulfide exchange reactions are highly reversible, displaying nucleophilic substitutions mechanism (S(N)2 type). For aliphatic, low molecular thiols, these reactions are slow, but can attain million times faster rates in enzymatic processes. Thioredoxin (Trx) proteins were the first enzymes described to accelerate thiol-disulfide exchange reactions and their high reactivity is related to the high nucleophilicity of the attacking thiol. Substrate specificity in Trx is achieved by several factors, including polar, hydrophobic, and topological interactions through a groove in the active site. Glutaredoxin (Grx) enzymes also contain the Trx fold, but they do not share amino acid sequence similarity with Trx. A conserved glutathione binding site is a typical feature of Grx that can reduce substrates by two mechanisms (mono and dithiol). The high reactivity of Grx enzymes is related to the very acid pK(a) values of reactive Cys that plays roles as good leaving groups. Therefore, although distinct oxidoreductases catalyze similar thiol–disulfide exchange reactions, their enzymatic mechanisms vary. PDI and DsbA are two other oxidoreductases, but they are involved in disulfide bond formation, instead of disulfide reduction, which is related to the oxidative environment where they are found. PDI enzymes and DsbC are endowed with disulfide isomerase activity, which is related with their tetra-domain architecture. As illustrative description of specificity in thiol-disulfide exchange, redox aspects of transcription activation in bacteria, yeast, and mammals are presented in an evolutionary perspective. Therefore, thiol-disulfide exchange reactions play important roles in conferring specificity to pathways, a required feature for signaling.

Graphene oxide as a protein matrix: influence on protein biophysical properties.

Science.gov (United States)

Hernández-Cancel, Griselle; Suazo-Dávila, Dámaris; Ojeda-Cruzado, Axel J; García-Torres, Desiree; Cabrera, Carlos R; Griebenow, Kai

2015-10-19

This study provides fundamental information on the influence of graphene oxide (GO) nanosheets and glycans on protein catalytic activity, dynamics, and thermal stability. We provide evidence of protein stabilization by glycans and how this strategy could be implemented when GO nanosheets is used as protein immobilization matrix. A series of bioconjugates was constructed using two different strategies: adsorbing or covalently attaching native and glycosylated bilirubin oxidase (BOD) to GO. Bioconjugate formation was followed by FT-IR, zeta-potential, and X-ray photoelectron spectroscopy measurements. Enzyme kinetic parameters (k(m) and k(cat)) revealed that the substrate binding affinity was not affected by glycosylation and immobilization on GO, but the rate of enzyme catalysis was reduced. Structural analysis by circular dichroism showed that glycosylation did not affect the tertiary or the secondary structure of BOD. However, GO produced slight changes in the secondary structure. To shed light into the biophysical consequence of protein glycosylation and protein immobilization on GO nanosheets, we studied structural protein dynamical changes by FT-IR H/D exchange and thermal inactivation. It was found that glycosylation caused a reduction in structural dynamics that resulted in an increase in thermostability and a decrease in the catalytic activity for both, glycoconjugate and immobilized enzyme. These results establish the usefulness of chemical glycosylation to modulate protein structural dynamics and stability to develop a more stable GO-protein matrix.

Hydrogen exchange kinetics changes upon formation of the soybean trypsin inhibitor: trypsin complex

International Nuclear Information System (INIS)

Woodward, C.K.; Ellis, L.M.

1975-01-01

The hydrogen exchange kinetics of the complex of trypsin--soybean trypsin inhibitor (Kunitz) have been compared to the calculated sum of the exchange kinetics for the inhibitor and trypsin measured separately. The exchange rates observed for the complex are substantially less than the sum of the exchange rates in the two individual proteins. These results cannot be accounted for by changes in intermolecular or intramolecular hydrogen bonding. The decrease in exchange rates in the complex are ascribed to changes in solvent accessibility in the component proteins. (U.S.)

Hydrogen Exchange Mass Spectrometry

Science.gov (United States)

Mayne, Leland

2018-01-01

Hydrogen exchange (HX) methods can reveal much about the structure, energetics, and dynamics of proteins. The addition of mass spectrometry (MS) to an earlier fragmentation-separation HX analysis now extends HX studies to larger proteins at high structural resolution and can provide information not available before. This chapter discusses experimental aspects of HX labeling, especially with respect to the use of MS and the analysis of MS data. PMID:26791986

The impact of loading approach and biological activity on NOM removal by ion exchange resins.

Science.gov (United States)

Winter, Joerg; Wray, Heather E; Schulz, Martin; Vortisch, Roman; Barbeau, Benoit; Bérubé, Pierre R

2018-05-01

The present study investigated the impact of different loading approaches and microbial activity on the Natural Organic Matter (NOM) removal efficiency and capacity of ion exchange resins. Gaining further knowledge on the impact of loading approaches is of relevance because laboratory-scale multiple loading tests (MLTs) have been introduced as a simpler and faster alternative to column tests for predicting the performance of IEX, but only anecdotal evidence exists to support their ability to forecast contaminant removal and runtime until breakthrough of IEX systems. The overall trends observed for the removal and the time to breakthrough of organic material estimated using MLTs differed from those estimated using column tests. The results nonetheless suggest that MLTs could best be used as an effective tool to screen different ion exchange resins in terms of their ability to remove various contaminants of interest from different raw waters. The microbial activity was also observed to impact the removal and time to breakthrough. In the absence of regeneration, a microbial community rapidly established itself in ion exchange columns and contributed to the removal of organic material. Biological ion exchange (BIEX) removed more organic material and enabled operation beyond the point when the resin capacity would have otherwise been exhausted using conventional (i.e. in the absence of a microbial community) ion exchange. Furthermore, significantly greater removal of organic matter could be achieved with BIEX than biological activated carbon (BAC) (i.e. 56 ± 7% vs. 15 ± 5%, respectively) when operated at similar loading rates. The results suggest that for some raw waters, BIEX could replace BAC as the technology of choice for the removal of organic material. Copyright © 2018 Elsevier Ltd. All rights reserved.

PDZ Protein Regulation of G Protein-Coupled Receptor Trafficking and Signaling Pathways.

Science.gov (United States)

Dunn, Henry A; Ferguson, Stephen S G

2015-10-01

G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and

Signals exchanged between legumes and Rhizobium: agricultural uses and perspectives

International Nuclear Information System (INIS)

Broughton, William J.; Perret, Xavier; Staehelin, Christian; Zhang Feng

2001-01-01

Legumes and rhizobia exchange at least three different, but sometimes complementary sets of signals. Amongst the variety of substances normally and continuously secreted into the rhizosphere by plants are phenolic compounds. Flavonoid components of these mixtures are especially active in inducing rhizobial nodulation genes. Many nodgenes exist. Some (e.g., nodD) serve as regulators of transcription, but most code for enzymes involved in the synthesis of a family of lipo-chito-oligosaccharides (LCOs) called Nod-factors. Nod-factors possess hormone-like properties, are key determinants in nodulation, and allow rhizobia to enter the plant. As Nod-factors also stimulate the synthesis and release of flavonoids from legume roots, the response to inoculation is amplified. Once the bacteria enter the plant, other sets of signals are exchanged between the symbionts. These include extra-cellular polysaccharides (EPSs) as well as proteins externalised via type-three secretion systems. These carbohydrates/proteins may be active in invasion of the root. At the time of writing, only flavonoids and Nodfactors have been chemically synthesised and of these only the former are available in large quantities. Field trials in North America show that seed application of flavonoids stimulates nodulation and nitrogen fixation in soybeans grown at low soil temperatures. The biological basis to these responses is discussed. (author)

Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.

Science.gov (United States)

Tong, X; Kono, T; Evans-Molina, C

2015-06-18

The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b

Speeding-up exchange-mediated saturation transfer experiments by Fourier transform

Energy Technology Data Exchange (ETDEWEB)

Carneiro, Marta G.; Reddy, Jithender G.; Griesinger, Christian; Lee, Donghan, E-mail: dole@nmr.mpibpc.mpg.de [Max-Planck Institute for Biophysical chemistry, Department of NMR-based Structural Biology (Germany)

2015-11-15

Protein motions over various time scales are crucial for protein function. NMR relaxation dispersion experiments play a key role in explaining these motions. However, the study of slow conformational changes with lowly populated states remained elusive. The recently developed exchange-mediated saturation transfer experiments allow the detection and characterization of such motions, but require extensive measurement time. Here we show that, by making use of Fourier transform, the total acquisition time required to measure an exchange-mediated saturation transfer profile can be reduced by twofold in case that one applies linear prediction. In addition, we demonstrate that the analytical solution for R{sub 1}ρ experiments can be used for fitting the exchange-mediated saturation transfer profile. Furthermore, we show that simultaneous analysis of exchange-mediated saturation transfer profiles with two different radio-frequency field strengths is required for accurate and precise characterization of the exchange process and the exchanging states.

Speeding-up exchange-mediated saturation transfer experiments by Fourier transform

International Nuclear Information System (INIS)

Carneiro, Marta G.; Reddy, Jithender G.; Griesinger, Christian; Lee, Donghan

2015-01-01

Protein motions over various time scales are crucial for protein function. NMR relaxation dispersion experiments play a key role in explaining these motions. However, the study of slow conformational changes with lowly populated states remained elusive. The recently developed exchange-mediated saturation transfer experiments allow the detection and characterization of such motions, but require extensive measurement time. Here we show that, by making use of Fourier transform, the total acquisition time required to measure an exchange-mediated saturation transfer profile can be reduced by twofold in case that one applies linear prediction. In addition, we demonstrate that the analytical solution for R 1 ρ experiments can be used for fitting the exchange-mediated saturation transfer profile. Furthermore, we show that simultaneous analysis of exchange-mediated saturation transfer profiles with two different radio-frequency field strengths is required for accurate and precise characterization of the exchange process and the exchanging states

Functional characterization of a wheat NHX antiporter gene TaNHX2 that encodes a K(+/H(+ exchanger.

Directory of Open Access Journals (Sweden)

Yuanyuan Xu

Full Text Available The subcellular localization of a wheat NHX antiporter, TaNHX2, was studied in Arabidopsis protoplasts, and its function was evaluated using Saccharomyces cerevisiae as a heterologous expression system. Fluorescence patterns of TaNHX2-GFP fusion protein in Arabidopsis cells indicated that TaNHX2 localized at endomembranes. TaNHX2 has significant sequence homology to NHX sodium exchangers from Arabidopsis, is abundant in roots and leaves and is induced by salt or dehydration treatments. Western blot analysis showed that TaNHX2 could be expressed in transgenic yeast cells. Expressed TaNHX2 protein suppressed the salt sensitivity of a yeast mutant strain by increasing its K(+ content when exposed to salt stress. TaNHX2 also increased the tolerance of the strain to potassium stress. However, the expression of TaNHX2 did not affect the sodium concentration in transgenic cells. Western blot analysis for tonoplast proteins indicated that the TaNHX2 protein localized at the tonoplast of transgenic yeast cells. The tonoplast vesicles from transgenic yeast cells displayed enhanced K(+/H(+ exchange activity but very little Na(+/H(+ exchange compared with controls transformed with the empty vector; Na(+/H(+ exchange was not detected with concentrations of less than 37.5 mM Na(+ in the reaction medium. Our data suggest that TaNHX2 is a endomembrane-bound protein and may primarily function as a K(+/H(+ antiporter, which is involved in cellular pH regulation and potassium nutrition under normal conditions. Under saline conditions, the protein mediates resistance to salt stress through the intracellular compartmentalization of potassium to regulate cellular pH and K(+ homeostasis.

Exchange of adsorbed serum proteins during adhesion of Staphylococcus aureus to an abiotic surface and Candida albicans hyphae--an AFM study.

Science.gov (United States)

Ovchinnikova, Ekaterina S; van der Mei, Henny C; Krom, Bastiaan P; Busscher, Henk J

2013-10-01

Staphylococcus aureus and Candida albicans are the second and third most commonly isolated microorganisms in hospital-related-infections, that are often multi-species in nature causing high morbidity and mortality. Here, adhesion forces between a S. aureus strain and abiotic (tissue-culture-polystyrene, TCPS) or partly biotic (TCPS with adhering hyphae of C. albicans) surfaces were investigated in presence of fetal-bovine-serum or individual serum proteins and related with staphylococcal adhesion. Atomic-force-microscopy was used to measure adhesion forces between S. aureus and the abiotic and biotic surfaces. Adsorption of individual serum proteins like albumin and apo-transferrin to abiotic TCPS surfaces during 60min, impeded development of strong adhesion forces as compared to fibronectin, while 60min adsorption of proteins from fetal-bovine-serum yielded a decrease in adhesion force from -5.7nN in phosphate-buffered-saline to -0.6nN. Adsorption of albumin and apo-transferrin also decreased staphylococcal adhesion forces to hyphae as compared with fibronectin. During 60min exposure to fetal-bovine-serum however, initial (5min protein adsorption) staphylococcal adhesion forces were low (-1.6nN), but strong adhesion forces of around -5.5nN were restored within 60min. This suggests for the first time that in whole fetal-bovine-serum exchange of non-adhesive proteins by fibronectin occurs on biotic C. albicans hyphal surfaces. No evidence was found for such protein exchange on abiotic TCPS surfaces. Staphylococcal adhesion of abiotic and biotic surfaces varied in line with the adhesion forces and was low on TCPS in presence of fetal-bovine-serum. On partly biotic TCPS, staphylococci aggregated in presence of fetal-bovine-serum around adhering C. albicans hyphae. Copyright © 2013 Elsevier B.V. All rights reserved.

Energy transfer at the active sites of heme proteins

International Nuclear Information System (INIS)

Dlott, D.D.; Hill, J.R.

1995-01-01

Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes

Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 μ1A (AP-1 mu1A)

International Nuclear Information System (INIS)

Sawasdee, Nunghathai; Junking, Mutita; Ngaojanlar, Piengpaga; Sukomon, Nattakan; Ungsupravate, Duangporn; Limjindaporn, Thawornchai; Akkarapatumwong, Varaporn; Noisakran, Sansanee; Yenchitsomanus, Pa-thai

2010-01-01

Research highlights: → Trafficking defect of kAE1 is a cause of dRTA but trafficking pathway of kAE1 has not been clearly described. → Adaptor-related protein complex 1 μ1A (AP-1 mu1A) was firstly reported to interact with kAE1. → The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. → AP-1 mu1A knockdown showed a marked reduction of kAE1 on the cell membrane and its accumulation in endoplasmic reticulum. → AP-1 mu1A has a critical role in kAE1 trafficking to the plasma membrane. -- Abstract: Kidney anion exchanger 1 (kAE1) mediates chloride (Cl - ) and bicarbonate (HCO 3 - ) exchange at the basolateral membrane of kidney α-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl - /HCO 3 - exchange at the basolateral membrane and failure of proton (H + ) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 μ1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1 trafficking of kidney α-intercalated cells.

Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 {mu}1A (AP-1 mu1A)

Energy Technology Data Exchange (ETDEWEB)

Sawasdee, Nunghathai; Junking, Mutita [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Ngaojanlar, Piengpaga [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Department of Immunology and Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Sukomon, Nattakan; Ungsupravate, Duangporn [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Limjindaporn, Thawornchai [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Akkarapatumwong, Varaporn [Institute of Molecular Biosciences, Mahidol University at Salaya Campus, Nakorn Pathom 73170 (Thailand); Noisakran, Sansanee [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand); Yenchitsomanus, Pa-thai, E-mail: grpye@mahidol.ac.th [Division of Medical Molecular Biology and BIOTEC-Medical Biotechnology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 (Thailand)

2010-10-08

Research highlights: {yields} Trafficking defect of kAE1 is a cause of dRTA but trafficking pathway of kAE1 has not been clearly described. {yields} Adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) was firstly reported to interact with kAE1. {yields} The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. {yields} AP-1 mu1A knockdown showed a marked reduction of kAE1 on the cell membrane and its accumulation in endoplasmic reticulum. {yields} AP-1 mu1A has a critical role in kAE1 trafficking to the plasma membrane. -- Abstract: Kidney anion exchanger 1 (kAE1) mediates chloride (Cl{sup -}) and bicarbonate (HCO{sub 3}{sup -}) exchange at the basolateral membrane of kidney {alpha}-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl{sup -}/HCO{sub 3}{sup -} exchange at the basolateral membrane and failure of proton (H{sup +}) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1

Protein corona between nanoparticles and bacterial proteins in activated sludge: Characterization and effect on nanoparticle aggregation.

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Zhang, Peng; Xu, Xiao-Yan; Chen, You-Peng; Xiao, Meng-Qian; Feng, Bo; Tian, Kai-Xun; Chen, Yue-Hui; Dai, You-Zhi

2018-02-01

In this work, the protein coronas of activated sludge proteins on TiO 2 nanoparticles (TNPs) and ZnO nanoparticles (ZNPs) were characterized. The proteins with high affinity to TNPs and ZNPs were identified by shotgun proteomics, and their effects of on the distributions of TNPs and ZNPs in activated sludge were concluded. In addition, the effects of protein coronas on the aggregations of TNPs and ZNPs were evaluated. Thirty and nine proteins with high affinities to TNPs and ZNPs were identified, respectively. The proteomics and adsorption isotherms demonstrated that activated sludge had a higher affinity to TNPs than to ZNPs. The aggregation percentages of ZNPs at 35, 53, and 106 mg/L of proteins were 13%, 14%, and 18%, respectively, whereas those of TNPs were 21%, 30%, 41%, respectively. The proteins contributed to ZNPs aggregation by dissolved Zn ion-bridging, whereas the increasing protein concentrations enhanced the TNPs aggregation through macromolecule bridging flocculation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Na+/H+ and Na+/NH4+ exchange activities of zebrafish NHE3b expressed in Xenopus oocytes

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Ito, Yusuke; Kato, Akira; Hirata, Taku; Hirose, Shigehisa

2014-01-01

Zebrafish Na+/H+ exchanger 3b (zNHE3b) is highly expressed in the apical membrane of ionocytes where Na+ is absorbed from ion-poor fresh water against a concentration gradient. Much in vivo data indicated that zNHE3b is involved in Na+ absorption but not leakage. However, zNHE3b-mediated Na+ absorption has not been thermodynamically explained, and zNHE3b activity has not been measured. To address this issue, we overexpressed zNHE3b in Xenopus oocytes and characterized its activity by electrophysiology. Exposure of zNHE3b oocytes to Na+-free media resulted in significant decrease in intracellular pH (pHi) and intracellular Na+ activity (aNai). aNai increased significantly when the cytoplasm was acidified by media containing CO2-HCO3− or butyrate. Activity of zNHE3b was inhibited by amiloride or 5-ethylisopropyl amiloride (EIPA). Although the activity was accompanied by a large hyperpolarization of ∼50 mV, voltage-clamp experiments showed that Na+/H+ exchange activity of zNHE3b is electroneutral. Exposure of zNHE3b oocytes to medium containing NH3/NH4+ resulted in significant decreases in pHi and aNai and significant increase in intracellular NH4+ activity, indicating that zNHE3b mediates the Na+/NH4+ exchange. In low-Na+ (0.5 mM) media, zNHE3b oocytes maintained aNai of 1.3 mM, and Na+-influx was observed when pHi was decreased by media containing CO2-HCO3− or butyrate. These results provide thermodynamic evidence that zNHE3b mediates Na+ absorption from ion-poor fresh water by its Na+/H+ and Na+/NH4+ exchange activities. PMID:24401990

Determination of glucose exchange rates and permeability of erythrocyte membrane in preeclampsia and subsequent oxidative stress-related protein damage using dynamic-{sup 19}F-NMR

Energy Technology Data Exchange (ETDEWEB)

Dickinson, Elizabeth, E-mail: elizabeth.dickinson@york.ac.uk [University of York, Department of Chemistry (United Kingdom); Arnold, John R. P. [Selby College (United Kingdom); Fisher, Julie [University of Leeds, School of Chemistry (United Kingdom)

2017-02-15

The cause of the pregnancy condition preeclampsia (PE) is thought to be endothelial dysfunction caused by oxidative stress. As abnormal glucose tolerance has also been associated with PE, we use a fluorinated-mimic of this metabolite to establish whether any oxidative damage to lipids and proteins in the erythrocyte membrane has increased cell membrane permeability. Data were acquired using {sup 19}F Dynamic-NMR (DNMR) to measure exchange of 3-fluoro-3-deoxyglucose (3-FDG) across the membrane of erythrocytes from 10 pregnant women (5 healthy control women, and 5 from women suffering from PE). Magnetisation transfer was measured using the 1D selective inversion and 2D EXSY pulse sequences, over a range of time delays. Integrated intensities from these experiments were used in matrix diagonalisation to estimate the values of the rate constants of exchange and membrane permeability. No significant differences were observed for the rate of exchange of 3-FDG and membrane permeability between healthy pregnant women and those suffering from PE, leading us to conclude that no oxidative damage had occurred at this carrier-protein site in the membrane.

Determination of glucose exchange rates and permeability of erythrocyte membrane in preeclampsia and subsequent oxidative stress-related protein damage using dynamic-"1"9F-NMR

International Nuclear Information System (INIS)

Dickinson, Elizabeth; Arnold, John R. P.; Fisher, Julie

2017-01-01

The cause of the pregnancy condition preeclampsia (PE) is thought to be endothelial dysfunction caused by oxidative stress. As abnormal glucose tolerance has also been associated with PE, we use a fluorinated-mimic of this metabolite to establish whether any oxidative damage to lipids and proteins in the erythrocyte membrane has increased cell membrane permeability. Data were acquired using "1"9F Dynamic-NMR (DNMR) to measure exchange of 3-fluoro-3-deoxyglucose (3-FDG) across the membrane of erythrocytes from 10 pregnant women (5 healthy control women, and 5 from women suffering from PE). Magnetisation transfer was measured using the 1D selective inversion and 2D EXSY pulse sequences, over a range of time delays. Integrated intensities from these experiments were used in matrix diagonalisation to estimate the values of the rate constants of exchange and membrane permeability. No significant differences were observed for the rate of exchange of 3-FDG and membrane permeability between healthy pregnant women and those suffering from PE, leading us to conclude that no oxidative damage had occurred at this carrier-protein site in the membrane.

The N54-αs Mutant Has Decreased Affinity for βγ and Suggests a Mechanism for Coupling Heterotrimeric G Protein Nucleotide Exchange with Subunit Dissociation.

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Cleator, John H; Wells, Christopher A; Dingus, Jane; Kurtz, David T; Hildebrandt, John D

2018-05-01

Ser54 of G s α binds guanine nucleotide and Mg 2+ as part of a conserved sequence motif in GTP binding proteins. Mutating the homologous residue in small and heterotrimeric G proteins generates dominant-negative proteins, but by protein-specific mechanisms. For α i/o , this results from persistent binding of α to βγ , whereas for small GTP binding proteins and α s this results from persistent binding to guanine nucleotide exchange factor or receptor. This work examined the role of βγ interactions in mediating the properties of the Ser54-like mutants of G α subunits. Unexpectedly, WT- α s or N54- α s coexpressed with α 1B -adrenergic receptor in human embryonic kidney 293 cells decreased receptor stimulation of IP3 production by a cAMP-independent mechanism, but WT- α s was more effective than the mutant. One explanation for this result would be that α s , like Ser47 α i/o , blocks receptor activation by sequestering βγ ; implying that N54- α S has reduced affinity for βγ since it was less effective at blocking IP3 production. This possibility was more directly supported by the observation that WT- α s was more effective than the mutant in inhibiting βγ activation of phospholipase C β 2. Further, in vitro synthesized N54- α s bound biotinylated- βγ with lower apparent affinity than did WT- α s The Cys54 mutation also decreased βγ binding but less effectively than N54- α s Substitution of the conserved Ser in α o with Cys or Asn increased βγ binding, with the Cys mutant being more effective. This suggests that Ser54 of α s is involved in coupling changes in nucleotide binding with altered subunit interactions, and has important implications for how receptors activate G proteins. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Evaluation of unrestrained replica-exchange simulations using dynamic walkers in temperature space for protein structure refinement.

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Mark A Olson

Full Text Available A central problem of computational structural biology is the refinement of modeled protein structures taken from either comparative modeling or knowledge-based methods. Simulations are commonly used to achieve higher resolution of the structures at the all-atom level, yet methodologies that consistently yield accurate results remain elusive. In this work, we provide an assessment of an adaptive temperature-based replica exchange simulation method where the temperature clients dynamically walk in temperature space to enrich their population and exchanges near steep energetic barriers. This approach is compared to earlier work of applying the conventional method of static temperature clients to refine a dataset of conformational decoys. Our results show that, while an adaptive method has many theoretical advantages over a static distribution of client temperatures, only limited improvement was gained from this strategy in excursions of the downhill refinement regime leading to an increase in the fraction of native contacts. To illustrate the sampling differences between the two simulation methods, energy landscapes are presented along with their temperature client profiles.

Gc protein-derived macrophage activating factor (GcMAF): isoelectric focusing pattern and tumoricidal activity.

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Mohamad, Saharuddin Bin; Nagasawa, Hideko; Sasaki, Hideyuki; Uto, Yoshihiro; Nakagawa, Yoshinori; Kawashima, Ken; Hori, Hitoshi

2003-01-01

Gc protein is the precursor for Gc protein-derived macrophage activating factor (GcMAF), with three phenotypes: Gc1f, Gc1s and Gc2, based on its electrophoretic mobility. The difference in electrophoretic mobility is because of the difference in its posttranslational sugar moiety composition. We compared the difference between Gc protein and GcMAF electrophoretic mobility using the isoelectric focusing (IEF) method. The tumoricidal activity of GcMAF-treated macrophage was evaluated after coculture with L-929 cell. The tumoricidal mechanism was investigated using TNF bioassay and nitric oxide (NO) release. The difference in Gc protein and GcMAF electrophoretic mobility was detected. The tumoricidal activity of GcMAF-treated macrophage was detected, but no release of TNF and NO was detected. The difference of isoelectric focusing mobility in Gc protein and GcMAF would be useful to develop a GcMAF detection method. GcMAF increased macrophage tumoricidal activity but TNF and NO release were not involved in the mechanism.

Electrochemical ion-exchange for active liquid waste treatment

International Nuclear Information System (INIS)

Turner, A.D.; Bridger, N.J.; Jones, C.P.

1992-10-01

Electrochemical ion exchange (EIX) has been firmly established as an effective process for the treatment of a wide range of liquid radioactive wastes. Both organic (for low specific activity streams) and inorganic systems (for higher activity wastes) have been demonstrated. A low cost current feeder electrode has also been developed, with a projected lifetime of > 6 years. While cation EIX can be used for the treatment of low salt content streams, combination with anion EIX to control the pH can extend its range of application. At the same time, it is also able to remove activity complexed in an anionic form. AEIX has also demonstrated its ability to remove radionuclides with insoluble hydroxides (eg Co, U and Pu) from both high and low salt content streams. EIX has been successfully scaled-up form the bench-top scale by increasing electrode size by a factor of 11, and then by operating five units in parallel. An improvement in performance of by a factor 3 was observed over a simple increase in area, due to the minimization of edge effects in the larger units. The most significant advantage of EIX is its compactness -with plant sizes of 1000). (Author)

NADH:ubiquinone reductase and succinate dehydrogenase activity in the liver of rats with acetaminophen-induced toxic hepatitis on the background of alimentary protein deficiency

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G. P. Kopylchuk

2015-02-01

Full Text Available The ratio between the redox forms of the nicotinamide coenzymes and key enzymatic activity of the I and II respiratory chain complexes in the liver cells mitochondria of rats with acetaminophen-induced hepatitis under the conditions of alimentary deprivation of protein was studied. It was estimated, that under the conditions of acute acetaminophen-induced hepatitis of rats kept on a low-protein diet during 4 weeks a significant decrease of the NADH:ubiquinone reductase and succinate dehydrogenase activity with simultaneous increase of the ratio between redox forms of the nicotinamide coenzymes (NAD+/NADН is observed compared to the same indices in the liver cells of animals with experimental hepatitis kept on the ration balanced by all nutrients. Results of research may become basic ones for the biochemical rationale for the approaches directed to the correction and elimination of the consequences­ of energy exchange in the toxic hepatitis, induced on the background of protein deficiency.

Activation of the polyomavirus enhancer by a murine activator protein 1 (AP1) homolog and two contiguous proteins.

OpenAIRE

Martin, M E; Piette, J; Yaniv, M; Tang, W J; Folk, W R

1988-01-01

The polyomavirus enhancer is composed of multiple DNA sequence elements serving as binding sites for proteins present in mouse nuclear extracts that activate transcription and DNA replication. We have identified three such proteins and their binding sites and correlate them with enhancer function. Mutation of nucleotide (nt) 5140 in the enhancer alters the binding site (TGACTAA, nt 5139-5145) for polyomavirus enhancer A binding protein 1 (PEA1), a murine homolog of the human transcription fac...

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)

Chemical exchange rotation transfer imaging of intermediate-exchanging amines at 2 ppm.

Science.gov (United States)

Zu, Zhongliang; Louie, Elizabeth A; Lin, Eugene C; Jiang, Xiaoyu; Does, Mark D; Gore, John C; Gochberg, Daniel F

2017-10-01

Chemical exchange saturation transfer (CEST) imaging of amine protons exchanging at intermediate rates and whose chemical shift is around 2 ppm may provide a means of mapping creatine. However, the quantification of this effect may be compromised by the influence of overlapping CEST signals from fast-exchanging amines and hydroxyls. We aimed to investigate the exchange rate filtering effect of a variation of CEST, named chemical exchange rotation transfer (CERT), as a means of isolating creatine contributions at around 2 ppm from other overlapping signals. Simulations were performed to study the filtering effects of CERT for the selection of transfer effects from protons of specific exchange rates. Control samples containing the main metabolites in brain, bovine serum albumin (BSA) and egg white albumen (EWA) at their physiological concentrations and pH were used to study the ability of CERT to isolate molecules with amines at 2 ppm that exchange at intermediate rates, and corresponding methods were used for in vivo rat brain imaging. Simulations showed that exchange rate filtering can be combined with conventional filtering based on chemical shift. Studies on samples showed that signal contributions from creatine can be separated from those of other metabolites using this combined filter, but contributions from protein amines may still be significant. This exchange filtering can also be used for in vivo imaging. CERT provides more specific quantification of amines at 2 ppm that exchange at intermediate rates compared with conventional CEST imaging. Copyright © 2017 John Wiley & Sons, Ltd.

Exploration of overloaded cation exchange chromatography for monoclonal antibody purification.

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Liu, Hui F; McCooey, Beth; Duarte, Tiago; Myers, Deanna E; Hudson, Terry; Amanullah, Ashraf; van Reis, Robert; Kelley, Brian D

2011-09-28

Cation exchange chromatography using conventional resins, having either diffusive or perfusive flow paths, operated in bind-elute mode has been commonly employed in monoclonal antibody (MAb) purification processes. In this study, the performance of diffusive and perfusive cation exchange resins (SP-Sepharose FF (SPSFF) and Poros 50HS) and a convective cation exchange membrane (Mustang S) and monolith (SO(3) Monolith) were compared. All matrices were utilized in an isocratic state under typical binding conditions with an antibody load of up to 1000 g/L of chromatographic matrix. The dynamic binding capacity of the cation exchange resins is typically below 100 g/L resin, so they were loaded beyond the point of anticipated MAb break through. All of the matrices performed similarly in that they effectively retained host cell protein and DNA during the loading and wash steps, while antibody flowed through each matrix after its dynamic binding capacity was reached. The matrices differed, though, in that conventional diffusive and perfusive chromatographic resins (SPSFF and Poros 50HS) demonstrated a higher binding capacity for high molecular weight species (HMW) than convective flow matrices (membrane and monolith); Poros 50HS displayed the highest HMW binding capacity. Further exploration of the conventional chromatographic resins in an isocratic overloaded mode demonstrated that the impurity binding capacity was well maintained on Poros 50HS, but not on SPSFF, when the operating flow rate was as high as 36 column volumes per hour. Host cell protein and HMW removal by Poros 50HS was affected by altering the loading conductivity. A higher percentage of host cell protein removal was achieved at a low conductivity of 3 mS/cm. HMW binding capacity was optimized at 5 mS/cm. Our data from runs on Poros 50HS resin also showed that leached protein A and cell culture additive such as gentamicin were able to be removed under the isocratic overloaded condition. Lastly, a MAb

The potent, indirect adenosine monophosphate-activated protein kinase activator R419 attenuates mitogen-activated protein kinase signaling, inhibits nociceptor excitability, and reduces pain hypersensitivity in mice

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Galo L. Mejia

2016-07-01

Full Text Available Abstract. There is a great need for new therapeutics for the treatment of pain. A possible avenue to development of such therapeutics is to interfere with signaling pathways engaged in peripheral nociceptors that cause these neurons to become hyperexcitable. There is strong evidence that mitogen-activated protein kinases and phosphoinositide 3-kinase (PI3K/mechanistic target of rapamycin signaling pathways are key modulators of nociceptor excitability in vitro and in vivo. Activation of adenosine monophosphate-activated protein kinase (AMPK can inhibit signaling in both of these pathways, and AMPK activators have been shown to inhibit nociceptor excitability and pain hypersensitivity in rodents. R419 is one of, if not the most potent AMPK activator described to date. We tested whether R419 activates AMPK in dorsal root ganglion (DRG neurons and if this leads to decreased pain hypersensitivity in mice. We find that R419 activates AMPK in DRG neurons resulting in decreased mitogen-activated protein kinase signaling, decreased nascent protein synthesis, and enhanced P body formation. R419 attenuates nerve growth factor (NGF-induced changes in excitability in DRG neurons and blocks NGF-induced mechanical pain amplification in vivo. Moreover, locally applied R419 attenuates pain hypersensitivity in a model of postsurgical pain and blocks the development of hyperalgesic priming in response to both NGF and incision. We conclude that R419 is a promising lead candidate compound for the development of potent and specific AMPK activation to inhibit pain hypersensitivity as a result of injury.

Interaction of C-terminal truncated human alphaA-crystallins with target proteins.

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Anbarasu Kumarasamy

2008-09-01

Full Text Available Significant portion of alphaA-crystallin in human lenses exists as C-terminal residues cleaved at residues 172, 168, and 162. Chaperone activity, determined with alcohol dehydrogenase (ADH and betaL-crystallin as target proteins, was increased in alphaA(1-172 and decreased in alphaA(1-168 and alphaA(1-162. The purpose of this study was to show whether the absence of the C-terminal residues influences protein-protein interactions with target proteins.Our hypothesis is that the chaperone-target protein binding kinetics, otherwise termed subunit exchange rates, are expected to reflect the changes in chaperone activity. To study this, we have relied on fluorescence resonance energy transfer (FRET utilizing amine specific and cysteine specific fluorescent probes. The subunit exchange rate (k for ADH and alphaA(1-172 was nearly the same as that of ADH and alphaA-wt, alphaA(1-168 had lower and alphaA(1-162 had the lowest k values. When betaL-crystallin was used as the target protein, alphaA(1-172 had slightly higher k value than alphaA-wt and alphaA(1-168 and alphaA(1-162 had lower k values. As expected from earlier studies, the chaperone activity of alphaA(1-172 was slightly better than that of alphaA-wt, the chaperone activity of alphaA(1-168 was similar to that of alphaA-wt and alphaA(1-162 had substantially decreased chaperone activity.Cleavage of eleven C-terminal residues including Arg-163 and the C-terminal flexible arm significantly affects the interaction with target proteins. The predominantly hydrophilic flexible arm appears to be needed to keep the chaperone-target protein complex soluble.

Protein C activity and antigen levels in childhood

NARCIS (Netherlands)

van Teunenbroek, A.; Peters, M.; Sturk, A.; Borm, J. J.; Breederveld, C.

1990-01-01

Hereditary protein C deficiency is an important risk factor for thrombosis. To enable its diagnosis shortly after birth, we determined reference values of protein C antigen and activity levels for the first 3 months of life. To establish an age-related range of protein C levels we also determined

Reconstitution of DNA strand exchange mediated by Rhp51 recombinase and two mediators.

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Yumiko Kurokawa

2008-04-01

Full Text Available In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that two mediators, Rad22 (the S. pombe Rad52 homolog and the Swi5-Sfr1 complex, participate in a common pathway of Rhp51 (the S. pombe Rad51 homolog-mediated homologous recombination (HR and HR repair. Here, we have demonstrated an in vitro reconstitution of the central step of DNA strand exchange during HR. Our system consists entirely of homogeneously purified proteins, including Rhp51, the two mediators, and replication protein A (RPA, which reflects genetic requirements in vivo. Using this system, we present the first robust biochemical evidence that concerted action of the two mediators directs the loading of Rhp51 onto single-stranded DNA (ssDNA precoated with RPA. Dissection of the reaction reveals that Rad22 overcomes the inhibitory effect of RPA on Rhp51-Swi5-Sfr1-mediated strand exchange. In addition, Rad22 negates the requirement for a strict order of protein addition to the in vitro system. However, despite the presence of Rad22, Swi5-Sfr1 is still essential for strand exchange. Importantly, Rhp51, but neither Rad22 nor the Swi5-Sfr1 mediator, is the factor that displaces RPA from ssDNA. Swi5-Sfr1 stabilizes Rhp51-ssDNA filaments in an ATP-dependent manner, and this stabilization is correlated with activation of Rhp51 for the strand exchange reaction. Rad22 alone cannot activate the Rhp51 presynaptic filament. AMP-PNP, a nonhydrolyzable ATP analog, induces a similar stabilization of Rhp51, but this stabilization is independent of Swi5-Sfr1. However, hydrolysis of ATP is required for processive strand transfer, which results in the formation of a long heteroduplex. Our in vitro reconstitution system has revealed that the two mediators have indispensable, but distinct, roles for mediating Rhp51 loading onto RPA-precoated ssDNA.

Novel Biosensor of Membrane Protein Proximity Based on Fluorogen Activated Proteins.

Science.gov (United States)

Vasilev, Kalin V; Gallo, Eugenio; Shank, Nathaniel; Jarvik, Jonathan W

2016-01-01

We describe a novel biosensor system for reporting proximity between cell surface proteins in live cultured cells. The biosensor takes advantage of recently developed fluorogen-activating proteins (FAPs) that display fluorescence only when bound to otherwise-nonfluorescent fluorogen molecules. To demonstrate feasibility for the approach, two recombinant rapamycin-binding proteins were expressed as single-pass plasma membrane proteins in HeLa cells; one of the proteins (scAvd- FRB) carried an extracellular avidin tag; the other (HL1-TO1-FKBP) carried an extracellular FAP. Cells were incubated with a membrane-impermeable bivalent ligand (biotin-PEG2000-DIR) consisting of biotin joined to a dimethyl-indole red (DIR) fluorogen by a polyethylene glycol linker, thus tethering the fluorogen to the scAvd-FRB fusion protein. Addition of rapamycin, which promotes FKBP-FRB dimerization and thereby brings the FAP in close proximity to the tethered fluorogen, led to a significant increase in DIR fluorescence. We call the new proximity assay TEFLA, for tethered fluorogen assay.

Protective features of resveratrol on human spermatozoa cryopreservation may be mediated through 5' AMP-activated protein kinase activation.

Science.gov (United States)

Shabani Nashtaei, M; Amidi, F; Sedighi Gilani, M A; Aleyasin, A; Bakhshalizadeh, Sh; Naji, M; Nekoonam, S

2017-03-01

Biochemical and physical modifications during the freeze-thaw process adversely influence the restoration of energy-dependent sperm functions required for fertilization. Resveratrol, a phytoalexin, has been introduced to activate 5' AMP-activated protein kinase which is a cell energy sensor and a cell metabolism regulator. The cryoprotection of resveratrol on sperm cryoinjury via activation of AMP-activated protein kinase also remains to be elucidated. Our aim, thus, was to investigate: (i) the presence and intracellular localization of AMP-activated protein kinase protein; (ii) whether resveratrol may exert a protective effect on certain functional properties of fresh and post-thaw human spermatozoa through modulation of AMP-activated protein kinase. Spermatozoa from normozoospermic men were incubated with or without different concentrations of Compound C as an AMP-activated protein kinase inhibitor or resveratrol as an AMP-activated protein kinase activator for different lengths of time and were then cryopreserved. AMP-activated protein kinase is expressed essentially in the entire flagellum and the post-equatorial region. Viability of fresh spermatozoa was not significantly affected by the presence of Compound C or resveratrol. However, although Compound C caused a potent inhibition of spermatozoa motility parameters, resveratrol did not induce negative effect, except a significant reduction in motility at 25 μm for 1 h. Furthermore, resveratrol significantly increased AMP-activated protein kinase phosphorylation and mitochondrial membrane potential and decreased reactive oxygen species and apoptosis-like changes in frozen-thawed spermatozoa. Nevertheless, it was not able to compensate decreased sperm viability and motility parameters following cryopreservation. In contrast, Compound C showed opposite effects to resveratrol on AMP-activated protein kinase phosphorylation, reactive oxygen species, apoptosis-like changes, mitochondrial membrane potential, and

Expression of the Sodium/Calcium/Potassium Exchanger, NCKX4, in Ameloblasts

Science.gov (United States)

Hu, Ping; Lacruz, Rodrigo S.; Smith, Charles E.; Smith, Susan M.; Kurtz, Ira; Paine, Michael L.

2012-01-01

Transcellular calcium transport is an essential activity in mineralized tissue formation, including dental hard tissues. In many organ systems, this activity is regulated by membrane-bound sodium/calcium (Na+/Ca2+) exchangers, which include the NCX and NCKX [sodium/calcium-potassium (Na+/Ca2+-K+ ) exchanger] proteins. During enamel maturation, when crystals expand in thickness, Ca2+ requirements vastly increase but exactly how Ca2+ traffics through ameloblasts remains uncertain. Previous studies have shown that several NCX proteins are expressed in ameloblasts, although no significant shifts in expression were observed during maturation which pointed to the possible identification of other Ca2+ membrane transporters. NCKX proteins are encoded by members of the solute carrier gene family, Slc24a, which include 6 different proteins (NCKX1–6). NCKX are bidirectional electrogenic transporters regulating Ca2+ transport in and out of cells dependent on the transmembrane ion gradient. In this study we show that all NCKX mRNAs are expressed in dental tissues. Real-time PCR indicates that of all the members of the NCKX group, NCKX4 is the most highly expressed gene transcript during the late stages of amelogenesis. In situ hybridization and immunolocalization analyses clearly establish that in the enamel organ, NCKX4 is expressed primarily by ameloblasts during the maturation stage. Further, during the mid-late maturation stages of amelogenesis, the expression of NCKX4 in ameloblasts is most prominent at the apical poles and at the lateral membranes proximal to the apical ends. These data suggest that NCKX4 might be an important regulator of Ca2+ transport during amelogenesis. PMID:22677781

Fast hydrogen exchange affects 15N relaxation measurements in intrinsically disordered proteins

International Nuclear Information System (INIS)

Kim, Seho; Wu, Kuen-Phon; Baum, Jean

2013-01-01

Unprotected amide protons can undergo fast hydrogen exchange (HX) with protons from the solvent. Generally, NMR experiments using the out-and-back coherence transfer with amide proton detection are affected by fast HX and result in reduced signal intensity. When one of these experiments, 1 H– 15 N HSQC, is used to measure the 15 N transverse relaxation rate (R 2 ), the measured R 2 rate is convoluted with the HX rate (k HX ) and has higher apparent R 2 values. Since the 15 N R 2 measurement is important for analyzing protein backbone dynamics, the HX effect on the R 2 measurement is investigated and described here by multi-exponential signal decay. We demonstrate these effects by performing 15 N R 2 CPMG experiments on α-synuclein, an intrinsically disordered protein, in which the amide protons are exposed to solvent. We show that the HX effect on R 2 CPMG can be extracted by the derived equation. In conclusion, the HX effect may be pulse sequence specific and results from various sources including the J coupling evolution, the change of steady state water proton magnetization, and the D 2 O content in the sample. To avoid the HX effect on the analysis of relaxation data of unprotected amides, it is suggested that NMR experimental conditions insensitive to the HX should be considered or that intrinsic R 2 CPMG values be obtained by methods described herein.

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

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.

Nullspace Sampling with Holonomic Constraints Reveals Molecular Mechanisms of Protein Gαs.

Directory of Open Access Journals (Sweden)

Dimitar V Pachov

2015-07-01

Full Text Available Proteins perform their function or interact with partners by exchanging between conformational substates on a wide range of spatiotemporal scales. Structurally characterizing these exchanges is challenging, both experimentally and computationally. Large, diffusional motions are often on timescales that are difficult to access with molecular dynamics simulations, especially for large proteins and their complexes. The low frequency modes of normal mode analysis (NMA report on molecular fluctuations associated with biological activity. However, NMA is limited to a second order expansion about a minimum of the potential energy function, which limits opportunities to observe diffusional motions. By contrast, kino-geometric conformational sampling (KGS permits large perturbations while maintaining the exact geometry of explicit conformational constraints, such as hydrogen bonds. Here, we extend KGS and show that a conformational ensemble of the α subunit Gαs of heterotrimeric stimulatory protein Gs exhibits structural features implicated in its activation pathway. Activation of protein Gs by G protein-coupled receptors (GPCRs is associated with GDP release and large conformational changes of its α-helical domain. Our method reveals a coupled α-helical domain opening motion while, simultaneously, Gαs helix α5 samples an activated conformation. These motions are moderated in the activated state. The motion centers on a dynamic hub near the nucleotide-binding site of Gαs, and radiates to helix α4. We find that comparative NMA-based ensembles underestimate the amplitudes of the motion. Additionally, the ensembles fall short in predicting the accepted direction of the full activation pathway. Taken together, our findings suggest that nullspace sampling with explicit, holonomic constraints yields ensembles that illuminate molecular mechanisms involved in GDP release and protein Gs activation, and further establish conformational coupling between key

New force replica exchange method and protein folding pathways probed by force-clamp technique.

Science.gov (United States)

Kouza, Maksim; Hu, Chin-Kun; Li, Mai Suan

2008-01-28

We have developed a new extended replica exchange method to study thermodynamics of a system in the presence of external force. Our idea is based on the exchange between different force replicas to accelerate the equilibrium process. This new approach was applied to obtain the force-temperature phase diagram and other thermodynamical quantities of the three-domain ubiquitin. Using the C(alpha)-Go model and the Langevin dynamics, we have shown that the refolding pathways of single ubiquitin depend on which terminus is fixed. If the N end is fixed then the folding pathways are different compared to the case when both termini are free, but fixing the C terminal does not change them. Surprisingly, we have found that the anchoring terminal does not affect the pathways of individual secondary structures of three-domain ubiquitin, indicating the important role of the multidomain construction. Therefore, force-clamp experiments, in which one end of a protein is kept fixed, can probe the refolding pathways of a single free-end ubiquitin if one uses either the polyubiquitin or a single domain with the C terminus anchored. However, it is shown that anchoring one end does not affect refolding pathways of the titin domain I27, and the force-clamp spectroscopy is always capable to predict folding sequencing of this protein. We have obtained the reasonable estimate for unfolding barrier of ubiquitin, using the microscopic theory for the dependence of unfolding time on the external force. The linkage between residue Lys48 and the C terminal of ubiquitin is found to have the dramatic effect on the location of the transition state along the end-to-end distance reaction coordinate, but the multidomain construction leaves the transition state almost unchanged. We have found that the maximum force in the force-extension profile from constant velocity force pulling simulations depends on temperature nonlinearly. However, for some narrow temperature interval this dependence becomes

A replica exchange Monte Carlo algorithm for protein folding in the HP model

Directory of Open Access Journals (Sweden)

Shmygelska Alena

2007-09-01

Full Text Available Abstract Background The ab initio protein folding problem consists of predicting protein tertiary structure from a given amino acid sequence by minimizing an energy function; it is one of the most important and challenging problems in biochemistry, molecular biology and biophysics. The ab initio protein folding problem is computationally challenging and has been shown to be NP MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaat0uy0HwzTfgDPnwy1egaryqtHrhAL1wy0L2yHvdaiqaacqWFneVtcqqGqbauaaa@3961@-hard even when conformations are restricted to a lattice. In this work, we implement and evaluate the replica exchange Monte Carlo (REMC method, which has already been applied very successfully to more complex protein models and other optimization problems with complex energy landscapes, in combination with the highly effective pull move neighbourhood in two widely studied Hydrophobic Polar (HP lattice models. Results We demonstrate that REMC is highly effective for solving instances of the square (2D and cubic (3D HP protein folding problem. When using the pull move neighbourhood, REMC outperforms current state-of-the-art algorithms for most benchmark instances. Additionally, we show that this new algorithm provides a larger ensemble of ground-state structures than the existing state-of-the-art methods. Furthermore, it scales well with sequence length, and it finds significantly better conformations on long biological sequences and sequences with a provably unique ground-state structure, which is believed to be a characteristic of real proteins. We also present evidence that our REMC algorithm can fold sequences which exhibit significant interaction between termini in the hydrophobic core relatively easily. Conclusion We demonstrate that REMC utilizing the pull move

Isolation and structure–function characterization of a signaling-active rhodopsin–G protein complex

Science.gov (United States)

Gao, Yang; Westfield, Gerwin; Erickson, Jon W.; Cerione, Richard A.; Skiniotis, Georgios; Ramachandran, Sekar

2017-01-01

The visual photo-transduction cascade is a prototypical G protein–coupled receptor (GPCR) signaling system, in which light-activated rhodopsin (Rho*) is the GPCR catalyzing the exchange of GDP for GTP on the heterotrimeric G protein transducin (GT). This results in the dissociation of GT into its component αT–GTP and β1γ1 subunit complex. Structural information for the Rho*–GT complex will be essential for understanding the molecular mechanism of visual photo-transduction. Moreover, it will shed light on how GPCRs selectively couple to and activate their G protein signaling partners. Here, we report on the preparation of a stable detergent-solubilized complex between Rho* and a heterotrimer (GT*) comprising a GαT/Gαi1 chimera (αT*) and β1γ1. The complex was formed on native rod outer segment membranes upon light activation, solubilized in lauryl maltose neopentyl glycol, and purified with a combination of affinity and size-exclusion chromatography. We found that the complex is fully functional and that the stoichiometry of Rho* to GαT* is 1:1. The molecular weight of the complex was calculated from small-angle X-ray scattering data and was in good agreement with a model consisting of one Rho* and one GT*. The complex was visualized by negative-stain electron microscopy, which revealed an architecture similar to that of the β2-adrenergic receptor–GS complex, including a flexible αT* helical domain. The stability and high yield of the purified complex should allow for further efforts toward obtaining a high-resolution structure of this important signaling complex. PMID:28655769

Protein and Peptide Gas-phase Structure Investigation Using Collision Cross Section Measurements and Hydrogen Deuterium Exchange

Science.gov (United States)

Khakinejad, Mahdiar

Protein and peptide gas-phase structure analysis provides the opportunity to study these species outside of their explicit environment where the interaction network with surrounding molecules makes the analysis difficult [1]. Although gas-phase structure analysis offers a unique opportunity to study the intrinsic behavior of these biomolecules [2-4], proteins and peptides exhibit very low vapor pressures [2]. Peptide and protein ions can be rendered in the gas-phase using electrospray ionization (ESI) [5]. There is a growing body of literature that shows proteins and peptides can maintain solution structures during the process of ESI and these structures can persist for a few hundred milliseconds [6-9]. Techniques for monitoring gas-phase protein and peptide ion structures are categorized as physical probes and chemical probes. Collision cross section (CCS) measurement, being a physical probe, is a powerful method to investigate gas-phase structure size [3, 7, 10-15]; however, CCS values alone do not establish a one to one relation with structure(i.e., the CCS value is an orientationally averaged value [15-18]. Here we propose the utility of gas-phase hydrogen deuterium exchange (HDX) as a second criterion of structure elucidation. The proposed approach incudes extensive MD simulations to sample biomolecular ion conformation space with the production of numerous, random in-silico structures. Subsequently a CCS can be calculated for these structures and theoretical CCS values are compared with experimental values to produce a pool of candidate structures. Utilizing a chemical reaction model based on the gas-phase HDX mechanism, the HDX kinetics behavior of these candidate structures are predicted and compared to experimental results to nominate the best in-silico structures which match (chemically and physically) with experimental observations. For the predictive approach to succeed, an extensive technique and method development is essential. To combine CCS

The analysis of false prolongation of the activated partial thromboplastin time (activator: silica): Interference of C-reactive protein.

Science.gov (United States)

Liu, Jie; Li, Fanfan; Shu, Kuangyi; Chen, Tao; Wang, Xiaoou; Xie, Yaoqi; Li, Shanshan; Zhang, Zhaohua; Jin, Susu; Jiang, Minghua

2018-05-13

To investigate the effect of C-reactive protein on the activated partial thromboplastin time (APTT) (different activators) in different detecting systems. The C-reactive protein and coagulation test of 112 patients with the infectious disease were determined by automation protein analyzer IMMAG 800 and automation coagulation analyzer STA-R Evolution, respectively. The pooled plasma APTT with different concentrations of C-reactive protein was measured by different detecting system: STA-R Evolution (activator: silica, kaolin), Sysmex CS-2000i (activator: ellagic acid), and ACL TOP 700 (activator: colloidal silica). In addition, the self-made platelet lysate (phospholipid) was added to correct the APTT prolonged by C-reactive protein (150 mg/L) on STA-R Evolution (activator: silica) system. The good correlation between C-reactive protein and APTT was found on the STA-R Evolution (activator: silica) system. The APTT on the STA-R Evolution (activator: silica) system was prolonged by 24.6 second, along with increasing C-reactive protein concentration. And the APTT of plasma containing 150 mg/L C-reactive protein was shortened by 3.4-6.9 second when the plasma was mixed with self-made platelet lysate. However, the APTT was prolonged unobviously on other detecting systems including STA-R Evolution (activator: kaolin), Sysmex CS-2000i, and ACL TOP 700. C-reactive protein interferes with the detection of APTT, especially in STA-R Evolution (activator: silica) system. The increasing in C-reactive protein results in a false prolongation of the APTT (activator: silica), and it is most likely that C-reactive protein interferes the coagulable factor binding of phospholipid. © 2018 Wiley Periodicals, Inc.

The functional significance of the autolysis loop in protein C and activated protein C.

Science.gov (United States)

Yang, Likui; Manithody, Chandrashekhara; Rezaie, Alireza R

2005-07-01

The autolysis loop of activated protein C (APC) is five residues longer than the autolysis loop of other vitamin K-dependent coagulation proteases. To investigate the role of this loop in the zymogenic and anticoagulant properties of the molecule, a protein C mutant was constructed in which the autolysis loop of the protein was replaced with the corresponding loop of factor X. The protein C mutant was activated by thrombin with approximately 5-fold higher rate in the presence of Ca2+. Both kinetics and direct binding studies revealed that the Ca2+ affinity of the mutant has been impaired approximately 3-fold. The result of a factor Va degradation assay revealed that the anticoagulant function of the mutant has been improved 4-5-fold in the absence but not in the presence of protein S. The improvement was due to a better recognition of both the P1-Arg506 and P1-Arg306 cleavage sites by the mutant protease. However, the plasma half-life of the mutant was markedly shortened due to faster inactivation by plasma serpins. These results suggest that the autolysis loop of protein C is critical for the Ca(2+)-dependence of activation by thrombin. Moreover, a longer autolysis loop in APC is not optimal for interaction with factor Va in the absence of protein S, but it contributes to the lack of serpin reactivity and longer half-life of the protease in plasma.

Baculoviral expression and characterization of human recombinant PGCP in the form of an active mature dimer and an inactive precursor protein.

Science.gov (United States)

Zajc, Tajana; Suban, Dejan; Rajković, Jelena; Dolenc, Iztok

2011-02-01

The human-blood plasma glutamate carboxypeptidase (PGCP) is a proteinase that acts on the unsubstituted N- and C-termini of dipeptides. It has been suggested that this PGCP is involved in the release of thyroxine. Furthermore, research has suggested that its activity is up-regulated in hepatitis-C-virus-infected patients with hepatocellular carcinoma. In this study expressed human PGCP in the baculovirus expression system was produced by a Sf9 insect cell line with aim to prepare sufficient amounts of active recombinant enzyme for a subsequent biological characterization. Recombinant PGCP was expressed and secreted into the medium in the form of an inactive proenzyme. It was gradually converted into an active form in the medium after three days, with the highest expression of the active form on day six. The protein was sequentially purified by a combination of various liquid chromatographies, such as hydroxyapatite, ion exchange, and gel chromatography, and as final step with affinity chromatography on Phe-Leu-Sepharose. The human PGCP was purified as an active enzyme in the dimer form and as inactive precursor protein. The dipeptidase activity was confirmed by measuring the hydrolysis of the Ser-Met dipeptide at a slightly acidic pH. Copyright © 2010 Elsevier Inc. All rights reserved.

Mitogen-activated protein kinase signaling in plants

DEFF Research Database (Denmark)

Rodriguez, Maria Cristina Suarez; Petersen, Morten; Mundy, John

2010-01-01

crossinhibition, feedback control, and scaffolding. Plant MAPK cascades regulate numerous processes, including stress and hormonal responses, innate immunity, and developmental programs. Genetic analyses have uncovered several predominant MAPK components shared by several of these processes including...... of substrate proteins, whose altered activities mediate a wide array of responses, including changes in gene expression. Cascades may share kinase components, but their signaling specificity is maintained by spaciotemporal constraints and dynamic protein-protein interactions and by mechanisms that include...

Simplified in vitro refolding and purification of recombinant human granulocyte colony stimulating factor using protein folding cation exchange chromatography.

Science.gov (United States)

Vemula, Sandeep; Dedaniya, Akshay; Thunuguntla, Rahul; Mallu, Maheswara Reddy; Parupudi, Pavani; Ronda, Srinivasa Reddy

2015-01-30

Protein folding-strong cation exchange chromatography (PF-SCX) has been employed for efficient refolding with simultaneous purification of recombinant human granulocyte colony stimulating factor (rhG-CSF). To acquire a soluble form of renatured and purified rhG-CSF, various chromatographic conditions, including the mobile phase composition and pH was evaluated. Additionally, the effects of additives such as urea, amino acids, polyols, sugars, oxidizing agents and their amalgamations were also investigated. Under the optimal conditions, rhG-CSF was efficaciously solubilized, refolded and simultaneously purified by SCX in a single step. The experimental results using ribose (2.0M) and arginine (0.6M) combination were found to be satisfactory with mass yield, purity and specific activity of 71%, ≥99% and 2.6×10(8)IU/mg respectively. Through this investigation, we concluded that the SCX refolding method was more efficient than conventional methods which has immense potential for the large-scale production of purified rhG-CSF. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of an activity-based probe for acyl-protein thioesterases

Science.gov (United States)

Garland, Megan; Schulze, Christopher J.; Foe, Ian T.; van der Linden, Wouter A.; Child, Matthew A.

2018-01-01

Protein palmitoylation is a dynamic post-translational modification (PTM) important for cellular functions such as protein stability, trafficking, localization, and protein-protein interactions. S-palmitoylation occurs via the addition of palmitate to cysteine residues via a thioester linkage, catalyzed by palmitoyl acyl transferases (PATs), with removal of the palmitate catalyzed by acyl protein thioesterases (APTs) and palmitoyl-protein thioesterases (PPTs). Tools that target the regulators of palmitoylation–PATs, APTs and PPTs–will improve understanding of this essential PTM. Here, we describe the synthesis and application of a cell-permeable activity-based probe (ABP) that targets APTs in intact mammalian cells and the parasite Toxoplasma gondii. Using a focused library of substituted chloroisocoumarins, we identified a probe scaffold with nanomolar affinity for human APTs (HsAPT1 and HsAPT2) and synthesized a fluorescent ABP, JCP174-BODIPY TMR (JCP174-BT). We use JCP174-BT to profile HsAPT activity in situ in mammalian cells, to detect an APT in T. gondii (TgPPT1). We show discordance between HsAPT activity levels and total protein concentration in some cell lines, indicating that total protein levels may not be representative of APT activity in complex systems, highlighting the utility of this probe. PMID:29364904

Acidosis-mediated regulation of the NHE1 isoform of the Na⁺/H⁺ exchanger in renal cells.

Science.gov (United States)

Odunewu, Ayodeji; Fliegel, Larry

2013-08-01

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is a ubiquitous plasma membrane protein that regulates intracellular pH by removing a proton in exchange for extracellular sodium. Renal tissues are subject to metabolic and respiratory acidosis, and acidosis has been shown to acutely activate NHE1 activity in other cell types. We examined if NHE1 is activated by acute acidosis in HEK293 and Madin-Darby canine kidney (MDCK) cells. Acute sustained intracellular acidosis (SIA) activated NHE1 in both cell types. We expressed wild-type and mutant NHE1 cDNAs in MDCK cells. All the cDNAs had a L163F/G174S mutation, which conferred a 100-fold resistance to EMD87580, an NHE1-specific inhibitor. We assayed exogenous NHE1 activity while inhibiting endogenous activity with EMD87580 and while inhibiting the NHE3 isoform of the Na⁺/H⁺ exchanger using the isoform-specific inhibitor S3226. We examined the activation and phosphorylation of the wild-type and mutant NHE1 proteins in response to SIA. In MDCK cells we demonstrated that the amino acids Ser⁷⁷¹, Ser⁷⁷⁶, Thr⁷⁷⁹, and Ser⁷⁸⁵ are important for NHE1 phosphorylation and activation after acute SIA. SIA activated ERK-dependent pathways in MDCK cells, and this was blocked by treatment with the MEK inhibitor U0126. Treatment with U0126 also blocked activation of NHE1 by SIA. These results suggest that acute acidosis activates NHE1 in mammalian kidney cells and that in MDCK cells this activation occurs through an ERK-dependent pathway affecting phosphorylation of a distinct set of amino acids in the cytosolic regulatory tail of NHE1.

The dopamine D2 receptor can directly recruit and activate GRK2 without G protein activation.

Science.gov (United States)

Pack, Thomas F; Orlen, Margo I; Ray, Caroline; Peterson, Sean M; Caron, Marc G

2018-04-20

The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that is critical for many central nervous system functions. The D2R carries out these functions by signaling through two transducers: G proteins and β-arrestins (βarrs). Selectively engaging either the G protein or βarr pathway may be a way to improve drugs targeting GPCRs. The current model of GPCR signal transduction posits a chain of events where G protein activation ultimately leads to βarr recruitment. GPCR kinases (GRKs), which are regulated by G proteins and whose kinase action facilitates βarr recruitment, bridge these pathways. Therefore, βarr recruitment appears to be intimately tied to G protein activation via GRKs. Here we sought to understand how GRK2 action at the D2R would be disrupted when G protein activation is eliminated and the effect of this on βarr recruitment. We used two recently developed biased D2R mutants that can preferentially interact either with G proteins or βarrs as well as a βarr-biased D2R ligand, UNC9994. With these functionally selective tools, we investigated the mechanism whereby the βarr-preferring D2R achieves βarr pathway activation in the complete absence of G protein activation. We describe how direct, G protein-independent recruitment of GRK2 drives interactions at the βarr-preferring D2R and also contributes to βarr recruitment at the WT D2R. Additionally, we found an additive interaction between the βarr-preferring D2R mutant and UNC9994. These results reveal that the D2R can directly recruit GRK2 without G protein activation and that this mechanism may have relevance to achieving βarr-biased signaling. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Chimeric microbial rhodopsins for optical activation of Gs-proteins

Science.gov (United States)

Yoshida, Kazuho; Yamashita, Takahiro; Sasaki, Kengo; Inoue, Keiichi; Shichida, Yoshinori; Kandori, Hideki

2017-01-01

We previously showed that the chimeric proteins of microbial rhodopsins, such as light-driven proton pump bacteriorhodopsin (BR) and Gloeobacter rhodopsin (GR) that contain cytoplasmic loops of bovine rhodopsin, are able to activate Gt protein upon light absorption. These facts suggest similar protein structural changes in both the light-driven proton pump and animal rhodopsin. Here we report two trials to engineer chimeric rhodopsins, one for the inserted loop, and another for the microbial rhodopsin template. For the former, we successfully activated Gs protein by light through the incorporation of the cytoplasmic loop of β2-adrenergic receptor (β2AR). For the latter, we did not observe any G-protein activation for the light-driven sodium pump from Indibacter alkaliphilus (IndiR2) or a light-driven chloride pump halorhodopsin from Natronomonas pharaonis (NpHR), whereas the light-driven proton pump GR showed light-dependent G-protein activation. This fact suggests that a helix opening motion is common to G protein coupled receptor (GPCR) and GR, but not to IndiR2 and NpHR. Light-induced difference FTIR spectroscopy revealed similar structural changes between WT and the third loop chimera for each light-driven pump. A helical structural perturbation, which was largest for GR, was further enhanced in the chimera. We conclude that similar structural dynamics that occur on the cytoplasmic side of GPCR are needed to design chimeric microbial rhodopsins. PMID:29362703

The influence of activation of heterogeneous ion-exchange membranes on their electrochemical properties

Czech Academy of Sciences Publication Activity Database

Brožová, Libuše; Křivčík, J.; Neděla, D.; Kysela, V.; Žitka, Jan

2015-01-01

Roč. 56, č. 12 (2015), s. 3228-3232 ISSN 1944-3994. [International Conference on Membrane and Electromembrane Processes - MELPRO 2014. Prague, 18.05.2014-21.05.2014] Institutional support: RVO:61389013 Keywords : heterogeneous ion-exchange membranes * electrochemical properties * activation Subject RIV: JP - Industrial Processing Impact factor: 1.272, year: 2015

A single disulfide bond disruption in the β3 integrin subunit promotes thiol/disulfide exchange, a molecular dynamics study.

Directory of Open Access Journals (Sweden)

Lihie Levin

Full Text Available The integrins are a family of membrane receptors that attach a cell to its surrounding and play a crucial function in cell signaling. The combination of internal and external stimuli alters a folded non-active state of these proteins to an extended active configuration. The β3 subunit of the platelet αIIbβ3 integrin is made of well-structured domains rich in disulfide bonds. During the activation process some of the disulfides are re-shuffled by a mechanism requiring partial reduction of some of these bonds; any disruption in this mechanism can lead to inherent blood clotting diseases. In the present study we employed Molecular Dynamics simulations for tracing the sequence of structural fluctuations initiated by a single cysteine mutation in the β3 subunit of the receptor. These simulations showed that in-silico protein mutants exhibit major conformational deformations leading to possible disulfide exchange reactions. We suggest that any mutation that prevents Cys560 from reacting with one of the Cys(567-Cys(581 bonded pair, thus disrupting its ability to participate in a disulfide exchange reaction, will damage the activation mechanism of the integrin. This suggestion is in full agreement with previously published experiments. Furthermore, we suggest that rearrangement of disulfide bonds could be a part of a natural cascade of thiol/disulfide exchange reactions in the αIIbβ3 integrin, which are essential for the native activation process.

Respiratory Mechanics and Gas Exchange: The Effect of Surfactants

Science.gov (United States)

Jbaily, Abdulrahman; Szeri, Andrew J.

2017-11-01

The purpose of the lung is to exchange gases, primarily oxygen and carbon dioxide, between the atmosphere and the circulatory system. To enable this exchange, the airways in the lungs terminate in some 300 million alveoli that provide adequate surface area for transport. During breathing, work must be done to stretch various tissues to accommodate a greater volume of gas. Considerable work must also be done to expand the liquid lining (hypophase) that coats the interior surfaces of the alveoli. This is enabled by a surface active lipo-protein complex, known as pulmonary surfactant, that modifies the surface tension at the hypophase-air interface. Surfactants also serve as physical barriers that modify the rate of gas transfer across interfaces. We develop a mathematical model to study the action of pulmonary surfactant and its determinative contributions to breathing. The model is used to explore the influence of surfactants on alveolar mechanics and on gas exchange: it relates the work of respiration at the level of the alveolus to the gas exchange rate through the changing influence of pulmonary surfactant over the breathing cycle. This work is motivated by a need to develop improved surfactant replacement therapies to treat serious medical conditions.

Active osmotic exchanger for advanced filtration at the nano scale

Science.gov (United States)

Marbach, Sophie; Bocquet, Lyderic

2015-11-01

One of the main functions of the kidney is to remove the waste products of an organism, mostly by excreting concentrated urea while reabsorbing water and other molecules. The human kidney is capable of recycling about 200 liters of water per day, at the relatively low cost of 0.5 kJ/L (standard dialysis requiring at least 150 kJ/L). Kidneys are constituted of millions of parallel filtration networks called nephrons. The nephrons of all mammalian kidneys present a specific loop geometry, the Loop of Henle, that is believed to play a key role in the urinary concentrating mechanism. One limb of the loop is permeable to water and the other contains sodium pumps that exchange with a common interstitium. In this work, we take inspiration from this osmotic exchanger design to propose new nanofiltration principles. We first establish simple analytical results to derive general operating principles, based on coupled water permeable pores and osmotic pumps. The best filtration geometry, in terms of power required for a given water recycling ratio, is comparable in many ways to the mammalian nephron. It is not only more efficient than traditional reverse osmosis systems, but can also work at much smaller pressures (of the order of the blood pressure, 0.13 bar, as compared to more than 30 bars for pressure-retarded osmosis systems). We anticipate that our proof of principle will be a starting point for the development of new filtration systems relying on the active osmotic exchanger principle.

Cyclic AMP signalling in Dictyostelium : G-proteins activate separate Ras pathways using specific RasGEFs

NARCIS (Netherlands)

Kae, Helmut; Kortholt, Arjan; Rehmann, Holger; Insall, RobertH.; Van Haastert, Peter J. M.; Spiegelman, George B.; Weeks, Gerald

In general, mammalian Ras guanine nucleotide exchange factors (RasGEFs) show little substrate specificity, although they are often thought to regulate specific pathways. Here, we provide in vitro and in vivo evidence that two RasGEFs can each act on specific Ras proteins. During Dictyostelium

Hydrogen exchange during cell-free incorporation of deuterated amino acids and an approach to its inhibition

Energy Technology Data Exchange (ETDEWEB)

Tonelli, Marco; Singarapu, Kiran K. [University of Wisconsin-Madison, National Magnetic Resonance Facility at Madison (NMRFAM), Department of Biochemistry (United States); Makino, Shin-ichi; Sahu, Sarata C.; Matsubara, Yuko [University of Wisconsin-Madison, Center for Eukaryotic Structural Genomics (CESG), Department of Biochemistry (United States); Endo, Yaeta [Ehime University, Cell-Free Science and Technology Research Center (Japan); Kainosho, Masatsune [Tokyo Metropolitan University, Center for Priority Areas (Japan); Markley, John L., E-mail: markley@nmrfam.wisc.edu [University of Wisconsin-Madison, National Magnetic Resonance Facility at Madison (NMRFAM), Department of Biochemistry (United States)

2011-12-15

Perdeuteration, selective deuteration, and stereo array isotope labeling (SAIL) are valuable strategies for NMR studies of larger proteins and membrane proteins. To minimize scrambling of the label, it is best to use cell-free methods to prepare selectively labeled proteins. However, when proteins are prepared from deuterated amino acids by cell-free translation in H{sub 2}O, exchange reactions can lead to contamination of {sup 2}H sites by {sup 1}H from the solvent. Examination of a sample of SAIL-chlorella ubiquitin prepared by Escherichia coli cell-free synthesis revealed that exchange had occurred at several residues (mainly at Gly, Ala, Asp, Asn, Glu, and Gln). We present results from a study aimed at identifying the exchanging sites and level of exchange and at testing a strategy for minimizing {sup 1}H contamination during wheat germ cell-free translation of proteins produced from deuterated amino acids by adding known inhibitors of transaminases (1 mM aminooxyacetic acid) and glutamate synthetase (0.1 mM l-methionine sulfoximine). By using a wheat germ cell-free expression system, we produced [U-{sup 2}H, {sup 15}N]-chlorella ubiquitin without and with added inhibitors, and [U-{sup 15}N]-chlorella ubiquitin as a reference to determine the extent of deuterium incorporation. We also prepared a sample of [U-{sup 13}C, {sup 15}N]-chlorella ubiquitin, for use in assigning the sites of exchange. The added inhibitors did not reduce the protein yield and were successful in blocking hydrogen exchange at C{sup {alpha}} sites, with the exception of Gly, and at C{sup {beta}} sites of Ala. We discovered, in addition, that partial exchange occurred with or without the inhibitors at certain side-chain methyl and methylene groups: Asn-H{sup {beta}}, Asp-H{sup {beta}}, Gln-H{sup {gamma}}, Glu-H{sup {gamma}}, and Lys-H{sup {epsilon}}. The side-chain labeling pattern, in particular the mixed chiral labeling resulting from partial exchange at certain sites, should be of

Bicarbonate uptake via an anion exchange protein is the main mechanism of inorganic carbon acquisition by the giant kelp Macrocystis pyrifera (Laminariales, Phaeophyceae) under variable pH.

Science.gov (United States)

Fernández, Pamela A; Hurd, Catriona L; Roleda, Michael Y

2014-12-01

Macrocystis pyrifera is a widely distributed, highly productive, seaweed. It is known to use bicarbonate (HCO3 (-) ) from seawater in photosynthesis and the main mechanism of utilization is attributed to the external catalyzed dehydration of HCO3 (-) by the surface-bound enzyme carbonic anhydrase (CAext ). Here, we examined other putative HCO3 (-) uptake mechanisms in M. pyrifera under pHT 9.00 (HCO3 (-) : CO2  = 940:1) and pHT 7.65 (HCO3 (-) : CO2  = 51:1). Rates of photosynthesis, and internal CA (CAint ) and CAext activity were measured following the application of AZ which inhibits CAext , and DIDS which inhibits a different HCO3 (-) uptake system, via an anion exchange (AE) protein. We found that the main mechanism of HCO3 (-) uptake by M. pyrifera is via an AE protein, regardless of the HCO3 (-) : CO2 ratio, with CAext making little contribution. Inhibiting the AE protein led to a 55%-65% decrease in photosynthetic rates. Inhibiting both the AE protein and CAext at pHT 9.00 led to 80%-100% inhibition of photosynthesis, whereas at pHT 7.65, passive CO2 diffusion supported 33% of photosynthesis. CAint was active at pHT 7.65 and 9.00, and activity was always higher than CAext , because of its role in dehydrating HCO3 (-) to supply CO2 to RuBisCO. Interestingly, the main mechanism of HCO3 (-) uptake in M. pyrifera was different than that in other Laminariales studied (CAext -catalyzed reaction) and we suggest that species-specific knowledge of carbon uptake mechanisms is required in order to elucidate how seaweeds might respond to future changes in HCO3 (-) :CO2 due to ocean acidification. © 2014 Phycological Society of America.

Imaging of endogenous exchangeable proton signals in the human brain using frequency labeled exchange transfer imaging.

Science.gov (United States)

Yadav, Nirbhay N; Jones, Craig K; Hua, Jun; Xu, Jiadi; van Zijl, Peter C M

2013-04-01

To image endogenous exchangeable proton signals in the human brain using a recently reported method called frequency labeled exchange transfer (FLEX) MRI. As opposed to labeling exchangeable protons using saturation (i.e., chemical exchange saturation transfer, or CEST), FLEX labels exchangeable protons with their chemical shift evolution. The use of short high-power frequency pulses allows more efficient labeling of rapidly exchanging protons, while time domain acquisition allows removal of contamination from semi-solid magnetization transfer effects. FLEX-based exchangeable proton signals were detected in human brain over the 1-5 ppm frequency range from water. Conventional magnetization transfer contrast and the bulk water signal did not interfere in the FLEX spectrum. The information content of these signals differed from in vivo CEST data in that the average exchange rate of these signals was 350-400 s(-1) , much faster than the amide signal usually detected using direct saturation (∼30 s(-1) ). Similarly, fast exchanging protons could be detected in egg white in the same frequency range where amide and amine protons of mobile proteins and peptides are known to resonate. FLEX MRI in the human brain preferentially detects more rapidly exchanging amide/amine protons compared to traditional CEST experiments, thereby changing the information content of the exchangeable proton spectrum. This has the potential to open up different types of endogenous applications as well as more easy detection of rapidly exchanging protons in diaCEST agents or fast exchanging units such as water molecules in paracest agents without interference of conventional magnetization transfer contrast. Copyright © 2013 Wiley Periodicals, Inc.

Stress-induced activation of protein kinase CK2 by direct interaction with p38 mitogen-activated protein kinase

DEFF Research Database (Denmark)

Sayed, M; Kim, S O; Salh, B S

2000-01-01

Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-alpha stimulate the specific enzyme activity of CK2...... in the human cervical carcinoma HeLa cells by up to 8-fold, and this could be blocked by the p38 MAP kinase inhibitor SB203580. We show that p38alpha MAP kinase, in a phosphorylation-dependent manner, can directly interact with the alpha and beta subunits of CK2 to activate the holoenzyme through what appears...

Histone acetyltransferase inhibitors antagonize AMP-activated protein kinase in postmortem glycolysis

Directory of Open Access Journals (Sweden)

Qiong Li

2017-06-01

Full Text Available Objective The purpose of this study was to investigate the influence of AMP-activated protein kinase (AMPK activation on protein acetylation and glycolysis in postmortem muscle to better understand the mechanism by which AMPK regulates postmortem glycolysis and meat quality. Methods A total of 32 mice were randomly assigned to four groups and intraperitoneally injected with 5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AICAR, a specific activator of AMPK, AICAR and histone acetyltransferase inhibitor II, or AICAR, Trichostatin A (TSA, an inhibitor of histone deacetylase I and II and Nicotinamide (NAM, an inhibitor of the Sirt family deacetylases. After mice were euthanized, the Longissimus dorsi muscle was collected at 0 h, 45 min, and 24 h postmortem. AMPK activity, protein acetylation and glycolysis in postmortem muscle were measured. Results Activation of AMPK by AICAR significantly increased glycolysis in postmortem muscle. At the same time, it increased the total acetylated proteins in muscle 45 min postmortem. Inhibition of protein acetylation by histone acetyltransferase inhibitors reduced AMPK activation induced increase in the total acetylated proteins and glycolytic rate in muscle early postmortem, while histone deacetylase inhibitors further promoted protein acetylation and glycolysis. Several bands of proteins were detected to be differentially acetylated in muscle with different glycolytic rates. Conclusion Protein acetylation plays an important regulatory role in postmortem glycolysis. As AMPK mediates the effects of pre-slaughter stress on postmortem glycolysis, protein acetylation is likely a mechanism by which antemortem stress influenced postmortem metabolism and meat quality though the exact mechanism is to be elucidated.

Activated Protein C Drives the Hyperfibrinolysis of Acute Traumatic Coagulopathy.

Science.gov (United States)

Davenport, Ross A; Guerreiro, Maria; Frith, Daniel; Rourke, Claire; Platton, Sean; Cohen, Mitchell; Pearse, Rupert; Thiemermann, Chris; Brohi, Karim

2017-01-01

Major trauma is a leading cause of morbidity and mortality worldwide with hemorrhage accounting for 40% of deaths. Acute traumatic coagulopathy exacerbates bleeding, but controversy remains over the degree to which inhibition of procoagulant pathways (anticoagulation), fibrinogen loss, and fibrinolysis drive the pathologic process. Through a combination of experimental study in a murine model of trauma hemorrhage and human observation, the authors' objective was to determine the predominant pathophysiology of acute traumatic coagulopathy. First, a prospective cohort study of 300 trauma patients admitted to a single level 1 trauma center with blood samples collected on arrival was performed. Second, a murine model of acute traumatic coagulopathy with suppressed protein C activation via genetic mutation of thrombomodulin was used. In both studies, analysis for coagulation screen, activated protein C levels, and rotational thromboelastometry (ROTEM) was performed. In patients with acute traumatic coagulopathy, the authors have demonstrated elevated activated protein C levels with profound fibrinolytic activity and early depletion of fibrinogen. Procoagulant pathways were only minimally inhibited with preservation of capacity to generate thrombin. Compared to factors V and VIII, proteases that do not undergo activated protein C-mediated cleavage were reduced but maintained within normal levels. In transgenic mice with reduced capacity to activate protein C, both fibrinolysis and fibrinogen depletion were significantly attenuated. Other recognized drivers of coagulopathy were associated with less significant perturbations of coagulation. Activated protein C-associated fibrinolysis and fibrinogenolysis, rather than inhibition of procoagulant pathways, predominate in acute traumatic coagulopathy. In combination, these findings suggest a central role for the protein C pathway in acute traumatic coagulopathy and provide new translational opportunities for management of

Stimulation through the T cell receptor induces Cbl association with Crk proteins and the guanine nucleotide exchange protein C3G

NARCIS (Netherlands)

Reedquist, K. A.; Fukazawa, T.; Panchamoorthy, G.; Langdon, W. Y.; Shoelson, S. E.; Druker, B. J.; Band, H.

1996-01-01

We and others have recently identified Cbl, the protein product of the c-cbl protooncogene, as an early tyrosine kinase substrate upon T cell activation and have shown that Cbl forms in vivo complexes with Src family tyrosine kinases, Grb2 adaptor protein, and the p85 subunit of PI-3 kinase. Here we

LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5

DEFF Research Database (Denmark)

John von Freyend, Simona; Rosenqvist, Heidi; Fink, Annette

2010-01-01

The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified...... LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric...... kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co...

Quantitative analysis of protein-ligand interactions by NMR.

Science.gov (United States)

Furukawa, Ayako; Konuma, Tsuyoshi; Yanaka, Saeko; Sugase, Kenji

2016-08-01

Protein-ligand interactions have been commonly studied through static structures of the protein-ligand complex. Recently, however, there has been increasing interest in investigating the dynamics of protein-ligand interactions both for fundamental understanding of the underlying mechanisms and for drug development. NMR is a versatile and powerful tool, especially because it provides site-specific quantitative information. NMR has widely been used to determine the dissociation constant (KD), in particular, for relatively weak interactions. The simplest NMR method is a chemical-shift titration experiment, in which the chemical-shift changes of a protein in response to ligand titration are measured. There are other quantitative NMR methods, but they mostly apply only to interactions in the fast-exchange regime. These methods derive the dissociation constant from population-averaged NMR quantities of the free and bound states of a protein or ligand. In contrast, the recent advent of new relaxation-based experiments, including R2 relaxation dispersion and ZZ-exchange, has enabled us to obtain kinetic information on protein-ligand interactions in the intermediate- and slow-exchange regimes. Based on R2 dispersion or ZZ-exchange, methods that can determine the association rate, kon, dissociation rate, koff, and KD have been developed. In these approaches, R2 dispersion or ZZ-exchange curves are measured for multiple samples with different protein and/or ligand concentration ratios, and the relaxation data are fitted to theoretical kinetic models. It is critical to choose an appropriate kinetic model, such as the two- or three-state exchange model, to derive the correct kinetic information. The R2 dispersion and ZZ-exchange methods are suitable for the analysis of protein-ligand interactions with a micromolar or sub-micromolar dissociation constant but not for very weak interactions, which are typical in very fast exchange. This contrasts with the NMR methods that are used

Site-specific incorporation of redox active amino acids into proteins

Science.gov (United States)

Alfonta, Lital [San Diego, CA; Schultz, Peter G [La Jolla, CA; Zhang, Zhiwen [San Diego, CA

2009-02-24

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Site-specific incorporation of redox active amino acids into proteins

Energy Technology Data Exchange (ETDEWEB)

Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

2017-10-10

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Hydrogen deuterium exchange mass spectrometry in biopharmaceutical discovery and development – A review

International Nuclear Information System (INIS)

Deng, Bin; Lento, Cristina; Wilson, Derek J.

2016-01-01

Protein therapeutics have emerged as a major class of biopharmaceuticals over the past several decades, a trend that has motivated the advancement of bioanalytical technologies for protein therapeutic characterization. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful and sensitive technique that can probe the higher order structure of proteins and has been used in the assessment and development of monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs) and biosimilar antibodies. It has also been used to quantify protein-ligand, protein-receptor and other protein-protein interactions involved in signaling pathways. In manufacturing and development, HDX-MS can validate storage formulations and manufacturing processes for various biotherapeutics. Currently, HDX-MS is being refined to provide additional coverage, sensitivity and structural specificity and implemented on the millisecond timescale to reveal residual structure and dynamics in disordered domains and intrinsically disordered proteins. - Highlights: • The pharmaceuticals industry is increasingly shifting to protein therapeutics. • Hydrogen deuterium exchange mass spectrometry is uniquely well suited to support biopharmaceutical development. • Applications for hydrogen deuterium exchange span drug discovery, development and manufacturing. • Future developments will allow improved sensitivity, structural resolution and a broader range of dynamics to be monitored.

Hydrogen deuterium exchange mass spectrometry in biopharmaceutical discovery and development – A review

Energy Technology Data Exchange (ETDEWEB)

Deng, Bin, E-mail: dengbin@yorku.ca [Chemistry Department, York University, 4700 Keele Street, Toronto, ON, M3J 1P3 (Canada); The Centre for Research in Mass Spectrometry, York University, Toronto, ON, M3J1P3 (Canada); Lento, Cristina, E-mail: clento@yorku.ca [Chemistry Department, York University, 4700 Keele Street, Toronto, ON, M3J 1P3 (Canada); The Centre for Research in Mass Spectrometry, York University, Toronto, ON, M3J1P3 (Canada); Wilson, Derek J., E-mail: dkwilson@yorku.ca [Chemistry Department, York University, 4700 Keele Street, Toronto, ON, M3J 1P3 (Canada); The Centre for Research in Mass Spectrometry, York University, Toronto, ON, M3J1P3 (Canada)

2016-10-12

Protein therapeutics have emerged as a major class of biopharmaceuticals over the past several decades, a trend that has motivated the advancement of bioanalytical technologies for protein therapeutic characterization. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful and sensitive technique that can probe the higher order structure of proteins and has been used in the assessment and development of monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs) and biosimilar antibodies. It has also been used to quantify protein-ligand, protein-receptor and other protein-protein interactions involved in signaling pathways. In manufacturing and development, HDX-MS can validate storage formulations and manufacturing processes for various biotherapeutics. Currently, HDX-MS is being refined to provide additional coverage, sensitivity and structural specificity and implemented on the millisecond timescale to reveal residual structure and dynamics in disordered domains and intrinsically disordered proteins. - Highlights: • The pharmaceuticals industry is increasingly shifting to protein therapeutics. • Hydrogen deuterium exchange mass spectrometry is uniquely well suited to support biopharmaceutical development. • Applications for hydrogen deuterium exchange span drug discovery, development and manufacturing. • Future developments will allow improved sensitivity, structural resolution and a broader range of dynamics to be monitored.

Kinetics of the homogeneous exchange of alpha-lactalbumin adsorbed on titanium oxide surface.

Science.gov (United States)

Bentaleb, A; Haïkel, Y; Voegel, J C; Schaaf, P

1998-06-05

The homogeneous exchange process whereby alpha-lactalbumine molecules adsorbed on hydrophilic titanium oxide particles are replaced by alpha-lactalbumine molecules in solution has been investigated by means of a 125I radio-labeling technique, alpha-lactalbumine is a compact and highly negatively charged protein, making this study complementary to previous work devoted to the general understanding of the exchange mechanisms of adsorbed proteins on solid surfaces. The isotherm of alpha-lactalbumine exhibits bimodal adsorption shape, and the exchange process whereby adsorbed proteins are replaced by new incoming ones from the bulk solution has been studied at both the upper and the lower plateau of the isotherm. In the upper plateau the exchange process was found to be of first order with respect to the bulk molecules, and the release rate constant was equal to 0.914 L. mol-1.s-1. This behavior is identical to what has been observed with other proteinic systems. In the lower plateau domain, in contrast, the protein release process is independent of the concentration of proteins in the bulk, but the release rates are higher than the pure desorption rates. This constitutes, to our knowledge, a behavior that never before has been observed and that remains to be explained.

Hydrogen-exchange kinetics of the indole NH proton of the buried tryptophan in the constant fragment of the immunoglobulin light chain

International Nuclear Information System (INIS)

Kawata, Y.; Goto, Y.; Hamaguchi, K.; Hayashi, F.; Kobayashi, Y.; Kyogoku, Y.

1988-01-01

The constant fragment of the immunoglobulin light chain (type λ) has two trytophyl residues at positions 150 and 187. Trp-150 is buried in the interior, and Trp-187 lies on the surface of the molecule. The hydrogen-deuterium exchange kinetics of the indole NH proton Trp-150 were studied at various pH values at 25 0 C by 1 H nuclear magnetic resonance. Exchange rates were approximately first order in hydroxyl ion dependence above pH 8, were relatively independent of pH between pH 7 and 8, and decreased below pH 7. On the assumption that the exchange above pH 8 proceeds through local fluctuations of the protein molecule, the exchange rates between pH 7 and 8 through global unfolding were estimated. The exchange rate constant within this pH range at 25 0 C thus estimated was consistent with that of the global unfolding of the constant fragment under the same conditions as those reported previously. The activation energy for the exchange process at pH 7.8 was the same as that for the unfolding process by 2 M guanidine hydrochloride. The exchange rates of backbone NH protons were almost the same as that of the indole NH proton of Trp-150 at pH 7.l. These observations also indicated that the exchange between pH 7 and 8 occurs through global unfolding of the protein molecule and is rate-limited by the unfolding. At around pH 9, on the other hand, the activation energy for the exchange process of the indole NH proton of Trp-150 was smaller than that for the unfolding process, and the exchange rates differed according to the different signals of backbone NH protons. These findings together with the pH dependence of the rate constant indicated that exchange due to local fluctuations is predominant above pH 8

The contact activation proteins: a structure/function overview

NARCIS (Netherlands)

Meijers, J. C.; McMullen, B. A.; Bouma, B. N.

1992-01-01

In recent years, extensive knowledge has been obtained on the structure/function relationships of blood coagulation proteins. In this overview, we present recent developments on the structure/function relationships of the contact activation proteins: factor XII, high molecular weight kininogen,

Specific deletion of AMP-activated protein kinase (α1AMPK in murine oocytes alters junctional protein expression and mitochondrial physiology.

Directory of Open Access Journals (Sweden)

Michael J Bertoldo

Full Text Available Oogenesis and folliculogenesis are dynamic processes that are regulated by endocrine, paracrine and autocrine signals. These signals are exchanged between the oocyte and the somatic cells of the follicle. Here we analyzed the role of AMP-activated protein kinase (AMPK, an important regulator of cellular energy homeostasis, by using transgenic mice deficient in α1AMPK specifically in the oocyte. We found a decrease of 27% in litter size was observed in ZP3-α1AMPK-/- (ZP3-KO female mice. Following in vitro fertilization, where conditions are stressful for the oocyte and embryo, ZP3-KO oocytes were 68% less likely to pass the 2-cell stage. In vivo and in cumulus-oocyte complexes, several proteins involved in junctional communication, such as connexin37 and N-cadherin were down-regulated in the absence of α1AMPK. While the two signalling pathways (PKA and MAPK involved in the junctional communication between the cumulus/granulosa cells and the oocyte were stimulated in control oocytes, ZP3-KO oocytes exhibited only low phosphorylation of MAPK or CREB proteins. In addition, MII oocytes deficient in α1AMPK had a 3-fold lower ATP concentration, an increase in abnormal mitochondria, and a decrease in cytochrome C and PGC1α levels, suggesting perturbed energy production by mitochondria. The absence of α1AMPK also induced a reduction in histone deacetylase activity, which was associated with an increase in histone H3 acetylation (K9/K14 residues. Together, the results of the present study suggest that absence of AMPK, modifies oocyte quality through energy processes and oocyte/somatic cell communication. The limited effect observed in vivo could be partly due to a favourable follicle microenvironment where nutrients, growth factors, and adequate cell interaction were present. Whereas in a challenging environment such as that of in vitro culture following IVF, the phenotype is revealed.

Pre-commissioning activities of exchange units of Heavy Water Plant, Manuguru (Paper No. 1.2)

International Nuclear Information System (INIS)

Kamath, H.S.; Sikaria, R.P.

1992-01-01

Heavy Water Plant, Manuguru (HWPM) comprises of two exchange units where enrichment of deuterium from natural concentration to 15% concentration is achieved using dual temperature H 2 S-H 2 O exchange process in a three stage cascade. In each exchange unit an inventory of around 200 MT of H 2 S gas is required under normal operating pressure and a feed water flow rate of 450 MT/Hr is required to be maintained. In view of the large flow rates of H 2 S circulating gas, water, cooling water, steam etc., the lines sizes in exchange unit are quite large. Further, H 2 S gas being highly toxic and corrosive, extreme care is required in order to ensure integrity and leak tightness. While due care is taken in material selection, design, quality surveillance etc., during fabrication, certain additional measures are required to be taken during precommissioning of the systems handling H 2 S under pressure. This paper deals with the activities from completion of plant erection including hydrotesting of piping by the piping contractor to the stage of trial production run. (author)

Modulation of Spc1 stress-activated protein kinase activity by methylglyoxal through inhibition of protein phosphatase in the fission yeast Schizosaccharomyces pombe

International Nuclear Information System (INIS)

Takatsume, Yoshifumi; Izawa, Shingo; Inoue, Yoshiharu

2007-01-01

Methylglyoxal, a ubiquitous metabolite derived from glycolysis has diverse physiological functions in yeast cells. Previously, we have reported that extracellularly added methylglyoxal activates Spc1, a stress-activated protein kinase (SAPK), in the fission yeast Schizosaccharomyces pombe [Y. Takatsume, S. Izawa, Y. Inoue, J. Biol. Chem. 281 (2006) 9086-9092]. Phosphorylation of Spc1 by treatment with methylglyoxal in S. pombe cells defective in glyoxalase I, an enzyme crucial for the metabolism of methylglyoxal, continues for a longer period than in wild-type cells. Here we show that methylglyoxal inhibits the activity of the protein phosphatase responsible for the dephosphorylation of Spc1 in vitro. In addition, we found that methylglyoxal inhibits human protein tyrosine phosphatase 1B (PTP1B) also. We propose a model for the regulation of the activity of the Spc1-SAPK signaling pathway by methylglyoxal in S. pombe

Vivo-morpholinos induced transient knockdown of physical activity related proteins.

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David P Ferguson

Full Text Available Physical activity is associated with disease prevention and overall wellbeing. Additionally there has been evidence that physical activity level is a result of genetic influence. However, there has not been a reliable method to silence candidate genes in vivo to determine causal mechanisms of physical activity regulation. Vivo-morpholinos are a potential method to transiently silence specific genes. Thus, the aim of this study was to validate the use of Vivo-morpholinos in a mouse model for voluntary physical activity with several sub-objectives. We observed that Vivo-morpholinos achieved between 60-97% knockdown of Drd1-, Vmat2-, and Glut4-protein in skeletal muscle, the delivery moiety of Vivo-morpholinos (scramble did not influence physical activity and that a cocktail of multiple Vivo-morpholinos can be given in a single treatment to achieve protein knockdown of two different targeted proteins in skeletal muscle simultaneously. Knocking down Drd1, Vmat2, or Glut4 protein in skeletal muscle did not affect physical activity. Vivo-morpholinos injected intravenously alone did not significantly knockdown Vmat2-protein expression in the brain (p = 0.28. However, the use of a bradykinin analog to increase blood-brain-barrier permeability in conjunction with the Vivo-morpholinos significantly (p = 0.0001 decreased Vmat2-protein in the brain with a corresponding later over-expression of Vmat2 coincident with a significant (p = 0.0016 increase in physical activity. We conclude that Vivo-morpholinos can be a valuable tool in determining causal gene-phenotype relationships in whole animal models.

A gratuitous β-Lactamase inducer uncovers hidden active site dynamics of the Staphylococcus aureus BlaR1 sensor domain.

Science.gov (United States)

Frederick, Thomas E; Peng, Jeffrey W

2018-01-01

Increasing evidence shows that active sites of proteins have non-trivial conformational dynamics. These dynamics include active site residues sampling different local conformations that allow for multiple, and possibly novel, inhibitor binding poses. Yet, active site dynamics garner only marginal attention in most inhibitor design efforts and exert little influence on synthesis strategies. This is partly because synthesis requires a level of atomic structural detail that is frequently missing in current characterizations of conformational dynamics. In particular, while the identity of the mobile protein residues may be clear, the specific conformations they sample remain obscure. Here, we show how an appropriate choice of ligand can significantly sharpen our abilities to describe the interconverting binding poses (conformations) of protein active sites. Specifically, we show how 2-(2'-carboxyphenyl)-benzoyl-6-aminopenicillanic acid (CBAP) exposes otherwise hidden dynamics of a protein active site that binds β-lactam antibiotics. When CBAP acylates (binds) the active site serine of the β-lactam sensor domain of BlaR1 (BlaRS), it shifts the time scale of the active site dynamics to the slow exchange regime. Slow exchange enables direct characterization of inter-converting protein and bound ligand conformations using NMR methods. These methods include chemical shift analysis, 2-d exchange spectroscopy, off-resonance ROESY of the bound ligand, and reduced spectral density mapping. The active site architecture of BlaRS is shared by many β-lactamases of therapeutic interest, suggesting CBAP could expose functional motions in other β-lactam binding proteins. More broadly, CBAP highlights the utility of identifying chemical probes common to structurally homologous proteins to better expose functional motions of active sites.

Association of the macrophage activating factor (MAF) precursor activity with polymorphism in vitamin D-binding protein.

Science.gov (United States)

Nagasawa, Hideko; Sasaki, Hideyuki; Uto, Yoshihiro; Kubo, Shinichi; Hori, Hitoshi

2004-01-01

Serum vitamin D-binding protein (Gc protein or DBP) is a highly expressed polymorphic protein, which is a precursor of the inflammation-primed macrophage activating factor, GcMAF, by a cascade of carbohydrate processing reactions. In order to elucidate the relationship between Gc polymorphism and GcMAF precursor activity, we estimated the phagocytic ability of three homotypes of Gc protein, Gc1F-1F, Gc1S-1S and Gc2-2, through processing of their carbohydrate moiety. We performed Gc typing of human serum samples by isoelectric focusing (IEF). Gc protein from human serum was purified by affinity chromatography with 25-hydroxyvitamin D3-sepharose. A phagocytosis assay of Gc proteins, modified using beta-glycosidase and sialidase, was carried out. The Gc1F-1F phenotype was revealed to possess Galbeta1-4GalNAc linkage by the analysis of GcMAF precursor activity using beta1-4 linkage-specific galactosidase from jack bean. The GcMAF precursor activity of the Gc1F-1F phenotype was highest among three Gc homotypes. The Gc polymorphism and carbohydrate diversity of Gc protein are significant for its pleiotropic effects.

Studies on neutron activation analysis using ion exchange preconcentration Pt. 6

International Nuclear Information System (INIS)

Hirose, A.; Ishii, D.

1978-01-01

Three convenient procedures are described for applying anion exchange resins to the preconcentration and irradiation support in neutron activation analysis. For each procedure the amounts of interferences were determined. The effect of the form, cross-linkage, pore size, and particle size of the resins on the elimination of interferences was investigated. Some other associated problems are also presented, viz. effect of concentration and temperature of washing solution, effect of irradiation time, and comparison with filter paper. Tabulated data are presented. (author)

Characterization of protein kinase CK2 protein subunits and p53 in F9 teratocarcinoma cells in the absence and presence of cisplatin

DEFF Research Database (Denmark)

Küpper, M; Köster, M; Schmidt-Spaniol, I

1994-01-01

cell extracts treated with and without cisplatin were analyzed by ion exchange chromatography for protein kinase CK2 alpha/beta subunits and p53 distribution. The following results were obtained: (a) in crude extracts of cisplatin-treated cells, CK2 activity was sometimes reduced by as much as 50%; (b......The effect of cis-diaminedichloroplatinum(II) (cisplatin) on the induction of p53 and protein kinase CK2 activity was studied in the mouse teratocarcinoma cell line F9. Treatment of the cells with the chemotherapeutic agent cisplatin led to the detection of p53 3 h after addition of the drug. F9...... by immunostaining, we have detected, at a concentration of approximately 200 mM NaCl, a protein of approximately 46 kDa which reacted with the CK2 alpha-specific antibody. This fraction was devoid of CK2 activity; and (d) cisplatin-treated cells exhibited p53 protein, which was mostly eluting ahead but also partly...

Animal and Plant Proteins as Precursors of Peptides with ACE Inhibitory Activity – An in silico Strategy of Protein Evaluation

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Anna Iwaniak

2009-01-01

Full Text Available This paper presents a modern in silico approach useful in the evaluation of proteins as a source of ACE inhibitors. All protein sequences analyzed were derived from the BIOPEP database. To determine the protein value, the following criteria of evaluation were applied: the profile of potential biological (ACE inhibitory activity of a protein, the frequency of the occurrence of fragments with ACE inhibitory activity (A and the potential biological activity of a protein (B. The results, based on a statistical analysis, indicate that milk proteins can be a better source of ACE inhibitors than wheat gliadins. Moreover, all analyzed gliadins possessed more potent ACE inhibitors than chicken meat proteins. No significant differences were observed when comparing A values between soy globulins and β-lactoglobulins. Although criteria such as the profile of potential biological activity of protein, as well as parameters A and B, can be suitable tools in protein evaluation, the proteolytic digestion of protein needs to be considered. Moreover, computerised methods of classifying proteins according to different algorithms are often subjective due to discretion in interpretation of the results.

PENURUNAN KADAR PROTEIN LIMBAH CAIR TAHU DENGAN PEMANFAATAN KARBON BAGASSE TERAKTIVASI (Protein Reduction of Tofu Wastewater Using Activated Carbon Bagasse

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Candra Purnawan

2014-10-01

Full Text Available ABSTRAK Penurunan kadar protein limbah tahu telah dilakukan dengan pemanfaatan karbon Bagasse teraktivasi. Tujuan dari penelitian ini adalah untuk mengetahui kondisi optimum dari karbon teraktivasi NaOH dan H2SO4 dalam menurunkan kadar protein limbah cair tahu dan mengetahui jenis isoterm adsorpsi dari karbon aktif yang digunakan untuk menyerap protein limbah cair tahu. Hasil penelitian menunjukkan konsentrasi NaOH yang optimum untuk aktivasi karbon aktif 15%, massa optimum karbon bagasse teraktivasi NaOH adalah 2 g dan penurunan kadar proteinnya 71,95%, sedangkan massa optimum karbon bagasse teraktivasi H2SO4 adalah 1 g dengan penurunan kadar protein sebesar 38,19%. Waktu kontak optimum karbon bagasse teraktivasi  NaOH dan H2SO4 adalah 12 jam. Adsorpsi protein oleh karbon bagasse teraktivasi NaOH mengikuti isoterm adsorpsi Langmuir dan Freundlich sedangkan karbon bagasse teraktivasi H2SO4 dominan mengikuti isoterm Freundlich.   ABSTRACT The protein reduction of tofu wastewater using activated carbon from bagasse  had been conducted. The purposes of this research were to analysis optimum condition of activated carbon bagsse using NaOH and H2SO4 for reduction protein in tofu wastewater, and analysis adsorption isotherm of activated carbon with protein. The result showed that optimum mass of carbon bagasse activated NaOH was  2 g with 71.95% protein reduction, while carbon bagasse activated H2SO4 has 1 g with 38.19% protein reduction. The optimum contact time between protein and activated carbon (with NaOH and H2SO4 was happened in 12 hours. Adsorption protein with carbon bagasse activated NaOH had followed Langmuir and Freundlich adsorption isotherm, while adsorption with carbon bagasse activated H2SO4 dominantlyhad followed Freundlich adsorption isotherm

Ion exchange kinetics of alkaline earths on Zr(IV) arsenosilicate cation exchanger

International Nuclear Information System (INIS)

Varshney, K.G.; Agrawal, S.; Varshney, K.

1984-01-01

A new approach based on the Nernst-Planck equations was applied to study the ion exchange kinetics for the exchange reactions of Mg(II), Ca(II), Sr(II) and Ba(II) with H + -ions at various temperatures on the zirconium(IV) arsenosilicate phase. Under the conditions of particle diffusion, the rate of exchange was found to be independent of the metal ion concentration at and above 0.1 M in aqueous medium. Energy and entropy of activation were determined and found to vary linearly with the ionic radii and mobilities of alkaline earths, a unique feature observed for an inorganic ion exchanger. The results are useful for predicting the ion exchange processes occurring on the surface of an inorganic material of the type studied. (author)

Identification of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH as a binding protein for a 68-kDa Bacillus thuringiensis parasporal protein cytotoxic against leukaemic cells

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Nadarajah Vishna

2010-11-01

Full Text Available Abstract Background Bacillus thuringiensis (Bt, an ubiquitous gram-positive spore-forming bacterium forms parasporal proteins during the stationary phase of its growth. Recent findings of selective human cancer cell-killing activity in non-insecticidal Bt isolates resulted in a new category of Bt parasporal protein called parasporin. However, little is known about the receptor molecules that bind parasporins and the mechanism of anti-cancer activity. A Malaysian Bt isolate, designated Bt18 produces parasporal protein that exhibit preferential cytotoxic activity for human leukaemic T cells (CEM-SS but is non-cytotoxic to normal T cells or other cancer cell lines such as human cervical cancer (HeLa, human breast cancer (MCF-7 and colon cancer (HT-29 suggesting properties similar to parasporin. In this study we aim to identify the binding protein for Bt18 in human leukaemic T cells. Methods Bt18 parasporal protein was separated using Mono Q anion exchange column attached to a HPLC system and antibody was raised against the purified 68-kDa parasporal protein. Receptor binding assay was used to detect the binding protein for Bt18 parasporal protein in CEM-SS cells and the identified protein was sent for N-terminal sequencing. NCBI protein BLAST was used to analyse the protein sequence. Double immunofluorescence staining techniques was applied to localise Bt18 and binding protein on CEM-SS cell. Results Anion exchange separation of Bt18 parasporal protein yielded a 68-kDa parasporal protein with specific cytotoxic activity. Polyclonal IgG (anti-Bt18 for the 68-kDa parasporal protein was successfully raised and purified. Receptor binding assay showed that Bt18 parasporal protein bound to a 36-kDa protein from the CEM-SS cells lysate. N-terminal amino acid sequence of the 36-kDa protein was GKVKVGVNGFGRIGG. NCBI protein BLAST revealed that the binding protein was Glyceraldehyde-3-phosphate dehydrogenase (GAPDH. Double immunofluorescence staining showed

Ligand and proton exchange dynamics in recombinant human myoglobin mutants.

Science.gov (United States)

Lambright, D G; Balasubramanian, S; Boxer, S G

1989-05-05

Site-specific mutants of human myoglobin have been prepared in which lysine 45 is replaced by arginine (K45R) and aspartate 60 by glutamate (D60E), in order to examine the influence of these residues and their interaction on the dynamics of the protein. These proteins were studied by a variety of methods, including one and two-dimensional proton nuclear magnetic resonance spectroscopy, exchange kinetics for the distal and proximal histidine NH protons as a function of pH in the met cyano forms, flash photolysis of the CO forms, and ligand replacement kinetics. The electronic absorption and proton nuclear magnetic resonance spectra of the CO forms of these proteins are virtually identical, indicating that the structure of the heme pocket is unaltered by these mutations. There are, however, substantial changes in the dynamics of both CO binding and proton exchange for the mutant K45R, whereas the mutant D60E exhibits behavior indistinguishable from the reference human myoglobin. K45R has a faster CO bimolecular recombination rate and slower CO off-rate relative to the reference. The kinetics for CO binding are independent of pH (6.5 to 10) as well as ionic strength (0 to 1 M-NaCl). The exchange rate for the distal histidine NH is substantially lower for K45R than the reference, whereas the proximal histidine NH exchange rate is unaltered. The exchange behavior of the human proteins is similar to that reported for a comparison of the exchange rates for myoglobins having lysine at position 45 with sperm whale myoglobin, which has arginine at this position. This indicates that the differences in exchange rates reflects largely the Lys----Arg substitution. The lack of a simple correlation for the CO kinetics with this substitution means that these are sensitive to other factors as well. Specific kinetic models, whereby substitution of arginine for lysine at position 45 can affect ligand binding dynamics, are outlined. These experiments demonstrate that a relatively

[Solid state isotope hydrogen exchange for deuterium and tritium in human gene-engineered insulin].

Science.gov (United States)

Zolotarev, Yu A; Dadayan, A K; Kozik, V S; Gasanov, E V; Nazimov, I V; Ziganshin, R Kh; Vaskovsky, B V; Murashov, A N; Ksenofontov, A L; Haribin, O N; Nikolaev, E N; Myasoedov, N F

2014-01-01

The reaction of high temperature solid state catalytic isotope exchange in peptides and proteins under the action of catalyst-activated spillover hydrogen was studied. The reaction of human gene-engineered insulin with deuterium and tritium was conducted at 120-140° C to produce insulin samples containing 2-6 hydrogen isotope atoms. To determine the distribution of the isotope label over tritium-labeled insulin's amino acid residues, oxidation of the S-S bonds of insulin by performic acid was performed and polypeptide chains isolated; then their acid hydrolysis, amino acid analysis and liquid scintillation counts of tritium in the amino acids were conducted. The isotope label was shown to be incorporated in all amino acids of the protein, with the peptide fragment FVNQHLCGSHLVE of the insulin β-chain showing the largest incorporation. About 45% of the total protein isotope label was incorporated in His5 and His10 of this fragment. For the analysis of isotope label distribution in labeled insulin's peptide fragments, the recovery of the S-S bonds by mercaptoethanol, the enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius and HPLC division of the resulting peptides were carried out. Attribution of the peptide fragments formed due to hydrolysis at the Glu-X bond in the β-chain was accomplished by mass spectrometry. Mass spectrometry analysis data of the deuterium-labeled insulin samples' isotopomeric composition showed that the studied solid state isotope exchange reaction equally involved all the protein molecules. Biological studying of tritium-labeled insulin showed its physiological activity to be completely retained.

Hypocholesterolaemic and antioxidant activities of chickpea (Cicer arietinum L.) protein hydrolysates.

Science.gov (United States)

Yust, María del Mar; Millán-Linares, María del Carmen; Alcaide-Hidalgo, Juan María; Millán, Francisco; Pedroche, Justo

2012-07-01

Some dietary proteins possess biological properties which make them potential ingredients of functional or health-promoting foods. Many of these properties are attributed to bioactive peptides that can be released by controlled hydrolysis using exogenous proteases. The aim of this work was to test the improvement of hypocholesterolaemic and antioxidant activities of chickpea protein isolate by means of hydrolysis with alcalase and flavourzyme. All hydrolysates tested exhibited better hypocholesterolaemic activity when compared with chickpea protein isolate. The highest cholesterol micellar solubility inhibition (50%) was found after 60 min of treatment with alcalase followed by 30 min of hydrolysis with flavourzyme. To test antioxidant activity of chickpea proteins three methods were used: β-carotene bleaching method, reducing power and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effect since antioxidant activity of protein hydrolysates may not be attributed to a single mechanism. Chickpea hydrolysates showed better antioxidant activity in all assays, especially reducing power and DPPH scavenging effect than chickpea protein isolate. The results of this study showed the good potential of chickpea protein hydrolysates as bioactive ingredients. The highest bioactive properties could be obtained by selecting the type of proteases and the hydrolysis time. Copyright © 2012 Society of Chemical Industry.

Protein crystallography and drug discovery: recollections of knowledge exchange between academia and industry

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Tom L. Blundell

2017-07-01

Full Text Available The development of structure-guided drug discovery is a story of knowledge exchange where new ideas originate from all parts of the research ecosystem. Dorothy Crowfoot Hodgkin obtained insulin from Boots Pure Drug Company in the 1930s and insulin crystallization was optimized in the company Novo in the 1950s, allowing the structure to be determined at Oxford University. The structure of renin was developed in academia, on this occasion in London, in response to a need to develop antihypertensives in pharma. The idea of a dimeric aspartic protease came from an international academic team and was discovered in HIV; it eventually led to new HIV antivirals being developed in industry. Structure-guided fragment-based discovery was developed in large pharma and biotechs, but has been exploited in academia for the development of new inhibitors targeting protein–protein interactions and also antimicrobials to combat mycobacterial infections such as tuberculosis. These observations provide a strong argument against the so-called `linear model', where ideas flow only in one direction from academic institutions to industry. Structure-guided drug discovery is a story of applications of protein crystallography and knowledge exhange between academia and industry that has led to new drug approvals for cancer and other common medical conditions by the Food and Drug Administration in the USA, as well as hope for the treatment of rare genetic diseases and infectious diseases that are a particular challenge in the developing world.

Knowledge Exchange and Management Research

DEFF Research Database (Denmark)

Bager, Torben

2018-01-01

for ‘interesting’ discoveries has a potential to lift off papers with a high level of scientific rigor as well as a high level of relevance for practice. Originality: An outcome focus on the relationship between knowledge exchange activities and management research is to our knowledge new in the debate about......Purpose: The growing involvement of management researchers in knowledge exchange activities and collaborative research does not seem to be reflected in a growing academic output. The purpose of this paper is to explore barriers for academic output from these activities as well as the potential...... derived from knowledge exchange activities and Mode 2 research into academic papers such as low priority of case study research in leading management journals, a growing practice orientation in the research funding systems, methodological challenges due to limited researcher control, and disincentives...

Chemical Denaturants Smoothen Ruggedness on the Free Energy Landscape of Protein Folding.

Science.gov (United States)

Malhotra, Pooja; Jethva, Prashant N; Udgaonkar, Jayant B

2017-08-08

To characterize experimentally the ruggedness of the free energy landscape of protein folding is challenging, because the distributed small free energy barriers are usually dominated by one, or a few, large activation free energy barriers. This study delineates changes in the roughness of the free energy landscape by making use of the observation that a decrease in ruggedness is accompanied invariably by an increase in folding cooperativity. Hydrogen exchange (HX) coupled to mass spectrometry was used to detect transient sampling of local energy minima and the global unfolded state on the free energy landscape of the small protein single-chain monellin. Under native conditions, local noncooperative openings result in interconversions between Boltzmann-distributed intermediate states, populated on an extremely rugged "uphill" energy landscape. The cooperativity of these interconversions was increased by selectively destabilizing the native state via mutations, and further by the addition of a chemical denaturant. The perturbation of stability alone resulted in seven backbone amide sites exchanging cooperatively. The size of the cooperatively exchanging and/or unfolding unit did not depend on the extent of protein destabilization. Only upon the addition of a denaturant to a destabilized mutant variant did seven additional backbone amide sites exchange cooperatively. Segmentwise analysis of the HX kinetics of the mutant variants further confirmed that the observed increase in cooperativity was due to the smoothing of the ruggedness of the free energy landscape of folding of the protein by the chemical denaturant.

The mechanics of motorised momentum exchange tethers when applied to active debris removal from LEO

Energy Technology Data Exchange (ETDEWEB)

Caldecott, Ralph; Kamarulzaman, Dayangku N. S.; Kirrane, James P.; Cartmell, Matthew P.; Ganilova, Olga A. [Department of Mechanical Engineering, University of Sheffield, Mappin St., Sheffield, S1 3JD, England (United Kingdom)

2014-12-10

The concept of momentum exchange when applied to space tethers for propulsion is well established, and a considerable body of literature now exists on the on-orbit modelling, the dynamics, and also the control of a large range of tether system applications. The authors consider here a new application for the Motorised Momentum Exchange Tether by highlighting three key stages of development leading to a conceptualisation that can subsequently be developed into a technology for Active Debris Removal. The paper starts with a study of the on-orbit mechanics of a full sized motorised tether in which it is shown that a laden and therefore highly massasymmetrical tether can still be forced to spin, and certainly to librate, thereby confirming its possible usefulness for active debris removal (ADR). The second part of the paper concentrates on the modelling of the centripetal deployment of a symmetrical MMET in order to get it initialized for debris removal operations, and the third and final part of the paper provides an entry into scale modelling for low cost mission design and testing. It is shown that the motorised momentum exchange tether offers a potential solution to the removal of large pieces of orbital debris, and that dynamic methodologies can be implemented to in order to optimise the emergent design.

The mechanics of motorised momentum exchange tethers when applied to active debris removal from LEO

International Nuclear Information System (INIS)

Caldecott, Ralph; Kamarulzaman, Dayangku N. S.; Kirrane, James P.; Cartmell, Matthew P.; Ganilova, Olga A.

2014-01-01

The concept of momentum exchange when applied to space tethers for propulsion is well established, and a considerable body of literature now exists on the on-orbit modelling, the dynamics, and also the control of a large range of tether system applications. The authors consider here a new application for the Motorised Momentum Exchange Tether by highlighting three key stages of development leading to a conceptualisation that can subsequently be developed into a technology for Active Debris Removal. The paper starts with a study of the on-orbit mechanics of a full sized motorised tether in which it is shown that a laden and therefore highly massasymmetrical tether can still be forced to spin, and certainly to librate, thereby confirming its possible usefulness for active debris removal (ADR). The second part of the paper concentrates on the modelling of the centripetal deployment of a symmetrical MMET in order to get it initialized for debris removal operations, and the third and final part of the paper provides an entry into scale modelling for low cost mission design and testing. It is shown that the motorised momentum exchange tether offers a potential solution to the removal of large pieces of orbital debris, and that dynamic methodologies can be implemented to in order to optimise the emergent design

Plasma Protein Turnover Rates in Rats Using Stable Isotope Labeling, Global Proteomics, and Activity-Based Protein Profiling

Energy Technology Data Exchange (ETDEWEB)

Smith, Jordan N.; Tyrrell, Kimberly J.; Hansen, Joshua R.; Thomas, Dennis G.; Murphree, Taylor A.; Shukla, Anil K.; Luders, Teresa; Madden, James M.; Li, Yunying; Wright, Aaron T.; Piehowski, Paul D.

2017-12-06

Protein turnover is important for general health on cellular and organism scales providing a strategy to replace old, damaged, or dysfunctional proteins. Protein turnover also informs of biomarker kinetics, as a better understanding of synthesis and degradation of proteins increases the clinical utility of biomarkers. Here, turnover rates of plasma proteins in rats were measured in vivo using a pulse-chase stable isotope labeling experiment. During the pulse, rats (n=5) were fed 13C6-labeled lysine (“heavy”) feed for 23 days to label proteins. During the chase, feed was changed to an unlabeled equivalent feed (“light”), and blood was repeatedly sampled from rats over 10 time points for 28 days. Plasma samples were digested with trypsin, and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant was used to identify peptides and proteins, and quantify heavy:light lysine ratios. A system of ordinary differential equations was used to calculate protein turnover rates. Using this approach, 273 proteins were identified, and turnover rates were quantified for 157 plasma proteins with half-lives ranging 0.3-103 days. For the ~70 most abundant proteins, variability in turnover rates among rats was low (median coefficient of variation: 0.09). Activity-based protein profiling was applied to pooled plasma samples to enrich serine hydrolases using a fluorophosphonate (FP2) activity-based probe. This enrichment resulted in turnover rates for an additional 17 proteins. This study is the first to measure global plasma protein turnover rates in rats in vivo, measure variability of protein turnover rates in any animal model, and utilize activity-based protein profiling for enhancing measurements of targeted, low-abundant proteins, such as those commonly used as biomarkers. Measured protein turnover rates will be important for understanding of the role of protein turnover in cellular and organism health as well as increasing the utility of protein

Protein phosphatase 5 promotes hepatocarcinogenesis through interaction with AMP-activated protein kinase.

Science.gov (United States)

Chen, Yao-Li; Hung, Man-Hsin; Chu, Pei-Yi; Chao, Tzu-I; Tsai, Ming-Hsien; Chen, Li-Ju; Hsiao, Yung-Jen; Shih, Chih-Ting; Hsieh, Feng-Shu; Chen, Kuen-Feng

2017-08-15

The serine-threonine protein phosphatase family members are known as critical regulators of various cellular functions, such as survival and transformation. Growing evidence suggests that pharmacological manipulation of phosphatase activity exhibits therapeutic benefits. Ser/Thr protein phosphatase 5 (PP5) is known to participate in glucocorticoid receptor (GR) and stress-induced signaling cascades that regulate cell growth and apoptosis, and has been shown to be overexpressed in various human malignant diseases. However, the role of PP5 in hepatocellular carcinoma (HCC) and whether PP5 may be a viable therapeutic target for HCC treatment are unknown. Here, by analyzing HCC clinical samples obtained from 215 patients, we found that overexpression of PP5 is tumor specific and associated with worse clinical outcomes. We further characterized the oncogenic properties of PP5 in HCC cells. Importantly, both silencing of PP5 with lentiviral-mediated short hairpin RNA (shRNA) and chemical inhibition of PP5 phosphatase activity using the natural compound cantharidin/norcantharidin markedly suppressed the growth of HCC cells and tumors in vitro and in vivo. Moreover, we identified AMP-activated protein kinase (AMPK) as a novel downstream target of oncogenic PP5 and demonstrated that the antitumor mechanisms underlying PP5 inhibition involve activation of AMPK signaling. Overall, our results establish a pathological function of PP5 in hepatocarcinogenesis via affecting AMPK signaling and suggest that PP5 inhibition is an attractive therapeutic approach for HCC. Copyright © 2017 Elsevier Inc. All rights reserved.

Activation of sea urchin sperm motility is accompanied by an increase in the creatine kinase exchange flux

NARCIS (Netherlands)

Dorsten, van F.A.; Wyss, M.; Wallimann, T.; Nicolaij, K.

1997-01-01

The kinetics of the creatine kinase (CK) reaction were studied in suspensions of quiescent and active, intact sea-urchin spermatozoa in artificial seawater, using 31P-NMR magnetization transfer. In inactive sperm, no CK-mediated exchange flux was detected, whereas in activated motile sperm, the

Preparation and characterization of zirconium phosphate ion exchanger samples with respect to the separation of highly active actinoid elements

International Nuclear Information System (INIS)

Treplan, J.

1972-01-01

Inorganic ion exchangers are of growing interest in connection with separation processes of α-radiators of high specific activity, or with high gamma doses, because they have a considerably higher radiation resistance at their disposal compared to the commonly used organic ion exchangers. In opposition to their use, however, are the worse properties regarding capacity, chemical resistivity, exchange rate and reproducibility of the ion exchange bed. In the present work, an attempt has been made to influence the properties of a typical representative of this group, zirconium phosphate (ZP), by systematic changing of the preparation parameters in such a manner that a sufficient capacity is obtained regarding tri-valent ions. In addition, information is to be gathered in order to clarify the connection between exchanger property and structure of the ZP. (orig./LH) [de

Preparation of Gc protein-derived macrophage activating factor (GcMAF) and its structural characterization and biological activities.

Science.gov (United States)

Mohamad, Saharuddin Bin; Nagasawa, Hideko; Uto, Yoshihiro; Hori, Hitoshi

2002-01-01

Gc protein has been reported to be a precursor of Gc protein-derived macrophage activation factor (GcMAF) in the inflammation-primed macrophage activation cascade. An inducible beta-galactosidase of B cells and neuraminidase of T cells convert Gc protein to GcMAF. Gc protein from human serum was purified using 25(OH)D3 affinity column chromatography and modified to GcMAF using immobilized glycosidases (beta-galactosidase and neuraminidase) The sugar moiety structure of GcMAF was characterized by lectin blotting by Helix pomatia agglutinin. The biological activities of GcMAF were evaluated by a superoxide generation assay and a phagocytosis assay. We successfully purified Gc protein from human serum. GcMAF was detected by lectin blotting and showed a high biological activity. Our results support the importance of the terminal N-acetylgalactosamine moiety in the GcMAF-mediated macrophage activation cascade, and the existence of constitutive GcMAF in human serum. These preliminary data are important for designing small molecular GcMAF mimics.

Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5

International Nuclear Information System (INIS)

Goda, Natsuko; Tenno, Takeshi; Inomata, Kosuke; Shirakawa, Masahiro; Tanaka, Toshiki; Hiroaki, Hidekazu

2008-01-01

Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20-150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

Science.gov (United States)

Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

1993-01-01

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein.

Science.gov (United States)

Khamina, Kseniya; Lercher, Alexander; Caldera, Michael; Schliehe, Christopher; Vilagos, Bojan; Sahin, Mehmet; Kosack, Lindsay; Bhattacharya, Anannya; Májek, Peter; Stukalov, Alexey; Sacco, Roberto; James, Leo C; Pinschewer, Daniel D; Bennett, Keiryn L; Menche, Jörg; Bergthaler, Andreas

2017-12-01

RNA-dependent RNA polymerases (RdRps) play a key role in the life cycle of RNA viruses and impact their immunobiology. The arenavirus lymphocytic choriomeningitis virus (LCMV) strain Clone 13 provides a benchmark model for studying chronic infection. A major genetic determinant for its ability to persist maps to a single amino acid exchange in the viral L protein, which exhibits RdRp activity, yet its functional consequences remain elusive. To unravel the L protein interactions with the host proteome, we engineered infectious L protein-tagged LCMV virions by reverse genetics. A subsequent mass-spectrometric analysis of L protein pulldowns from infected human cells revealed a comprehensive network of interacting host proteins. The obtained LCMV L protein interactome was bioinformatically integrated with known host protein interactors of RdRps from other RNA viruses, emphasizing interconnected modules of human proteins. Functional characterization of selected interactors highlighted proviral (DDX3X) as well as antiviral (NKRF, TRIM21) host factors. To corroborate these findings, we infected Trim21-/- mice with LCMV and found impaired virus control in chronic infection. These results provide insights into the complex interactions of the arenavirus LCMV and other viral RdRps with the host proteome and contribute to a better molecular understanding of how chronic viruses interact with their host.

An Empirical Investigation into Exchange Rate Regime Choice and Exchange Rate Volatility

OpenAIRE

Helge Berger; Jan-Egbert Sturm; Jakob de Haan

2000-01-01

We test a simple model of exchange rate regime choice with data for 65 non-OECD countries covering the period 1980-94. We find that the variance of output at home and in potential target c ountries as well as the correlation between home and foreign real activity are powerful and robust predictors of exchange rate regime choice. Surprisingly, a more volatile foreign economy can be an argument in favor of a fixed exchange rate regime once similarities in the business cycle are taken into accou...

Theory of the Protein Equilibrium Population Snapshot by H/D Exchange Electrospray Ionization Mass Spectrometry (PEPS-HDX-ESI-MS) Method used to obtain Protein Folding Energies/Rates and Selected Supporting Experimental Evidence.

Science.gov (United States)

Liyanage, Rohana; Devarapalli, Nagarjuna; Pyland, Derek B; Puckett, Latisha M; Phan, N H; Starch, Joel A; Okimoto, Mark R; Gidden, Jennifer; Stites, Wesley E; Lay, Jackson O

2012-12-15

Protein equilibrium snapshot by hydrogen/deuterium exchange electrospray ionization mass spectrometry (PEPS-HDX-ESI-MS or PEPS) is a method recently introduced for estimating protein folding energies and rates. Herein we describe the basis for this method using both theory and new experiments. Benchmark experiments were conducted using ubiquitin because of the availability of reference data for folding and unfolding rates from NMR studies. A second set of experiments was also conducted to illustrate the surprising resilience of the PEPS to changes in HDX time, using staphylococcal nuclease and time frames ranging from a few seconds to several minutes. Theory suggests that PEPS experiments should be conducted at relatively high denaturant concentrations, where the protein folding/unfolding rates are slow with respect to HDX and the life times of both the closed and open states are long enough to be sampled experimentally. Upon deliberate denaturation, changes in folding/unfolding are correlated with associated changes in the ESI-MS signal upon fast HDX. When experiments are done quickly, typically within a few seconds, ESI-MS signals, corresponding to the equilibrium population of the native (closed) and denatured (open) states can both be detected. The interior of folded proteins remains largely un-exchanged. Amongst MS methods, the simultaneous detection of both states in the spectrum is unique to PEPS and provides a "snapshot" of these populations. The associated ion intensities are used to estimate the protein folding equilibrium constant (or the free energy change, ΔG). Linear extrapolation method (LEM) plots of derived ΔG values for each denaturant concentration can then be used to calculate ΔG in the absence of denaturant, ΔG(H(2)O). In accordance with the requirement for detection of signals for both the folded and unfolded states, this theoretical framework predicts that PEPS experiments work best at the middle of the denaturation curve where natured

Investing in International Information Exchange Activities to Improve the Safety, Cost Effectiveness and Schedule of Cleanup - 13281

International Nuclear Information System (INIS)

Seed, Ian; James, Paula; Mathieson, John; Judd, Laurie; Elmetti-Ramirez, Rosa; Han, Ana

2013-01-01

With decreasing budgets and increasing pressure on completing cleanup missions as quickly, safely and cost-effectively as possible, there is significant benefit to be gained from collaboration and joint efforts between organizations facing similar issues. With this in mind, the US Department of Energy (DOE) and the UK Nuclear Decommissioning Authority (NDA) have formally agreed to share information on lessons learned on the development and application of new technologies and approaches to improve the safety, cost effectiveness and schedule of the cleanup legacy wastes. To facilitate information exchange a range of tools and methodologies were established. These included tacit knowledge exchange through facilitated meetings, conference calls and Site visits as well as explicit knowledge exchange through document sharing and newsletters. A DOE web-based portal has been established to capture these exchanges and add to them via discussion boards. The information exchange is operating at the Government-to-Government strategic level as well as at the Site Contractor level to address both technical and managerial topic areas. This effort has resulted in opening a dialogue and building working relationships. In some areas joint programs of work have been initiated thus saving resource and enabling the parties to leverage off one another activities. The potential benefits of high quality information exchange are significant, ranging from cost avoidance through identification of an approach to a problem that has been proven elsewhere to cost sharing and joint development of a new technology to address a common problem. The benefits in outcomes significantly outweigh the costs of the process. The applicability of the tools and methods along with the lessons learned regarding some key issues is of use to any organization that wants to improve value for money. In the waste management marketplace, there are a multitude of challenges being addressed by multiple organizations and

Investing in International Information Exchange Activities to Improve the Safety, Cost Effectiveness and Schedule of Cleanup - 13281

Energy Technology Data Exchange (ETDEWEB)

Seed, Ian; James, Paula [Cogentus Consulting (United States); Mathieson, John [NDA United Kingdom (United Kingdom); Judd, Laurie [NuVision Engineering, Inc. (United States); Elmetti-Ramirez, Rosa; Han, Ana [US DOE (United States)

2013-07-01

With decreasing budgets and increasing pressure on completing cleanup missions as quickly, safely and cost-effectively as possible, there is significant benefit to be gained from collaboration and joint efforts between organizations facing similar issues. With this in mind, the US Department of Energy (DOE) and the UK Nuclear Decommissioning Authority (NDA) have formally agreed to share information on lessons learned on the development and application of new technologies and approaches to improve the safety, cost effectiveness and schedule of the cleanup legacy wastes. To facilitate information exchange a range of tools and methodologies were established. These included tacit knowledge exchange through facilitated meetings, conference calls and Site visits as well as explicit knowledge exchange through document sharing and newsletters. A DOE web-based portal has been established to capture these exchanges and add to them via discussion boards. The information exchange is operating at the Government-to-Government strategic level as well as at the Site Contractor level to address both technical and managerial topic areas. This effort has resulted in opening a dialogue and building working relationships. In some areas joint programs of work have been initiated thus saving resource and enabling the parties to leverage off one another activities. The potential benefits of high quality information exchange are significant, ranging from cost avoidance through identification of an approach to a problem that has been proven elsewhere to cost sharing and joint development of a new technology to address a common problem. The benefits in outcomes significantly outweigh the costs of the process. The applicability of the tools and methods along with the lessons learned regarding some key issues is of use to any organization that wants to improve value for money. In the waste management marketplace, there are a multitude of challenges being addressed by multiple organizations and

Orally active-targeted drug delivery systems for proteins and peptides.

Science.gov (United States)

Li, Xiuying; Yu, Miaorong; Fan, Weiwei; Gan, Yong; Hovgaard, Lars; Yang, Mingshi

2014-09-01

In the past decade, extensive efforts have been devoted to designing 'active targeted' drug delivery systems (ATDDS) to improve oral absorption of proteins and peptides. Such ATDDS enhance cellular internalization and permeability of proteins and peptides via molecular recognition processes such as ligand-receptor or antigen-antibody interaction, and thus enhance drug absorption. This review focuses on recent advances with orally ATDDS, including ligand-protein conjugates, recombinant ligand-protein fusion proteins and ligand-modified carriers. In addition to traditional intestinal active transport systems of substrates and their corresponding receptors, transporters and carriers, new targets such as intercellular adhesion molecule-1 and β-integrin are also discussed. ATDDS can improve oral absorption of proteins and peptides. However, currently, no clinical studies on ATDDS for proteins and peptides are underway, perhaps due to the complexity and limited knowledge of transport mechanisms. Therefore, more research is warranted to optimize ATDDS efficiency.

NOx reduction over metal-ion exchanged novel zeolite under lean conditions. Activity and hydrothermal stability

International Nuclear Information System (INIS)

Subbiah, Ayyappan; Gujar, Amit; Price, Geoffrey L.; Cho, Byong K.; Blint, Richard J.; Yie, Jae E.

2003-01-01

Zeolite SUZ-4 was synthesized and tested for its hydrothermal stability using a standard aging procedure coupled with NMR spectroscopy, and was identified as a promising support for lean-NO x catalysts for high temperature applications. Various metals such as Cu, Ag, Fe, Co were ion exchanged onto the SUZ-4 zeolite, and their catalytic activity for NO/NO x conversion was measured in the presence of excess oxygen using ethylene as the reducing agent. Among the metal-ions exchanged, copper proved to be the best metal cation for lean-NO x catalysis with the optimum level of exchange at 29-42%. The optimized, fresh Cu/SUZ-4 catalyst achieved 70-80% of NO/NO x conversion activity over a wide range of temperature from 350 to 600C with the maximum conversion temperature at 450C. The presence of H 2 O and SO 2 reduced the NO/NO x conversion by about 30% of the fresh Cu/SUZ-4 catalyst due possibly to the blocking of active sites for NO/NO x adsorption. Substitution of gasoline vapor for ethylene as the reductant improved the NO x reduction activity of the fresh Cu/SUZ-4 catalyst at high temperatures above 350C. Aging the Cu/SUZ-4 catalyst resulted in a slight shift of activity profile toward higher temperatures, yielding an increase of NO conversion by 16% and a decrease of NO x conversion by 15% at 525C. The effect of H 2 O and SO 2 on the aged catalyst was to reduce the NO activity by 20% and NO x activity by 30% at 500C. The effect of space velocity change was not significant except in the low temperature range where the reaction light-off occurs. Adsorption/desorption measurements indicate that aging Cu/SUZ-4 results in partial migration/agglomeration of Cu particles in the pores thereby reducing the NO/NO x activity. Overall, the NO x conversion efficiency of Cu/SUZ-4, for both fresh and aged, is much better than the benchmark Cu/ZSM-5 in the presence of H 2 O and/or SO 2