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The Effect of Different Force Applications on the Protein-Protein Complex Barnase-Barstar

Neumann, Jan; Gottschalk, Kay-Eberhard
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: text
Publicado em 16/09/2009 Português
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Steered molecular dynamics simulations are a tool to examine the energy landscape of protein-protein complexes by applying external forces. Here, we analyze the influence of the velocity and geometry of the probing forces on a protein complex using this tool. With steered molecular dynamics, we probe the stability of the protein-protein complex Barnase-Barstar. The individual proteins are mechanically labile. The Barnase-Barstar binding site is more stable than the folds of the individual proteins. By using different force protocols, we observe a variety of responses of the system to the applied tension.

Hypoxia-inducible Factor Prolyl-4-hydroxylase PHD2 Protein Abundance Depends on Integral Membrane Anchoring of FKBP38*

Barth, Sandra; Edlich, Frank; Berchner-Pfannschmidt, Utta; Gneuss, Silke; Jahreis, Günther; Hasgall, Philippe A.; Fandrey, Joachim; Wenger, Roland H.; Camenisch, Gieri
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: text
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Prolyl-4-hydroxylase domain (PHD) proteins are 2-oxoglutarate and dioxygen-dependent enzymes that mediate the rapid destruction of hypoxia-inducible factor ? subunits. Whereas PHD1 and PHD3 proteolysis has been shown to be regulated by Siah2 ubiquitin E3 ligase-mediated polyubiquitylation and proteasomal destruction, protein regulation of the main oxygen sensor responsible for hypoxia-inducible factor ? regulation, PHD2, remained unknown. We recently reported that the FK506-binding protein (FKBP) 38 specifically interacts with PHD2 and determines PHD2 protein stability in a peptidyl-prolyl cis-trans isomerase-independent manner. Using peptide array binding assays, fluorescence spectroscopy, and fluorescence resonance energy transfer analysis, we defined a minimal linear glutamate-rich PHD2 binding domain in the N-terminal part of FKBP38 and showed that this domain forms a high affinity complex with PHD2. Vice versa, PHD2 interacted with a non-linear N-terminal motif containing the MYND (myeloid, Nervy, and DEAF-1)-type Zn2+ finger domain with FKBP38. Biochemical fractionation and immunofluorescence analysis demonstrated that PHD2 subcellular localization overlapped with FKBP38 in the endoplasmic reticulum and mitochondria. An additional fraction of PHD2 was found in the cytoplasm. In cellulo PHD2/FKBP38 association...

Phosphorylation Status of Nuclear Ribosomal Protein S3 Is Reciprocally Regulated by Protein Kinase C? and Protein Phosphatase 2A*

Kim, Tae-Sung; Kim, Hag Dong; Shin, Hyun-Seock; Kim, Joon
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: text
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It has been shown previously that ribosomal protein S3 (rpS3) has an endonuclease activity, which is increased by protein kinase C? (PKC?)-dependent phosphorylation. However, the reciprocal mechanism for rpS3 dephosphorylation is not known. In this study, we examined phosphatases involved in rpS3 dephosphorylation, and we determined that rpS3 is specifically dephosphorylated by protein phosphatase 2A (PP2A). By immunoprecipitation assay, rpS3 only interacted with PP2Ac but not with protein phosphatase 1. The interaction between rpS3 and PP2Ac occurred only in the nuclear fraction. Moreover, the PP2Ac association with rpS3 was identified in cells transfected with wild-type rpS3 but not with mutant rpS3 lacking PKC? phosphorylation sites. PP2A inhibition using okadaic acid induced rpS3 phosphorylation. The level of phosphorylated rpS3 in cells was decreased by the overexpression of PP2Ac and was increased by the down-regulation of PP2Ac. Taken together, these results suggest that oxidative stress regulates the phosphorylation status of nonribosomal rpS3 by both activating PKC? and blocking the PP2A interaction with rpS3.

Electrophilic Affibodies Forming Covalent Bonds to Protein Targets*

Holm, Lotta; Moody, Paul; Howarth, Mark
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: text
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Antibody affinity limits sensitivity of detection in many areas of biology and medicine. High affinity usually depends on achieving the optimal combination of the natural 20 amino acids in the antibody binding site. Here, we investigate the effect on recognition of protein targets of placing an unnatural electrophile adjacent to the target binding site. We positioned a weak electrophile, acrylamide, near the binding site between an affibody, a non-immunoglobulin binding scaffold, and its protein target. The proximity between cysteine, lysine, or histidine on the target protein drove covalent bond formation to the electrophile on the affibody. Covalent bonds did not form to a non-interacting point mutant of the target, and there was minimal cross-reactivity with serum, cell lysate, or when imaging at the cell surface. Electrophilic affibodies showed more stable protein imaging at the surface of mammalian cells, and the sensitivity of protein detection in an immunoassay improved by two orders of magnitude. Thus electrophilic affibodies combined good specificity with improved detection of protein targets.

Ligand Binding Turns Moth Pheromone-binding Protein into a pH Sensor: EFFECT ON THE ANTHERAEA POLYPHEMUS PBP1 CONFORMATION*

Katre, Uma V.; Mazumder, Suman; Prusti, Rabi K.; Mohanty, Smita
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: text
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In moths, pheromone-binding proteins (PBPs) are responsible for the transport of the hydrophobic pheromones to the membrane-bound receptors across the aqueous sensillar lymph. We report here that recombinant Antheraea polyphemus PBP1 (ApolPBP1) picks up hydrophobic molecule(s) endogenous to the Escherichia coli expression host that keeps the protein in the “open” (bound) conformation at high pH but switches to the “closed” (free) conformation at low pH. This finding has bearing on the solution structures of undelipidated lepidopteran moth PBPs determined thus far. Picking up a hydrophobic molecule from the host expression system could be a common feature for lipid-binding proteins. Thus, delipidation is critical for bacterially expressed lipid-binding proteins. We have shown for the first time that the delipidated ApolPBP1 exists primarily in the closed form at all pH levels. Thus, current views on the pH-induced conformational switch of PBPs hold true only for the ligand-bound open conformation of the protein. Binding of various ligands to delipidated ApolPBP1 studied by solution NMR revealed that the protein in the closed conformation switches to the open conformation only at or above pH 6.0 with a protein to ligand stoichiometry of ?1:1. Mutation of His70 and His95 to alanine drives the equilibrium toward the open conformation even at low pH for the ligand-bound protein by eliminating the histidine-dependent pH-induced conformational switch. Thus...

The Importance of Protein-Protein Interactions on the pH-Induced Conformational Changes of Bovine Serum Albumin: A Small-Angle X-Ray Scattering Study

Barbosa, Leandro R.S.; Ortore, Maria Grazia; Spinozzi, Francesco; Mariani, Paolo; Bernstorff, Sigrid; Itri, Rosangela
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: text
Publicado em 06/01/2010 Português
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The combined effects of concentration and pH on the conformational states of bovine serum albumin (BSA) are investigated by small-angle x-ray scattering. Serum albumins, at physiological conditions, are found at concentrations of ?35–45 mg/mL (42 mg/mL in the case of humans). In this work, BSA at three different concentrations (10, 25, and 50 mg/mL) and pH values (2.0–9.0) have been studied. Data were analyzed by means of the Global Fitting procedure, with the protein form factor calculated from human serum albumin (HSA) crystallographic structure and the interference function described, considering repulsive and attractive interaction potentials within a random phase approximation. Small-angle x-ray scattering data show that BSA maintains its native state from pH 4.0 up to 9.0 at all investigated concentrations. A pH-dependence of the absolute net protein charge is shown and the charge number per BSA is quantified to 10(2), 8(1), 13(2), 20(2), and 26(2) for pH values 4.0, 5.4, 7.0, 8.0, and 9.0, respectively. The attractive potential diminishes as BSA concentration increases. The coexistence of monomers and dimers is observed at 50 mg/mL and pH 5.4, near the BSA isoelectric point. Samples at pH 2.0 show a different behavior, because BSA overall shape changes as a function of concentration. At 10 mg/mL...

Water and Backbone Dynamics in a Hydrated Protein

Diakova, Galina; Goddard, Yanina A.; Korb, Jean-Pierre; Bryant, Robert G.
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: text
Publicado em 06/01/2010 Português
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Rotational immobilization of proteins permits characterization of the internal peptide and water molecule dynamics by magnetic relaxation dispersion spectroscopy. Using different experimental approaches, we have extended measurements of the magnetic field dependence of the proton-spin-lattice-relaxation rate by one decade from 0.01 to 300 MHz for 1H and showed that the underlying dynamics driving the protein 1H spin-lattice relaxation is preserved over 4.5 decades in frequency. This extension is critical to understanding the role of 1H2O in the total proton-spin-relaxation process. The fact that the protein-proton-relaxation-dispersion profile is a power law in frequency with constant coefficient and exponent over nearly 5 decades indicates that the characteristics of the native protein structural fluctuations that cause proton nuclear spin-lattice relaxation are remarkably constant over this wide frequency and length-scale interval. Comparison of protein-proton-spin-lattice-relaxation rate constants in protein gels equilibrated with 2H2O rather than 1H2O shows that water protons make an important contribution to the total spin-lattice relaxation in the middle of this frequency range for hydrated proteins because of water molecule dynamics in the time range of tens of ns. This water contribution is with the motion of relatively rare...

Multiple Molecules of Hsc70 and a Dimer of DjA1 Independently Bind to an Unfolded Protein*

Terada, Kazutoyo; Oike, Yuichi
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: text
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Protein folding is a prominent chaperone function of the Hsp70 system. Refolding of an unfolded protein is efficiently mediated by the Hsc70 system with either type 1 DnaJ protein, DjA1 or DjA2, and a nucleotide exchange factor. A surface plasmon resonance technique was applied to investigate substrate recognition by the Hsc70 system and demonstrated that multiple Hsc70 proteins and a dimer of DjA1 initially bind independently to an unfolded protein. The association rate of the Hsc70 was faster than that of DjA1 under folding-compatible conditions. The Hsc70 binding involved a conformational change, whereas the DjA1 binding was bivalent and substoichiometric. Consistently, we found that the bound 14C-labeled Hsc70 to the unfolded protein became more resistant to tryptic digestion. The gel filtration and cross-linking experiments revealed the predominant presence of the DjA1 dimer. Furthermore, the Hsc70 and DjA1 bound to distinct sets of peptide array sequences. All of these findings argue against the generality of the widely proposed hypothesis that the DnaJ-bound substrate is targeted and transferred to Hsp70. Instead, these results suggest the importance of the bivalent binding of DjA1 dimer that limits unfavorable transitions of substrate conformations in protein folding.

The Plastic Energy Landscape of Protein Folding: A TRIANGULAR FOLDING MECHANISM WITH AN EQUILIBRIUM INTERMEDIATE FOR A SMALL PROTEIN DOMAIN*

Haq, S. Raza; Jürgens, Maike C.; Chi, Celestine N.; Koh, Cha-San; Elfström, Lisa; Selmer, Maria; Gianni, Stefano; Jemth, Per
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: text
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Protein domains usually fold without or with only transiently populated intermediates, possibly to avoid misfolding, which could result in amyloidogenic disease. Whether observed intermediates are productive and obligatory species on the folding reaction pathway or dispensable by-products is a matter of debate. Here, we solved the crystal structure of a small protein domain, SAP97 PDZ2 I342W C378A, and determined its folding pathway. The presence of a folding intermediate was demonstrated both by single and double-mixing kinetic experiments using urea-induced (un)folding as well as ligand-induced folding. This protein domain was found to fold via a triangular scheme, where the folding intermediate could be either on- or off-pathway, depending on the experimental conditions. Furthermore, we found that the intermediate was present at equilibrium, which is rarely seen in folding reactions of small protein domains. The folding mechanism observed here illustrates the roughness and plasticity of the protein folding energy landscape, where several routes may be employed to reach the native state. The results also reconcile the folding mechanisms of topological variants within the PDZ domain family.

The Novel Membrane Protein TMEM59 Modulates Complex Glycosylation, Cell Surface Expression, and Secretion of the Amyloid Precursor Protein*

Ullrich, Sylvia; Münch, Anna; Neumann, Stephanie; Kremmer, Elisabeth; Tatzelt, Jörg; Lichtenthaler, Stefan F.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: text
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Ectodomain shedding of the amyloid precursor protein (APP) by the two proteases ?- and ?-secretase is a key regulatory event in the generation of the Alzheimer disease amyloid ? peptide (A?). At present, little is known about the cellular mechanisms that control APP shedding and A? generation. Here, we identified a novel protein, transmembrane protein 59 (TMEM59), as a new modulator of APP shedding. TMEM59 was found to be a ubiquitously expressed, Golgi-localized protein. TMEM59 transfection inhibited complex N- and O-glycosylation of APP in cultured cells. Additionally, TMEM59 induced APP retention in the Golgi and inhibited A? generation as well as APP cleavage by ?- and ?-secretase cleavage, which occur at the plasma membrane and in the endosomes, respectively. Moreover, TMEM59 inhibited the complex N-glycosylation of the prion protein, suggesting a more general modulation of Golgi glycosylation reactions. Importantly, TMEM59 did not affect the secretion of soluble proteins or the ?-secretase like shedding of tumor necrosis factor ?, demonstrating that TMEM59 did not disturb the general Golgi function. The phenotype of TMEM59 transfection on APP glycosylation and shedding was similar to the one observed in cells lacking conserved oligomeric Golgi (COG) proteins COG1 and COG2. Both proteins are required for normal localization and activity of Golgi glycosylation enzymes. In summary...

Attractive Protein-Polymer Interactions Markedly Alter the Effect of Macromolecular Crowding on Protein Association Equilibria

Jiao, Ming; Li, Hong-Tao; Chen, Jie; Minton, Allen P.; Liang, Yi
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: text
Publicado em 04/08/2010 Português
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The dependence of the fluorescence of catalase upon the concentration of added superoxide dismutase (SOD) indicates that SOD binds to saturable sites on catalase. The affinity of SOD for these sites varies with temperature, and with the concentration of each of three nominally inert polymeric additives—dextran 70, Ficoll 70, and polyethylene glycol 2000. At room temperature (25.0°C) and higher, the addition of high concentrations of polymer is found to significantly enhance the affinity of SOD for catalase, but with decreasing temperature the enhancing effect of polymer addition diminishes, and at 8.0°C, addition of polymer has little or no effect on the affinity of SOD for catalase. The results presented here provide the first experimental evidence for the existence of competition between a repulsive excluded volume interaction between protein and polymer, which tends to enhance association of dilute protein, and an attractive interaction between protein and polymer, which tends to inhibit protein association. The net effect of high concentrations of polymer upon protein associations depends upon the relative strength of these two types of interactions at the temperature of measurement, and may vary significantly between different proteins and/or polymers.

Experimental Evolution of Adenylate Kinase Reveals Contrasting Strategies toward Protein Thermostability

Miller, Corwin; Davlieva, Milya; Wilson, Corey; White, Kristopher I.; Couñago, Rafael; Wu, Gang; Myers, Jeffrey C.; Wittung-Stafshede, Pernilla; Shamoo, Yousif
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: text
Publicado em 04/08/2010 Português
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Success in evolution depends critically upon the ability of organisms to adapt, a property that is also true for the proteins that contribute to the fitness of an organism. Successful protein evolution is enhanced by mutational pathways that generate a wide range of physicochemical mechanisms to adaptation. In an earlier study, we used a weak-link method to favor changes to an essential but maladapted protein, adenylate kinase (AK), within a microbial population. Six AK mutants (a single mutant followed by five double mutants) had success within the population, revealing a diverse range of adaptive strategies that included changes in nonpolar packing, protein folding dynamics, and formation of new hydrogen bonds and electrostatic networks. The first mutation, AKBSUB Q199R, was essential in defining the structural context that facilitated subsequent mutations as revealed by a considerable mutational epistasis and, in one case, a very strong dependence upon the order of mutations. Namely, whereas the single mutation AKBSUB G213E decreases protein stability by >25°C, the same mutation in the background of AKBSUB Q199R increases stability by 3.4°C, demonstrating that the order of mutations can play a critical role in favoring particular molecular pathways to adaptation. In turn...

Interaction Energy Based Protein Structure Networks

Vijayabaskar, M.S.; Vishveshwara, Saraswathi
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: text
Publicado em 01/12/2010 Português
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The three-dimensional structure of a protein is formed and maintained by the noncovalent interactions among the amino-acid residues of the polypeptide chain. These interactions can be represented collectively in the form of a network. So far, such networks have been investigated by considering the connections based on distances between the amino-acid residues. Here we present a method of constructing the structure network based on interaction energies among the amino-acid residues in the protein. We have investigated the properties of such protein energy-based networks (PENs) and have shown correlations to protein structural features such as the clusters of residues involved in stability, formation of secondary and super-secondary structural units. Further we demonstrate that the analysis of PENs in terms of parameters such as hubs and shortest paths can provide a variety of biologically important information, such as the residues crucial for stabilizing the folded units and the paths of communication between distal residues in the protein. Finally, the energy regimes for different levels of stabilization in the protein structure have clearly emerged from the PEN analysis.

Biophysical Characterization of the Complex between Human Papillomavirus E6 Protein and Synapse-associated Protein 97*?

Chi, Celestine N.; Bach, Anders; Engström, Åke; Strømgaard, Kristian; Lundström, Patrik; Ferguson, Neil; Jemth, Per
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: text
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The E6 protein of human papillomavirus (HPV) exhibits complex interaction patterns with several host proteins, and their roles in HPV-mediated oncogenesis have proved challenging to study. Here we use several biophysical techniques to explore the binding of E6 to the three PDZ domains of the tumor suppressor protein synapse-associated protein 97 (SAP97). All of the potential binding sites in SAP97 bind E6 with micromolar affinity. The dissociation rate constants govern the different affinities of HPV16 and HPV18 E6 for SAP97. Unexpectedly, binding is not mutually exclusive, and all three PDZ domains can simultaneously bind E6. Intriguingly, this quaternary complex has the same apparent hydrodynamic volume as the unliganded PDZ region, suggesting that a conformational change occurs in the PDZ region upon binding, a conclusion supported by kinetic experiments. Using NMR, we discovered a new mode of interaction between E6 and PDZ: a subset of residues distal to the canonical binding pocket in the PDZ2 domain exhibited noncanonical interactions with the E6 protein. This is consistent with a larger proportion of the protein surface defining binding specificity, as compared with that reported previously.

Crystal structure of the Leishmania major MIX protein: A scaffold protein that mediates protein–protein interactions

Gorman, Michael A; Uboldi, Alex D; Walsh, Peter J; Tan, Kher Shing; Hansen, Guido; Huyton, Trevor; Ji, Hong; Curtis, Joan; Kedzierski, Lukasz; Papenfuss, Anthony T; Dogovski, Con; Perugini, Matthew A; Simpson, Richard J; Handman, Emanuela; Parker, Michael
Fonte: Wiley Subscription Services, Inc., A Wiley Company Publicador: Wiley Subscription Services, Inc., A Wiley Company
Tipo: text
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Infection by Leishmania and Trypanosoma causes severe disease and can be fatal. The reduced effectiveness of current treatments is largely due to drug resistance, hence the urgent need to develop new drugs, preferably against novel targets. We have recently identified a mitochondrial membrane-anchored protein, designated MIX, which occurs exclusively in these parasites and is essential for virulence. We have determined the crystal structure of Leishmania major MIX to a resolution of 2.4 Å. MIX forms an all ?-helical fold comprising seven ?-helices that fold into a single domain. The distribution of helices is similar to a number of scaffold proteins, namely HEAT repeats, 14-3-3, and tetratricopeptide repeat proteins, suggesting that MIX mediates protein–protein interactions. Accordingly, using copurification and mass spectroscopy we were able to identify several proteins that may interact with MIX in vivo. Being parasite specific, MIX is a promising new drug target and, thus, the structure and potential interacting partners provide a basis for structure-guided drug discovery.

Enhanced Biosynthesis of Coagulation Factor VIII through Diminished Engagement of the Unfolded Protein Response*

Brown, Harrison C.; Gangadharan, Bagirath; Doering, Christopher B.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: text
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Human and porcine coagulation factor VIII (fVIII) display a biosynthetic efficiency differential that is being exploited for the development of new protein and gene transfer-based therapies for hemophilia A. The cellular and/or molecular mechanism(s) responsible for this phenomenon have yet to be uncovered, although it has been temporally localized to post-translational biosynthetic steps. The unfolded protein response (UPR) is a cellular adaptation to structurally distinct (e.g. misfolded) or excess protein in the endoplasmic reticulum and is known to be induced by heterologous expression of recombinant human fVIII. Therefore, it is plausible that the biosynthetic differential between human and porcine fVIII results from differential UPR activation. In the current study, UPR induction was examined in the context of ongoing fVIII expression. UPR activation was greater during human fVIII expression when compared with porcine fVIII expression as determined by ER response element (ERSE)-luciferase reporter activity, X-box-binding protein 1 (XBP1) splicing, and immunoglobulin-binding protein (BiP) up-regulation. Immunofluorescence microscopy of fVIII expressing cells revealed that human fVIII was notably absent in the Golgi apparatus, confirming that endoplasmic reticulum to Golgi transport is rate-limiting. In contrast...

Moving Iron through Ferritin Protein Nanocages Depends on Residues throughout Each Four ?-Helix Bundle Subunit*

Haldar, Suranjana; Bevers, Loes E.; Tosha, Takehiko; Theil, Elizabeth C.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: text
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Eukaryotic H ferritins move iron through protein cages to form biologically required, iron mineral concentrates. The biominerals are synthesized during protein-based Fe2+/O2 oxidoreduction and formation of [Fe3+O]n multimers within the protein cage, en route to the cavity, at sites distributed over ?50 ?. Recent NMR and Co2+-protein x-ray diffraction (XRD) studies identified the entire iron path and new metal-protein interactions: (i) lines of metal ions in 8 Fe2+ ion entry channels with three-way metal distribution points at channel exits and (ii) interior Fe3+O nucleation channels. To obtain functional information on the newly identified metal-protein interactions, we analyzed effects of amino acid substitution on formation of the earliest catalytic intermediate (diferric peroxo-A650 nm) and on mineral growth (Fe3+O-A350 nm), in A26S, V42G, D127A, E130A, and T149C. The results show that all of the residues influenced catalysis significantly (p < 0.01), with effects on four functions: (i) Fe2+ access/selectivity to the active sites (Glu130), (ii) distribution of Fe2+ to each of the three active sites near each ion channel (Asp127), (iii) product (diferric oxo) release into the Fe3+O nucleation channels (Ala26), and (iv) [Fe3+O]n transit through subunits (Val42...

Torsion Stiffness of a Protein Pair Determined by Magnetic Particles

Janssen, X.J.A.; van Noorloos, J.M.; Jacob, A.; van IJzendoorn, L.J.; de Jong, A.M.; Prins, M.W.J.
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: text
Publicado em 04/05/2011 Português
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We demonstrate the ability to measure torsion stiffness of a protein complex by applying a controlled torque on a magnetic particle. As a model system we use protein G bound to an IgG antibody. The protein pair is held between a magnetic particle and a polystyrene substrate. The angular orientation of the magnetic particle shows an oscillating behavior upon application of a rotating magnetic field. The amplitude of the oscillation increases with a decreasing surface coverage of antibodies on the substrate and with an increasing magnitude of the applied field. For decreasing antibody coverage, the torsion spring constant converges to a minimum value of 1.5 × 103 pN·nm/rad that corresponds to a torsion modulus of 4.5 × 104 pN·nm2. This torsion stiffness is an upper limit for the molecular bond between the particle and the surface that is tentatively assigned to a single protein G–IgG protein pair. This assignment is supported by interpreting the measured stiffness with a simple mechanical model that predicts a two orders of magnitude larger stiffness for the protein G–IgG complex than values found for micrometer length dsDNA. This we understand from the structural properties of the molecules, i.e., DNA is a long and flexible chain-like molecule...

A Histidine-rich and Cysteine-rich Metal-binding Domain at the C Terminus of Heat Shock Protein A from Helicobacter pylori: IMPLICATION FOR NICKEL HOMEOSTASIS AND BISMUTH SUSCEPTIBILITY*S?

Cun, Shujian; Li, Hongyan; Ge, Ruiguang; Lin, Marie C. M.; Sun, Hongzhe
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: text
Publicado em 30/05/2008 Português
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HspA, a member of the GroES chaperonin family, is a small protein found in Helicobacter pylori with a unique histidine- and cysteine-rich domain at the C terminus. In this work, we overexpressed, purified, and characterized this protein both in vitro and in vivo. The apo form of the protein binds 2.10 ± 0.07 Ni2+ or 1.98 ± 0.08 Bi3+ ions/monomer with a dissociation constant (Kd) of 1.1 or 5.9 × 10-19 ?m, respectively. Importantly, Ni2+ can reversibly bind to the protein, as the bound nickel can be released either in the presence of a chelating ligand, e.g. EDTA, or at an acidic pH (pH½ 3.8 ± 0.2). In contrast, Bi3+ binds almost irreversibly to the protein. Both gel filtration chromatography and native electrophoresis demonstrated that apo-HspA exists as a heptamer in solution. Unexpectedly, binding of Bi3+ to the protein altered its quaternary structure from a heptamer to a dimer, indicating that bismuth may interfere with the biological functions of HspA. When cultured in Ni2+-supplemented M9 minimal medium, Escherichia coli BL21(DE3) cells expressing wild-type HspA or the C-terminal deletion mutant clearly indicated that the C terminus might protect cells from high concentrations of external Ni2+. However, an opposite phenomenon was observed when the same E. coli hosts were grown in Bi3+-supplemented medium. HspA may therefore play a dual role: to facilitate nickel acquisition by donating Ni2+ to appropriate proteins in a nickel-deficient environment and to carry out detoxification via sequestration of excess nickel. Meanwhile...

Solution Model of the Intrinsically Disordered Polyglutamine Tract-Binding Protein-1

Rees, Martin; Gorba, Christian; de Chiara, Cesira; Bui, Tam T.T.; Garcia-Maya, Mitla; Drake, Alex F.; Okazawa, Hitoshi; Pastore, Annalisa; Svergun, Dmitri; Chen, Yu Wai
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: text
Publicado em 04/04/2012 Português
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Polyglutamine tract-binding protein-1 (PQBP-1) is a 265-residue nuclear protein that is involved in transcriptional regulation. In addition to its role in the molecular pathology of the polyglutamine expansion diseases, mutations of the protein are associated with X-linked mental retardation. PQBP-1 binds specifically to glutamine repeat sequences and proline-rich regions, and interacts with RNA polymerase II and the spliceosomal protein U5-15kD. In this work, we obtained a biophysical characterization of this protein by employing complementary structural methods. PQBP-1 is shown to be a moderately compact but largely disordered molecule with an elongated shape, having a Stokes radius of 3.7 nm and a maximum molecular dimension of 13 nm. The protein is monomeric in solution, has residual ?-structure, and is in a premolten globule state that is unaffected by natural osmolytes. Using small-angle x-ray scattering data, we were able to generate a low-resolution, three-dimensional model of PQBP-1.