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ABSTRACT: 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.
PLoS ONE 01/2013; 8(3):e59175. · 4.09 Impact Factor
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ABSTRACT: Key synaptic proteins from the soluble SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) family, among many others, are organized at the plasma membrane of cells as clusters containing dozens to hundreds of protein copies. However, the exact membranal distribution of proteins into clusters or as single molecules, the organization of molecules inside the clusters, and the clustering mechanisms are unclear due to limitations of the imaging and analytical tools. Focusing on syntaxin 1 and SNAP-25, we implemented direct stochastic optical reconstruction microscopy together with quantitative clustering algorithms to demonstrate a novel approach to explore the distribution of clustered and nonclustered molecules at the membrane of PC12 cells with single-molecule precision. Direct stochastic optical reconstruction microscopy images reveal, for the first time, solitary syntaxin/SNAP-25 molecules and small clusters as well as larger clusters. The nonclustered syntaxin or SNAP-25 molecules are mostly concentrated in areas adjacent to their own clusters. In the clusters, the density of the molecules gradually decreases from the dense cluster core to the periphery. We further detected large clusters that contain several density gradients. This suggests that some of the clusters are formed by unification of several clusters that preserve their original organization or reorganize into a single unit. Although syntaxin and SNAP-25 share some common distributional features, their clusters differ markedly from each other. SNAP-25 clusters are significantly larger, more elliptical, and less dense. Finally, this study establishes methodological tools for the analysis of single-molecule-based super-resolution imaging data and paves the way for revealing new levels of membranal protein organization.
Journal of Biological Chemistry 06/2012; 287(32):27158-67. · 4.77 Impact Factor
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ABSTRACT: The small GTPase proteins are components of the intracellular signaling system, alternating between active (membrane-bound and GTP-loaded) and inactive (GDP-loaded and cytosolic) states. In the inactive state, the proteins are soluble in the cytoplasm. To compensate for the energetic penalty of extraction of the hydrophobic moiety from the membrane phase, the inactive state is stabilized via formation of a complex with the RhoGDI proteins that provide a hydrophobic pocket for the binding of the hydrophobic moieties. The signals delivered by the Rho subfamily involve a specific, short, highly exposed α-helix (Rho-insert), located close to the GDP binding site. Upon simulating the complex in solution, we observed that the Rho-insert domain of Cdc42 can assume two basic orientations. One is the canonical one, as detected in both crystals and NMR spectra of concentrated protein solutions. The second orientation appears only in the RhoGDI-Cdc42 complex where the GER moiety of Cdc42 is properly inserted into the specific binding site of RhoGDI. Any impairment of the GER-RhoGDI interactions, such as insertion of specific mutations in the hydrophobic binding site, abolished the coupling between the proteins and the Rho-insert domain, preserving its canonical orientation as in the crystalline structure. The noncanonical conformation of the Rho-insert domain is not a simulation artifact, as it appears in crystals of plant Rho proteins (ROP4, ROP5, and ROP7). In accord with the notion that the Rho-insert domain participates in downstream signaling, we propose that the deformation of the Rho-insert is part of the signal transmissions.
Biochemistry 12/2011; 51(2):715-23. · 3.42 Impact Factor
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ABSTRACT: Time-resolved fluorescence decay of flavin adenine dinucleotide (FAD) was studied at room temperature in water and water-methanol mixtures by a fluorescence upconversion technique. The observations were focused on the most initial decay phase (200 ps), before the residual fluorescence assumes a single exponential decay, typical for an extended conformation of the fluorophore. Within the first few picoseconds, where most of the electron transfer coupled quenching takes place, the emission decay curves could be fitted by a stretched exponent, compatible with the inhomogeneous distance dependent electron transfer model. This implies that the population of the excited FAD molecules exhibits a large number of non-identical states, each with its own separation between the donor (adenine) and acceptor (isoalloxazine) moieties, having its own rate of electron transfer. To evaluate the distribution of the separation between the donor-acceptor pair, we carried out molecular dynamics simulations of closed conformation of the FAD in water and water-methanol mixtures, sampling the structure at 10 fs intervals. The analysis of the dynamics reveals that within the 4 ps time frame, where most of the nonexponential fluorescence relaxation takes place, the relative motion of the donor-acceptor pair is consistent with a one-dimensional Brownian motion, where the diffusion coefficient and the shape of the confining potential well are solvent dependent. The presence of methanol enhances the diffusion constant and widens the width of the potential well. On the basis of these parameters, the relaxation dynamics was accurately reconstructed as an electron transfer reaction in an inhomogeneous system where the reactants are diffusing within the time frame of the observation.
Physical Chemistry Chemical Physics 07/2011; 13(25):12058-66. · 3.57 Impact Factor
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ABSTRACT: The Sec1/munc18 protein family is essential for vesicle fusion in eukaryotic cells via binding to SNARE proteins. Protein kinase C modulates these interactions by phosphorylating munc18a thereby reducing its affinity to one of the central SNARE members, syntaxin-1a. The established hypothesis is that the reduced affinity of the phosphorylated munc18a to syntaxin-1a is a result of local electrostatic repulsion between the two proteins, which interferes with their compatibility. The current study challenges this paradigm and offers a novel mechanistic explanation by revealing a syntaxin-non-binding conformation of munc18a that is induced by the phosphomimetic mutations. In the present study, using molecular dynamics simulations, we explored the dynamics of the wild-type munc18a versus phosphomimetic mutant munc18a. We focused on the structural changes that occur in the cavity between domains 3a and 1, which serves as the main syntaxin-binding site. The results of the simulations suggest that the free wild-type munc18a exhibits a dynamic equilibrium between several conformations differing in the size of its cavity (the main syntaxin-binding site). The flexibility of the cavity's size might facilitate the binding or unbinding of syntaxin. In silico insertion of phosphomimetic mutations into the munc18a structure induces the formation of a conformation where the syntaxin-binding area is rigid and blocked as a result of interactions between residues located on both sides of the cavity. Therefore, we suggest that the reduced affinity of the phosphomimetic mutant/phosphorylated munc18a is a result of the closed-cavity conformation, which makes syntaxin binding energetically and sterically unfavorable. The current study demonstrates the potential of phosphorylation, an essential biological process, to serve as a driving force for dramatic conformational changes of proteins modulating their affinity to target proteins.
PLoS Computational Biology 03/2011; 7(3):e1001097. · 5.22 Impact Factor
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ABSTRACT: The encounter of a Ca(2+) ion with a protein and its subsequent binding to specific binding sites is an intricate process that cannot be fully elucidated from experimental observations. We have applied Molecular Dynamics to study this process with atomistic details, using Calbindin D9k (CaB) as a model protein. The simulations show that in most of the time the Ca(2+) ion spends within the Debye radius of CaB, it is being detained at the 1st and 2nd solvation shells. While being detained near the protein, the diffusion coefficient of the ion is significantly reduced. However, due to the relatively long period of detainment, the ion can scan an appreciable surface of the protein. The enhanced propagation of the ion on the surface has a functional role: significantly increasing the ability of the ion to scan the protein's surface before being dispersed to the bulk. The contribution of this mechanism to Ca(2+) binding becomes significant at low ion concentrations, where the intervals between successive encounters with the protein are getting longer. The efficiency of the surface diffusion is affected by the distribution of charges on the protein's surface. Comparison of the Ca(2+) binding dynamics in CaB and its E60D mutant reveals that in the wild type (WT) protein the carboxylate of E60 function as a preferred landing-site for the Ca(2+) arriving from the bulk, followed by delivering it to the final binding site. Replacement of the glutamate by aspartate significantly reduced the ability to transfer Ca(2+) ions from D60 to the final binding site, explaining the observed decrement in the affinity of the mutated protein to Ca(2+).
PLoS ONE 01/2011; 6(2):e14718. · 4.09 Impact Factor
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ABSTRACT: The structural and the dynamic features of the Calbindin (CaB) protein in its holo and apo states are compared using molecular dynamics simulations under nine different force fields (FFs) (G43a1, G53a6, Opls-AA, Amber94, Amber99, Amber99p, AmberGS, AmberGSs, and Amber99sb). The results show that most FFs reproduce reasonably well the majority of the experimentally derived features of the CaB protein. However, in several cases, there are significant differences in secondary structure properties, root mean square deviations (RMSDs), root mean square fluctuations (RMSFs), and S(2) order parameters among the various FFs. What is more, in certain cases, these parameters differed from the experimentally derived values. Some of these deviations became noticeable only after 50 ns. A comparison with experimental data indicates that, for CaB, the Amber94 shows overall best agreement with the measured values, whereas several others seem to deviate from both crystal and nuclear magnetic resonance data.
Journal of Computational Chemistry 07/2010; 31(9):1864-72. · 4.58 Impact Factor
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ABSTRACT: The excited state of flavin adenine dinucleotide (FAD), dissolved in water, is subjected to intensive quenching due to electron transfer from the adenine moiety to the excited isoalloxazine ring. Increasing the methanol concentration in the solution enhances the quantum yield of the fluorescence. In the present study, we carried out molecular dynamics simulations of FAD in explicit water and water-methanol mixtures over time frames of hundreds of nanoseconds. The simulations record rapid structural fluctuations of the molecule, where the distance between the centers of mass (COMs) of the two nucleotides varied from contact distance (folded) up to fully extended (open) structure. The methanol affected the dynamics of the FAD by enhancing the frequency of unfolding events without any effect on the lifetime of the open state. The correlation of the molecular dynamics simulations with fluorescence titration of the FAD in water/methanol mixtures indicates that the internal quenching takes place when the distance between COMs is <5.5 +/- 0.5 A.
The Journal of Physical Chemistry A 12/2009; 114(2):1017-22. · 2.95 Impact Factor
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ABSTRACT: The translocation of proteins from the cytosol into the mitochondrial matrix is mediated by the coordinated action of the TOM complex in the outer membrane, as well as the TIM23 complex and its associated protein import motor in the inner membrane. The focus of this work is the peripheral inner membrane protein Tim44. Tim44 is a vital component of the mitochondrial protein translocation motor that anchors components of the motor to the TIM23 complex. For this purpose, Tim44 associates with the import channel by direct interaction with the Tim23 protein. Additionally, it was shown in vitro that Tim44 associates with acidic model membranes, in particular those containing cardiolipin. The latter interaction was shown to be mediated by the carboxy-terminal domain of Tim44 [Weiss, C., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8890-8894]. The aim of this study was to determine the precise recognition site for negative lipids in the C-terminal domain of Tim44. In particular, we wanted to examine the recently suggested hypothesis that acidic phospholipids associate with Tim44 via a hydrophobic cavity that is observed in the high-resolution structure of the C-terminal domain of the protein [Josyula, R., et al. (2006) J. Mol. Biol. 359, 798-804]. Molecular dynamics simulations suggest that (i) the hydrophobic tail of lipids may interact with Tim44 via the latter's hydrophobic cavity and (ii) a region, located in the N-terminal alpha-helix of the C-terminal domain (helices A1 and A2), may serve as a membrane attachment site. To validate this assumption, N-terminal truncations of yeast Tim44 were examined for their ability to bind cardiolipin-containing phospholipid vesicles. The results indicate that removal of the N-terminal alpha-helix (helix A1) abolishes the capacity of Tim44 to associate with cardiolipin-containing liposomes. We suggest that helices A1 and A2, in Tim44, jointly promote the association of the protein with acidic phospholipids.
Biochemistry 11/2009; 48(47):11185-95. · 3.42 Impact Factor
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ABSTRACT: Fatty acid binding proteins (FABPs), are evolutionarily conserved small cytoplasmic proteins that occur in many tissue-specific types. One of their primary functions is to facilitate the clearance of the cytoplasmic matrix from free fatty acids and of other detergent-like compounds. Crystallographic studies of FABP proteins have revealed a well defined binding site located deep inside their beta-clam structure that is hardly exposed to the bulk solution. However, NMR measurements revealed that, when the protein is equilibrated with its ligands, residues that are clearly located on the outer surface of the protein do interact with the ligand. To clarify this apparent contradiction we applied molecular dynamics simulations to follow the initial steps associated with the FABP-fatty acid interaction using, as a model, the interaction of toad liver basic FABP, or chicken liver bile acid binding protein, with a physiological concentration of palmitate ions. The simulations (approximately 200 ns of accumulated time) show that fatty acid molecules interact, unevenly, with various loci on the protein surface, with the favored regions being the portal and the anti-portal domains. Random encounters with palmitate at these regions led to lasting adsorption to the surface, while encounters at the outer surface of the beta-clam were transient. Therefore, we suggest that the protein surface is capable of sequestering free fatty acids from solution, where brief encounters evolve into adsorbed states, which later mature by migration of the ligand into a more specific binding site.
Journal of Molecular Modeling 10/2009; 16(5):929-38. · 1.80 Impact Factor
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ABSTRACT: The soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein (SNAP) receptor (SNARE) protein syntaxin 1A forms nano-sized clusters (membrane rafts) on the plasma membrane (PM) that are in equilibrium with freely diffusing syntaxin molecules. SNARE-complex formation between syntaxin 1A and SNAP-25 (synaptosome-associated protein of 25 kDa) on the PM and synaptobrevin 2 on the vesicles (trans-SNAREs) is crucial for vesicle priming and fusion. This process might be impeded by the spontaneous accumulation of non-fusogenic cis-SNARE complexes formed when all three SNARE proteins reside on the PM. We investigated the kinetics of cis-SNARE complex assembly and disassembly and both exhibited biphasic behavior. The experimental measurements were analyzed through integration of differential rate equations pertinent to the reaction mechanism and through the application of a heuristic search for time constants and concentrations using a genetic algorithm. Reconstruction of the measurements necessitated the partitioning of syntaxin into two phases that might represent the syntaxin clusters and free syntaxin outside the clusters. The analysis suggests that most of the syntaxin in the clusters is concentrated in a nonreactive form. Consequently, cis-SNARE complex assembly in the clusters is substantially slower than outside the rafts. Interestingly, the clusters also mediate efficient disassembly of cis-SNARE complexes possibly attributable to the high local concentration of complexes in the clusters area that allows efficient disassembly by the enzymatic reaction of NSF.
Journal of Neuroscience 09/2009; 29(39):12292-301. · 7.11 Impact Factor
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ABSTRACT: Calcium/calmodulin (Ca/CaM) binds to the intracellular juxtamembrane domain (JMD) of the epidermal growth factor receptor (EGFR). The basic JMD also binds to acidic lipids in the inner leaflet of the plasma membrane, and this interaction may contribute an extra level of autoinhibition to the receptor. Binding of a ligand to the EGFR produces a rapid increase in intracellular calcium, [Ca2+]i, and thus Ca/CaM. How does Ca/CaM compete with the plasma membrane for the JMD? Does Ca/CaM directly pull the JMD off the membrane or does Ca/CaM only bind to the JMD after it has dissociated spontaneously from the bilayer? To answer this question, we studied the effect of Ca/CaM on the rate of dissociation of fluorescent JMD peptides from phospholipid vesicles by making kinetic stop-flow measurements. Ca/CaM increases the rate of dissociation: an analysis of the differential equations that describe the dissociation shows that Ca/CaM must directly pull the basic JMD peptide off the membrane surface. These measurements lead to a detailed atomic-level mechanism for EGFR activation that reconciles the existence of preformed EGFR dimers/oligomers with the Kuriyan allosteric model for activation of the EGFR kinase domains.
Biophysical Journal 07/2009; 96(12):4887-95. · 3.65 Impact Factor
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ABSTRACT: The fatty acid binding proteins (FAPBs) function as intracellular carriers of fatty acid (FA) and related compounds. During the digestion of lipids, the local concentration of FA exceeds their critical micellar concentration; the excess ratio of FA/FABP can be as high as approximately 1,000/1, consequently building micelles. Considering that the micelle formation is a rapid process, the FABP must be able to remove the mini-micelle. In this study, we describe the results of molecular dynamics simulations of liver basic FABP (Lb-FABP), carried out in the presence of approximately 20 mM palmitate ions, all in the presence of explicit water and at ionic strength of approximately 100 mM, approximating physiological conditions. The Lb-FABP appears to react, along with a free FA, with mini-micelle creating a stable complex (on the time scale of the simulations), which is attached to the anti-portal domain of the protein. The complex may be formed by the stepwise addition of free FA or through the interaction of a pre-formed mini-micelle with the free protein. The driving force of the mini-micelle-FABP complex is a combination of electrostatic attraction between the negative carboxylates of the mini-micelle with the positive charge of the N terminal amine residues and Lennard-Jones FA-protein interactions. The preferred tendency of the mini-micelle to react with the anti-portal domain retains the alpha-helixes of the portal region free for its electrostatic interaction with the membrane, ensuring a rapid unloading of the cargo on the membrane.
Molecular and Cellular Biochemistry 02/2009; 326(1-2):29-33. · 2.06 Impact Factor
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ABSTRACT: Mild sonication of eukaryotic cells produces native plasma membrane sheets that retain their docked organelles, cytoskeleton structures and cytoplasmic complexes. While the delicate organization of membranous protein complexes remains undisturbed, their inner plasmalemmel leaflet can be rapidly exposed to bathing solutions, enabling specific biochemical manipulations. Here, we apply this system to track membrane-biochemistry kinetics. We monitor soluble NSF-attachment protein receptor (SNARE) complex assembly and disassembly on the plasma membrane at high time resolution. The results suggest two-phase kinetics for the assembly process and dependence of the disassembly kinetics on both N-ethyl maleimide-sensitive factor (NSF) and soluble NSF-attachment protein (alpha-SNAP) concentrations.
FEBS Letters 10/2008; 582(23-24):3563-8. · 3.54 Impact Factor
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ABSTRACT: The calnexin/calreticulin cycle is a quality control system responsible for promoting the folding of newly synthesized glycoproteins entering the endoplasmic reticulum (ER). The association of calnexin and calreticulin with the glycoproteins is regulated by ER glucosidase II, which hydrolyzes Glc 2Man X GlcNAc 2 glycans to Glc 1Man X GlcNAc 2 and further to Glc 0Man X GlcNAc 2 ( X represents any number between 5 and 9). To gain new insights into the reaction mechanism of glucosidase II, we developed a kinetic model that describes the interactions between glucosidase II, calnexin/calreticulin, and the glycans. Our model accurately reconstructed the hydrolysis of glycans with nine mannose residues and glycans with seven mannose residues, as measured by Totani et al. [Totani, K., Ihara, Y., Matsuo, I., and Ito, Y. (2006) J. Biol. Chem. 281, 31502-31508]. Intriguingly, our model predicted that glucosidase II was inhibited by its nonglucosylated end products, where the inhibitory effect of Glc 0Man 7GlcNAc 2 was much stronger than that of Glc 0Man 9GlcNAc 2. These predictions were confirmed experimentally. Moreover, our model suggested that glycans with a different number of mannose residues can be equivalent substrates of glucosidase II, in contrast to what had been previously thought. We discuss the possibility that nonglucosylated glycans, existing in the ER, might regulate the entry of newly synthesized glycoproteins into the calnexin/calreticulin cycle. Our model also shows that glucosidase II does not interact with monoglucosylated glycans while they are bound to calnexin or calreticulin.
Biochemistry 10/2008; 47(41):10970-80. · 3.42 Impact Factor
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ABSTRACT: Molecular dynamics simulations of Ca+2 ions near protein were performed with three force fields: GROMOS96, OPLS-AA, and CHARMM22. The simulations reveal major, force-field dependent, inconsistencies in the interaction between the Ca+2 ions with the protein. The variations are attributed to the nonbonded parameterizations of the Ca+2-carboxylates interactions. The simulations results were compared to experimental data, using the Ca+2-HCOO- equilibrium as a model. The OPLS-AA force field grossly overestimates the binding affinity of the Ca+2 ions to the carboxylate whereas the GROMOS96 and CHARMM22 force fields underestimate the stability of the complex. Optimization of the Lennard-Jones parameters for the Ca+2-carboxylate interactions were carried out, yielding new parameters which reproduce experimental data.
Journal of Computational Chemistry 06/2008; 29(7):1163-9. · 4.58 Impact Factor
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ABSTRACT: Ca2+ regulates multiple processes in nerve terminals, including synaptic vesicle recruitment, priming, and fusion. Munc13s, the mammalian homologs of Caenorhabditis elegans Unc13, are essential vesicle-priming proteins and contain multiple regulatory domains that bind second messengers such as diacylglycerol and Ca2+/calmodulin (Ca2+/CaM). Binding of Ca2+/CaM is necessary for the regulatory effect that allows Munc13-1 and ubMunc13-2 to promote short-term synaptic plasticity. However, the relative contributions of Ca2+ and Ca2+/CaM to vesicle priming and recruitment by Munc13 are not known. Here, we investigated the effect of Ca2+/CaM binding on ubMunc13-2 activity in chromaffin cells via membrane-capacitance measurements and a detailed simulation of the exocytotic machinery. Stimulating secretion under various basal Ca2+ concentrations from cells overexpressing either ubMunc13-2 or a ubMunc13-2 mutant deficient in CaM binding enabled a distinction between the effects of Ca2+ and Ca2+/CaM. We show that vesicle priming by ubMunc13-2 is Ca2+ dependent but independent of CaM binding to ubMunc13-2. However, Ca2+/CaM binding to ubMunc13-2 specifically promotes vesicle recruitment during ongoing stimulation. Based on the experimental data and our simulation, we propose that ubMunc13-2 is activated by two Ca2+-dependent processes: a slow activation mode operating at low Ca2+ concentrations, in which ubMunc13-2 acts as a priming switch, and a fast mode at high Ca2+ concentrations, in which ubMunc13-2 is activated in a Ca2+/CaM-dependent manner and accelerates vesicle recruitment and maturation during stimulation. These different Ca2+ activation steps determine the kinetic properties of exocytosis and vesicle recruitment and can thus alter plasticity and efficacy of transmitter release.
Journal of Neuroscience 03/2008; 28(8):1949-60. · 7.11 Impact Factor
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ABSTRACT: Myosin V moves along actin filaments by an arm-over-arm motion, known as the lever mechanism. Each of its arms is composed of six consecutive IQ peptides that bind light chain proteins, such as calmodulin or calmodulin-like proteins. We have employed a multistage approach in order to investigate the mechanochemical structural basis of the movement of myosin V from the budding yeast Saccharomyces cerevisiae. For that purpose, we previously carried out molecular dynamics simulations of the Mlc1p-IQ2 and the Mlc1p-IQ4 protein-peptide complexes, and the present study deals with the structures of the IQ peptides when stripped from the Mlc1p protein. We have found that the crystalline structure of the IQ2 peptide retains a stable rodlike configuration in solution, whereas that of the IQ4 peptide grossly deviates from its X-ray conformation exhibiting an intrinsic tendency to curve and bend. The refolding process of the IQ4 peptide is initially driven by electrostatic interactions followed by nonpolar stabilization. Its bending appears to be affected by the ionic strength, when ionic strength higher than approximately 300 mM suppresses it from flexing. Considering that a poly-IQ sequence is the lever arm of myosin V, we suggest that the arm may harbor a joint, localized within the IQ4 sequence, enabling the elasticity of the neck of myosin V. Given that a poly-IQ sequence is present at the entire class of myosin V and the possibility that the yeast's myosin V molecule can exist either as a nonprocessive monomer or as a processive dimer depending on conditions (Krementsova, E. B., Hodges, A. R., Lu, H., and Trybus, K. M. (2006) J. Biol. Chem. 281, 6079-6086), our observations may account for a general structural feature for the myosins' arm embedded flexibility.
Biochemistry 01/2008; 46(50):14524-36. · 3.42 Impact Factor
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Journal of Computational Chemistry. 01/2008; 29:1163-1169.
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ABSTRACT: The capacity to transfer protons between surface groups is an innate property of many proteins. The transfer of a proton between donor and acceptor, located as far as 6-7 A apart, necessitates the participation of water molecules in the process. In a previous study we investigated the mechanism of proton transfer (PT) between bulk exposed sites, a few ångströms apart, using as a model the proton exchange between the proton-binding sites of the fluorescein molecule in dilute aqueous solution.1 The present study expands the understanding of PT reactions between adjacent sites exposed to water through the calculation the minimum energy pathways (MEPs) by the conjugate peak refinement algorithm2 and a quantum-mechanical potential. The PT reaction trajectories were calculated for the fluorescein system with an increasing number of water molecules. The MEP calculations reveal that the transition state is highly strained and involves a supramolecular structure in which fluorescein and the interconnecting water molecules are covalently bonded together and the protons are shared between neighboring oxygens. These findings are in accord with the high activation energy, as measured for the reaction, and indicate that PT reactions on the surface proceed by a semi- or fully concerted rather than stepwise mechanism. A similar mechanism is assumed to be operative on the surface of proteins and renders water-mediated PT reactions as highly efficient as they are.
The Journal of Physical Chemistry B 06/2007; 111(21):6059-70. · 3.70 Impact Factor
