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Nikolay V Dokholyan

Publications and source records attributed to Nikolay V Dokholyan.

At least 19 recordsLinked to original sources
PubMed2006

Combining fluorescence detection and mass spectrometric analysis for comprehensive and quantitative analysis of redox-sensitive cysteines in native membrane proteins.

Monobromobimane (MBB) is a lipophilic reagent that selectively modifies free cysteine residues in proteins. Because of its lipophilic character, MBB is capable of labeling cysteine residues in membrane proteins under native conditions. Reaction of MBB with the sulfhydryl groups of free cysteines leads to formation of highly fluorescent derivatives. Here we describe a procedure for the detection and relative quantitation of MBB-labeled cysteines using fluorescence and mass spectrometric analyses, which allow determination of free cysteine content and unambiguous identification of MBB-modified cysteine residues. We have applied this approach to the analysis of the free and redox-sensitive cysteine residues of a large membrane protein, the sarcoplasmic reticulum Ca2+ release channel with a molecular mass of 2.2 million Da. Labeling was performed under physiologic conditions where the channel complex is in its native environment and is functionally active. The purified MBB-labeled channel complex was enzymatically digested, and the resulting peptides were separated by reversed-phase high-performance chromatography. MBB-labeled peptides were detected by fluorescence and identified by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Under MALDI conditions, partial photolytic fragmentation of the MBB-peptide bound occurred, thus allowing convenient screening for the MBB-modified peptides in the MS spectrum by detection of the specific mass increment of 190.07 Da for MBB-modified cysteine residues. Modification of the peptides was further confirmed by tandem mass spectrometric analysis, utilizing sequencing information and the presence of the specific immonium ion for the MBB-modified cysteine residues at m/z 266.6. Quantitative information was obtained by comparison of both fluorescence and MS signal intensities of MBB-modified peptides. Combination of fluorescence with MS detection and analysis of MBB-labeled peptides supported by a customized software program provides a convenient method for identifying and quantifying redox-sensitive cysteines in membrane proteins of native biological systems. Identification of one redox-sensitive cysteine (2327) in the native membrane-bound sarcoplasmic reticulum Ca2+ release channel is described.

Animals↗
PubMed2006

Multiscale modeling of nucleosome dynamics.

Nucleosomes form the fundamental building blocks of chromatin. Subtle modifications of the constituent histone tails mediate chromatin stability and regulate gene expression. For this reason, it is important to understand structural dynamics of nucleosomes at atomic levels. We report a novel multiscale model of the fundamental chromatin unit, a nucleosome, using a simplified model for rapid discrete molecular dynamics simulations and an all-atom model for detailed structural investigation. Using a simplified structural model, we perform equilibrium simulations of a single nucleosome at various temperatures. We further reconstruct all-atom nucleosome structures from simulation trajectories. We find that histone tails bind to nucleosomal DNA via strong salt-bridge interactions over a wide range of temperatures, suggesting a mechanism of chromatin structural organization whereby histone tails regulate inter- and intranucleosomal assemblies via binding with nucleosomal DNA. We identify specific regions of the histone core H2A/H2B-H4/H3-H3/H4-H2B/H2A, termed "cold sites", which retain a significant fraction of contacts with adjoining residues throughout the simulation, indicating their functional role in nucleosome organization. Cold sites are clustered around H3-H3, H2A-H4 and H4-H2A interhistone interfaces, indicating the necessity of these contacts for nucleosome stability. Essential dynamics analysis of simulation trajectories shows that bending across the H3-H3 is a prominent mode of intranucleosomal dynamics. We postulate that effects of salts on mononucleosomes can be modeled in discrete molecular dynamics by modulating histone-DNA interaction potentials. Local fluctuations in nucleosomal DNA vary significantly along the DNA sequence, suggesting that only a fraction of histone-DNA contacts make strong interactions dominating mononucleosomal dynamics. Our findings suggest that histone tails have a direct functional role in stabilizing higher-order chromatin structure, mediated by salt-bridge interactions with adjacent DNA.

Chromatin↗
PubMed2006

A structural model reveals energy transduction in dynein.

Intracellular active transport is driven by ATP-hydrolyzing motor proteins that move along cytoskeletal filaments. In particular, the microtubule-associated dynein motor is involved in the transport of organelles and vesicles, the maintenance of the Golgi, and mitosis. However, unlike kinesin and myosin, the mechanism by which dynein converts chemical energy into mechanical force remains largely a mystery, due primarily to the lack of a high-resolution molecular structure. Using homology modeling and normal mode analysis, we propose a complete atomic structure and a mechanism for force generation by the motor protein dynein. In agreement with very recent electron microscopy (EM) reconstructions showing dynein as a ring-shaped heptamer, our model consists of six ATPases of the AAA (ATPases associated with various cellular activities) superfamily and a C-terminal domain, which is experimentally known to control motor function. Our model shows a coiled coil spanning the diameter of the motor that accounts for previously unidentified structures in EM studies and provides a potential mechanism for long-range communication between the AAA domains. Furthermore, normal mode analysis reveals that the subunits of the motor that contain the nucleotide binding sites exhibit minimal movement, whereas the rest of the motor is very mobile. Our analysis suggests the likely domain rearrangements of the motor unit that generate its power stroke. This study provides insights into the structure and function of dynein that can guide further experimental investigations into energy transduction in dynein.

Amino Acid Sequence↗
PubMed2006

iFold: a platform for interactive folding simulations of proteins.

UNLABELLED: We built a novel web-based platform for performing discrete molecular dynamics simulations of proteins. In silico protein folding involves searching for minimal frustration in the vast conformational landscape. Conventional approaches for simulating protein folding insufficiently address the problem of simulations in relevant time and length scales necessary for a mechanistic understanding of underlying biomolecular phenomena. Discrete molecular dynamics (DMD) offers an opportunity to bridge the size and timescale gaps and uncover the structural and biological properties of experimentally undetectable protein dynamics. The iFold server supports large-scale simulations of protein folding, thermal denaturation, thermodynamic scan, simulated annealing and p(fold) analysis using DMD and coarse-grained protein model with structure-based Gō-interactions between amino acids. AVAILABILITY: http://ifold.dokhlab.org

Amino Acid Sequence↗
PubMed2006

Insights into allosteric control of vinculin function from its large scale conformational dynamics.

Vinculin is an important constituent of both cell-cell and cell-matrix junctions, where it plays crucial roles in the regulation of cell adhesion and migration. When activated, it mediates the linkage between cadherins (cell-cell) or integrins (cell-matrix) and the actin cytoskeleton through interactions with various proteins. The activation of vinculin requires structural conversions from an autoinhibited conformation to the "open" conformations in which the occluded binding sites of its different ligands become exposed, while the structural dynamics underlying the vinculin activation remains largely unknown. Here we report the first computational study of large scale conformational dynamics of full-length vinculin. We find that the "holding" and "releasing" motions between vinculin tail and pincer-like structure formed by first three domains of vinculin are the dominant motions near the native state of vinculin, indicating that an inherent flexibility of vinculin has a large influence on its allostery. We also find a cooperative dissociation between the head and tail domains of vinculin with increasing temperature in both thermodynamic and kinetic simulations, implying that vinculin may function as an allosteric switch in response to external signals. We show that the kinetics of vinculin unfolding exhibits specific sequential patterns, suggesting that a sophisticated interplay between domains may synergistically contribute to vinculin activation. We further find that the interaction between vinculin-binding site peptide from talin and vinculin significantly destabilizes the intramolecular head-tail interactions, suggesting a direct role of talin binding in vinculin activation.

Actins↗
PubMed2006

The coordinated evolution of yeast proteins is constrained by functional modularity.

Functional modularity is a key attribute of cellular systems and has important roles in evolution. However, the extent to which functional modularity affects protein evolution is largely unknown. Here, we analyzed the evolution of both sequence and expression level of proteins in the yeast Saccharomyces cerevisiae and found that proteins within the same functional modules evolve at more similar rates than those between different modules. We also found stronger co-evolution of expression levels between proteins within functional modules than between them. These results suggest that a coordinated evolution of both the sequence and expression level of proteins is constrained by functional modularity.

Evolution, Molecular↗
PubMed2006

Emergence of protein fold families through rational design.

Diverse proteins with similar structures are grouped into families of homologs and analogs, if their sequence similarity is higher or lower, respectively, than 20%-30%. It was suggested that protein homologs and analogs originate from a common ancestor and diverge in their distinct evolutionary time scales, emerging as a consequence of the physical properties of the protein sequence space. Although a number of studies have determined key signatures of protein family organization, the sequence-structure factors that differentiate the two evolution-related protein families remain unknown. Here, we stipulate that subtle structural changes, which appear due to accumulating mutations in the homologous families, lead to distinct packing of the protein core and, thus, novel compositions of core residues. The latter process leads to the formation of distinct families of homologs. We propose that such differentiation results in the formation of analogous families. To test our postulate, we developed a molecular modeling and design toolkit, Medusa, to computationally design protein sequences that correspond to the same fold family. We find that analogous proteins emerge when a backbone structure deviates only 1-2 angstroms root-mean-square deviation from the original structure. For close homologs, core residues are highly conserved. However, when the overall sequence similarity drops to approximately 25%-30%, the composition of core residues starts to diverge, thereby forming novel families of protein homologs. This direct observation of the formation of protein homologs within a specific fold family supports our hypothesis. The conservation of amino acids in designed sequences recapitulates that of the naturally occurring sequences, thereby validating our computational design methodology.

Computational Biology↗
PubMed2006

Common dynamical signatures of familial amyotrophic lateral sclerosis-associated structurally diverse Cu, Zn superoxide dismutase mutants.

More than 100 structurally diverse point mutations leading to aggregation in the dimeric enzyme Cu, Zn superoxide dismutase (SOD1) are implicated in familial amyotrophic lateral sclerosis (FALS). Although SOD1 dimer dissociation is a known requirement for its aggregation, the common structural basis for diverse FALS mutations resulting in aggregation is not fully understood. In molecular dynamics simulations of wild-type SOD1 and three structurally diverse FALS mutants (A4V, G37R, and H46R), we find that a common effect of mutations on SOD1 dimer is the mutation-induced disruption of dynamic coupling between monomers. In the wild-type dimer, the principal coupled motion corresponds to a "breathing motion" of the monomers around an axis parallel to the dimer interface, and an opening-closing motion of the distal metal-binding loops. These coupled motions are disrupted in all three mutants independent of the mutation location. Loss of coupled motions in mutant dimers occurs with increased disruption of a key stabilizing structural element (the beta-plug) leading to the de-protection of edge strands. To rationalize disruption of coupling, which is independent of the effect of the mutation on global SOD1 stability, we analyze the residue-residue interaction network formed in SOD1. We find that the dimer interface and metal-binding loops, both involved in coupled motions, are regions of high connectivity in the network. Our results suggest that independent of the effect on protein stability, altered protein dynamics, due to long-range communication within its structure, may underlie the aggregation of mutant SOD1 in FALS.

Amyotrophic Lateral Sclerosis↗
PubMed2006

Fast complementation of split fluorescent protein triggered by DNA hybridization.

Fluorescent proteins have proven to be excellent reporters and biochemical sensors with a wide range of applications. In a split form, they are not fluorescent, but their fluorescence can be restored by supplementary protein-protein or protein-nucleic acid interactions that reassemble the split polypeptides. However, in prior studies, it took hours to restore the fluorescence of a split fluorescent protein because the formation of the protein chromophore slowly occurred de novo concurrently with reassembly. Here we provide evidence that a fluorogenic chromophore can self-catalytically form within an isolated N-terminal fragment of the enhanced green fluorescent protein (EGFP). We show that restoration of the split protein fluorescence can be driven by nucleic acid complementary interactions. In our assay, fluorescence development is fast (within a few minutes) when complementary oligonucleotide-linked fragments of the split EGFP are combined. The ability of our EGFP system to respond quickly to DNA hybridization should be useful for detecting the kinetics of many other types of pairwise interactions both in vitro and in living cells.

DNA↗
PubMed2006

Studies of folding and misfolding using simplified models.

Computer simulations are as vital to our studies of biological systems as experiments. They bridge and rationalize experimental observations, extend the experimental "field of view", which is often limited to a specific time or length scale, and, most importantly, provide novel insights into biological systems, offering hypotheses about yet-to-be uncovered phenomena. These hypotheses spur further experimental discoveries. Simplified molecular models have a special place in the field of computational biology. Branded as less accurate than all-atom protein models, they have offered what all-atom molecular dynamics simulations could not--the resolution of the length and time scales of biological phenomena. Not only have simplified models proven to be accurate in explaining or reproducing several biological phenomena, they have also offered a novel multiscale computational strategy for accessing a broad range of time and length scales upon integration with traditional all-atom simulations. Recent computer simulations of simplified models have shaken or advanced the established understanding of biological phenomena. It was demonstrated that simplified models can be as accurate as traditional molecular dynamics approaches in identifying native conformations of proteins. Their application to protein structure prediction yielded phenomenal accuracy in recapitulating native protein conformations. New studies that utilize the synergy of simplified protein models with all-atom models and experiments yielded novel insights into complex biological processes, such as protein folding, aggregation and the formation of large protein complexes.

Computational Biology↗
PubMed2006

Topological determinants of protein domain swapping.

Protein domain swapping has been repeatedly observed in a variety of proteins and is believed to result from destabilization due to mutations or changes in environment. Based on results from our studies and others, we propose that structures of the domain-swapped proteins are mainly determined by their native topologies. We performed molecular dynamics simulations of seven different proteins, known to undergo domain swapping experimentally, under mildly denaturing conditions and found in all cases that the domain-swapped structures can be recapitulated by using protein topology in a simple protein model. Our studies further indicated that, in many cases, domain swapping occurs at positions around which the protein tends to unfold prior to complete unfolding. This, in turn, enabled prediction of protein structural elements that are responsible for domain swapping. In particular, two distinct domain-swapped dimer conformations of the focal adhesion targeting domain of focal adhesion kinase were predicted computationally and were supported experimentally by data obtained from NMR analyses.

Animals↗
PubMed2006

FALS mutations in Cu, Zn superoxide dismutase destabilize the dimer and increase dimer dissociation propensity: a large-scale thermodynamic analysis.

Mutations in the dimeric enzyme Cu, Zn superoxide dismutase (SOD1) leading to its aggregation are implicated in the toxicity in familial amyotrophic lateral sclerosis (FALS). We and others have previously shown that aggregation occurs by a pathway involving dimer dissociation, metal-loss from monomers and multimeric assembly of apo-SOD1 monomers. We postulate that FALS mutations cause enhanced aggregation by affecting one or more steps in the pathway, and computationally test this postulate for 75 known mis-sense FALS mutants of SOD1. Based on an extensive thermodynamic analysis of the stability of apo-dimer and apo-monomer forms of these mutants, we classify the mutations into the following groups: 70 out of 75 mutations in SOD1 lead to (i) decreased dimer stability, and/or (ii) increased dimer dissociation, compared to wild type, and four mutations lead to (iii) decreased monomer stability compared to wild type. Our results suggest that enhanced aggregation of SOD1 in FALS occurs due to an increased population of mutant SOD1 apo-monomers compared to wild type. The dissociation of multimeric proteins induced by diverse mutations may be a common theme in several human diseases.

Amyotrophic Lateral Sclerosis↗
PubMed2005

Sequence and structural determinants of Cu, Zn superoxide dismutase aggregation.

Diverse point mutations in the enzyme Cu, Zn superoxide dismutase (SOD1) are linked to its aggregation in the familial form of the disease amyotrophic lateral sclerosis. The disease-associated mutations are known to destabilize the protein, but the structural basis of the aggregation of the destabilized protein and the structure of aggregates are not well understood. Here, we investigate in silico the sequence and structural determinants of SOD1 aggregation: (1) We identify sequence fragments in SOD1 that have a high aggregation propensity, using only the sequence of SOD1, and (2) we perform molecular dynamics simulations of the SOD1 dimer folding and misfolding. In both cases, we identify identical regions of the protein as having high propensity to form intermolecular interactions. These regions correspond to the N- and C-termini, and two crossover loops and two beta-strands in the Greek-key native fold of SOD1. Our results suggest that the high aggregation propensity of mutant SOD1 may result from a synergy of two factors: the presence of highly amyloidogenic sequence fragments ("hot spots"), and the presence of these fragments in regions of the protein that are structurally most likely to form intermolecular contacts under destabilizing conditions. Therefore, we postulate that the balance between the self-association of aggregation-prone sequences and the specific structural context of these sequences in the native state determines the aggregation propensity of proteins.

Amyloid↗
PubMed2005

A single disulfide bond differentiates aggregation pathways of beta2-microglobulin.

Deposition of wild-type beta2-microglobulin (beta2m) into amyloid fibrils is a complication in patients undergoing long-term hemodialysis. The native beta-sandwich fold of beta2m has a highly conserved disulfide bond linking Cys25 and Cys80. Oxidized beta2m forms needle-like amyloid fibrils at pH 2.5 in vitro, whereas reduced beta2m, at acid pH, in which the intra-chain disulfide bond is disrupted, cannot form typical fibrils. Instead, reduced beta2m forms thinner and more flexible filaments. To uncover the difference in molecular mechanisms underlying the aggregation of the oxidized and reduced beta2m, we performed molecular dynamics simulations of beta2m oligomerization under oxidized and reduced conditions. We show that, consistent with experimental observations, the oxidized beta2m forms domain-swapped dimer, in which the two proteins exchange their N-terminal segments complementing each other. In contrast, both dimers and trimers, formed by reduced beta2m, are comprised of parallel beta-sheets between monomers and stabilized by the hydrogen bond network along the backbone. The oligomerized monomers are in extended conformations, capable of further aggregation. We find that both reduced and oxidized dimers are thermodynamically less stable than their corresponding monomers, indicating that beta2m oligomerization is not accompanied by the formation of a thermodynamically stable dimer. Our studies suggest that the different aggregation pathways of oxidized and reduced beta2m are dictated by the formation of distinct precursor oligomeric species that are modulated by Cys25-Cys80 disulfide-bonds. We propose that the propagation of domain swapping is the aggregation mechanism for the oxidized beta2m, while "parallel stacking" of partially unfolded beta2m is the aggregation mechanism for the reduced beta2m.

Amyloid↗
PubMed2005

Direct observation of protein folding, aggregation, and a prion-like conformational conversion.

Protein conformational transition from alpha-helices to beta-sheets precedes aggregation of proteins implicated in many diseases, including Alzheimer and prion diseases. Direct characterization of such transitions is often hindered by the complicated nature of the interaction network among amino acids. A recently engineered small protein-like peptide with a simple amino acid composition features a temperature-driven alpha-helix to beta-sheet conformational change. Here we studied the conformational transition of this peptide by molecular dynamics simulations. We observed a critical temperature, below which the peptide folds into an alpha-helical coiled-coil state and above which the peptide misfolds into beta-rich structures with a high propensity to aggregate. The structures adopted by this peptide during low temperature simulations have a backbone root mean square deviation less than 2 A from the crystal structure. At high temperatures, this peptide adopts an amyloid-like structure, which is mainly composed of coiled anti-parallel beta-sheets with the cross-beta-signature of amyloid fibrils. Most strikingly, we observed conformational conversions in which an alpha-helix is converted into a beta-strand by proximate stable beta-sheets with exposed hydrophobic surfaces and unsaturated hydrogen bonds. Our study suggested a possible generic molecular mechanism of the template-mediated aggregation process, originally proposed by Prusiner (Prusiner, S. B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13363-13383) to account for prion infectivity.

Amyloid↗
PubMed2005

Molecular origin of polyglutamine aggregation in neurodegenerative diseases.

Expansion of polyglutamine (polyQ) tracts in proteins results in protein aggregation and is associated with cell death in at least nine neurodegenerative diseases. Disease age of onset is correlated with the polyQ insert length above a critical value of 35-40 glutamines. The aggregation kinetics of isolated polyQ peptides in vitro also shows a similar critical-length dependence. While recent experimental work has provided considerable insights into polyQ aggregation, the molecular mechanism of aggregation is not well understood. Here, using computer simulations of isolated polyQ peptides, we show that a mechanism of aggregation is the conformational transition in a single polyQ peptide chain from random coil to a parallel beta-helix. This transition occurs selectively in peptides longer than 37 glutamines. In the beta-helices observed in simulations, all residues adopt beta-strand backbone dihedral angles, and the polypeptide chain coils around a central helical axis with 18.5 +/- 2 residues per turn. We also find that mutant polyQ peptides with proline-glycine inserts show formation of antiparallel beta-hairpins in their ground state, in agreement with experiments. The lower stability of mutant beta-helices explains their lower aggregation rates compared to wild type. Our results provide a molecular mechanism for polyQ-mediated aggregation.

Journal Article↗
PubMed2005

Reconstruction of the src-SH3 protein domain transition state ensemble using multiscale molecular dynamics simulations.

We use an integrated computational approach to reconstruct accurately the transition state ensemble (TSE) for folding of the src-SH3 protein domain. We first identify putative TSE conformations from free energy surfaces generated by importance sampling molecular dynamics for a fully atomic, solvated model of the src-SH3 protein domain. These putative TSE conformations are then subjected to a folding analysis using a coarse-grained representation of the protein and rapid discrete molecular dynamics simulations. Those conformations that fold to the native conformation with a probability (P(fold)) of approximately 0.5, constitute the true transition state. Approximately 20% of the putative TSE structures were found to have a P(fold) near 0.5, indicating that, although correct TSE conformations are populated at the free energy barrier, there is a critical need to refine this ensemble. Our simulations indicate that the true TSE conformations are compact, with a well-defined central beta sheet, in good agreement with previous experimental and theoretical studies. A structured central beta sheet was found to be present in a number of pre-TSE conformations, however, indicating that this element, although required in the transition state, does not define it uniquely. An additional tight cluster of contacts between highly conserved residues belonging to the diverging turn and second beta-sheet of the protein emerged as being critical elements of the folding nucleus. A number of commonly used order parameters to identify the transition state for folding were investigated, with the number of native Cbeta contacts displaying the most satisfactory correlation with P(fold) values.

Models, Molecular↗