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Michele Vendruscolo

Publications and source records attributed to Michele Vendruscolo.

At least 19 recordsLinked to original sources
PubMed2007

The MUMO (minimal under-restraining minimal over-restraining) method for the determination of native state ensembles of proteins.

While reliable procedures for determining the conformations of proteins are available, methods for generating ensembles of structures that also reflect their flexibility are much less well established. Here we present a systematic assessment of the ability of ensemble-averaged molecular dynamics simulations with ensemble-averaged NMR restraints to simultaneously reproduce the average structure of proteins and their associated dynamics. We discuss the effects that under-restraining (overfitting) and over-restraining (underfitting) have on the structures generated in ensemble-averaged molecular simulations. We then introduce the MUMO (minimal under-restraining minimal over-restraining) method, a procedure in which different observables are averaged over a different number of molecules. As both over-restraining and under-restraining are significantly reduced in the MUMO method, it is possible to generate ensembles of conformations that accurately characterize both the structure and the dynamics of native states of proteins. The application of the MUMO method to the protein ubiquitin yields a high-resolution structural ensemble with an RDC Q-factor of 0.19.

Amino Acid Sequence↗
PubMed2006

A PDZ domain recapitulates a unifying mechanism for protein folding.

A unifying view has been recently proposed according to which the classical diffusion-collision and nucleation-condensation models may represent two extreme manifestations of an underlying common mechanism for the folding of small globular proteins. We report here the characterization of the folding process of the PDZ domain, a protein that recapitulates the three canonical steps involved in this unifying mechanism, namely: (i) the early formation of a weak nucleus that determines the native-like topology of a large portion of the structure, (ii) a global collapse of the entire polypeptide chain, and (iii) the consolidation of the remaining partially structured regions to achieve the native state conformation. These steps, which are clearly detectable in the PDZ domain investigated here, may be difficult to distinguish experimentally in other proteins, which would thus appear to follow one of the two limiting mechanisms. The analysis of the (un)folding kinetics for other three-state proteins (when available) appears consistent with the predictions ensuing from this unifying mechanism, thus providing a powerful validation of its general nature.

Protein Folding↗
PubMed2006

Geometry, energetics, and dynamics of hydrogen bonds in proteins: structural information derived from NMR scalar couplings.

An accurate description of hydrogen bonds is essential to identify the determinants of protein stability and function as well as folding and misfolding behavior. We describe a method of using J couplings through hydrogen bonds as ensemble-averaged restraints in molecular dynamics simulations. Applications to the cases of ubiquitin and protein G show that these scalar couplings provide powerful structural information that, when used through the methodology that we present here, enables the description of the geometry and energetics of hydrogen bonds with an accuracy approaching that of high-resolution X-ray structures.

Computer Simulation↗
PubMed2006

BPPred: a Web-based computational tool for predicting biophysical parameters of proteins.

We exploit the availability of recent experimental data on a variety of proteins to develop a Web-based prediction algorithm (BPPred) to calculate several biophysical parameters commonly used to describe the folding process. These parameters include the equilibrium m-values, the length of proteins, and the changes upon unfolding in the solvent-accessible surface area, in the heat capacity, and in the radius of gyration. We also show that the knowledge of any one of these quantities allows an estimate of the others to be obtained, and describe the confidence limits with which these estimations can be made. Furthermore, we discuss how the kinetic m-values, or the Beta Tanford values, may provide an estimate of the solvent-accessible surface area and the radius of gyration of the transition state for protein folding. Taken together, these results suggest that BPPred should represent a valuable tool for interpreting experimental measurements, as well as the results of molecular dynamics simulations.

Algorithms↗
PubMed2006

Characterization of the residual structure in the unfolded state of the Delta131Delta fragment of staphylococcal nuclease.

The determination of the conformational preferences in unfolded states of proteins constitutes an important challenge in structural biology. We use inter-residue distances estimated from site-directed spin-labeling NMR experimental measurements as ensemble-averaged restraints in all-atom molecular dynamics simulations to characterise the residual structure of the Delta131Delta fragment of staphylococcal nuclease under physiological conditions. Our findings indicate that Delta131Delta under these conditions shows a tendency to form transiently hydrophobic clusters similar to those present in the native state of wild-type staphylococcal nuclease. Only rarely, however, all these interactions are simultaneously realized to generate conformations with an overall native topology.

Computer Simulation↗
PubMed2006

Relation between native ensembles and experimental structures of proteins.

Different experimental structures of the same protein or of proteins with high sequence similarity contain many small variations. Here we construct ensembles of "high-sequence similarity Protein Data Bank" (HSP) structures and consider the extent to which such ensembles represent the structural heterogeneity of the native state in solution. We find that different NMR measurements probing structure and dynamics of given proteins in solution, including order parameters, scalar couplings, and residual dipolar couplings, are remarkably well reproduced by their respective high-sequence similarity Protein Data Bank ensembles; moreover, we show that the effects of uncertainties in structure determination are insufficient to explain the results. These results highlight the importance of accounting for native-state protein dynamics in making comparisons with ensemble-averaged experimental data and suggest that even a modest number of structures of a protein determined under different conditions, or with small variations in sequence, capture a representative subset of the true native-state ensemble.

Animals↗
PubMed2006

A protein evolution model with independent sites that reproduces site-specific amino acid distributions from the Protein Data Bank.

BACKGROUND: Since thermodynamic stability is a global property of proteins that has to be conserved during evolution, the selective pressure at a given site of a protein sequence depends on the amino acids present at other sites. However, models of molecular evolution that aim at reconstructing the evolutionary history of macromolecules become computationally intractable if such correlations between sites are explicitly taken into account. RESULTS: We introduce an evolutionary model with sites evolving independently under a global constraint on the conservation of structural stability. This model consists of a selection process, which depends on two hydrophobicity parameters that can be computed from protein sequences without any fit, and a mutation process for which we consider various models. It reproduces quantitatively the results of Structurally Constrained Neutral (SCN) simulations of protein evolution in which the stability of the native state is explicitly computed and conserved. We then compare the predicted site-specific amino acid distributions with those sampled from the Protein Data Bank (PDB). The parameters of the mutation model, whose number varies between zero and five, are fitted from the data. The mean correlation coefficient between predicted and observed site-specific amino acid distributions is larger than = 0.70 for a mutation model with no free parameters and no genetic code. In contrast, considering only the mutation process with no selection yields a mean correlation coefficient of = 0.56 with three fitted parameters. The mutation model that best fits the data takes into account increased mutation rate at CpG dinucleotides, yielding = 0.90 with five parameters. CONCLUSION: The effective selection process that we propose reproduces well amino acid distributions as observed in the protein sequences in the PDB. Its simplicity makes it very promising for likelihood calculations in phylogenetic studies. Interestingly, in this approach the mutation process influences the effective selection process, i.e. selection and mutation must be entangled in order to obtain effectively independent sites. This interdependence between mutation and selection reflects the deep influence that mutation has on the evolutionary process: The bias in the mutation influences the thermodynamic properties of the evolving proteins, in agreement with comparative studies of bacterial proteomes, and it also influences the rate of accepted mutations.

Algorithms↗
PubMed2006

Structural comparison of the two alternative transition states for folding of TI I27.

TI I27, a beta-sandwich domain from the human muscle protein titin, has been shown to fold via two alternative pathways, which correspond to a change in the folding mechanism. Under physiological conditions, TI I27 folds by a classical nucleation-condensation mechanism (diffuse transition state), whereas at extreme conditions of temperature and denaturant it switches to having a polarized transition state. We have used experimental Phi-values as restraints in ensemble-averaged molecular dynamics simulations to determine the ensembles of structures representing the two transition states. The comparison of these ensembles indicates that when native interactions are substantially weakened, a protein may still be able to fold if it can access an alternative transition state characterized by a much larger entropic contribution. Analysis of the probability distribution of Phi-values derived from ensemble averaged simulations, enables us to identify residues that form contacts in some members of the ensemble but not in others illustrating that many interactions present in transition states are not strictly required for the successful completion of the folding process.

Computer Simulation↗
PubMed2006

Structural interpretation of hydrogen exchange protection factors in proteins: characterization of the native state fluctuations of CI2.

Protection factors obtained from equilibrium hydrogen exchange experiments are an important source of structural information on both native and nonnative states of proteins. We present a method for determining ensembles of protein structures by using hydrogen exchange data as restraints in molecular dynamics simulations in conjunction with an empirical force-field. The method is applied to determine the ensemble of structures representing the native state of chymotrypsin inhibitor 2 (CI2), including the rare, large fluctuations responsible for hydrogen exchange.

Chymotrypsin↗
PubMed2006

Theoretical approaches to protein aggregation.

The process of protein misfolding and aggregation has been associated with an increasing number of pathological conditions that include Alzheimer's and Parkinson's diseases, and type II diabetes. In addition, the discovery that proteins unrelated to any known disorder can be converted into aggregates of morphologies similar to those found in diseased tissue has lead to the recognition that this type of assemblies represents a generic state of polypeptide chains. Therefore, despite the enormous complexity of the in vivo mechanisms that have evolved in living organisms to prevent and control the formation of protein aggregates, the process of aggregation itself appears ultimately to be caused by intrinsic properties of polypeptide chains, in particular by the tendency of the backbone to form hydrogen bonds, and be modulated by the presence of specific patterns of hydrophobic and charged residues. Theoreticians have just recently started to respond to the challenge of identifying the determinants of the aggregation process. In this review, we provide an account of the theoretical results obtained so far.

Alzheimer Disease↗
PubMed2005

Determination of an ensemble of structures representing the intermediate state of the bacterial immunity protein Im7.

We present a detailed structural characterization of the intermediate state populated during the folding and unfolding of the bacterial immunity protein Im7. We achieve this result by incorporating a variety of experimental data available for this species in molecular dynamics simulations. First, we define the structure of the exchange-competent intermediate state of Im7 by using equilibrium hydrogen-exchange protection factors. Second, we use this ensemble to predict Phi-values and compare the results with the experimentally determined Phi-values of the kinetic refolding intermediate. Third, we predict chemical-shift measurements and compare them with the measured chemical shifts of a mutational variant of Im7 for which the kinetic folding intermediate is the most stable state populated at equilibrium. Remarkably, we found that the properties of the latter two species are predicted with high accuracy from the exchange-competent intermediate that we determined, suggesting that these three states are characterized by a similar architecture in which helices I, II, and IV are aligned in a native-like, but reorganized, manner. Furthermore, the structural ensemble that we obtained enabled us to rationalize the results of tryptophan fluorescence experiments in the WT protein and a series of mutational variants. The results show that the integration of diverse sets of experimental data at relatively low structural resolution is a powerful approach that can provide insights into the structural organization of this conformationally heterogeneous three-helix intermediate with unprecedented detail and highlight the importance of both native and non-native interactions in stabilizing its structure.

Biophysical Phenomena↗
PubMed2005

Effective interactions between chaotropic agents and proteins.

Chaotropic agents are cosolutes that can disrupt the hydrogen bonding network between water molecules and reduce the stability of the native state of proteins by weakening the hydrophobic effect. In this work, we represent the chaotropic agent as a factor that reduces the amount of order in the structures formed by water molecules, both in the bulk and the hydration shells around hydrophobic amino acids. In this framework we show that low chaotrope concentrations lead to a destabilization of the native state of proteins, and that high concentrations induce complete denaturation. We also find that the reduction of the number of bulk ordered states of water molecules can give origin to an effective interaction between chaotropic molecules and proteins.

Entropy↗
PubMed2005

Transition state contact orders correlate with protein folding rates.

We have used molecular dynamics simulations restrained by experimental phi values derived from protein engineering experiments to determine the structures of the transition state ensembles of ten proteins that fold with two-state kinetics. For each of these proteins we then calculated the average contact order in the transition state ensemble and compared it with the corresponding experimental folding rate. The resulting correlation coefficient is similar to that computed for the contact orders of the native structures, supporting the use of native state contact orders for predicting folding rates. The native contacts in the transition state also correlate with those of the native state but are found to be about 30% lower. These results show that, despite the high levels of heterogeneity in the transition state ensemble, the large majority of contributing structures have native-like topologies and that the native state contact order captures this phenomenon.

Kinetics↗
PubMed2005

Determination of the folding transition states of barnase by using PhiI-value-restrained simulations validated by double mutant PhiIJ-values.

The protein barnase folds from the denatured state into its native conformation via a high-energy intermediate. Using PhiI-values determined experimentally from single-point mutations as restraints in all-atom molecular dynamics simulations, we have determined ensembles of structures corresponding to the transition states for the formation of the folding intermediate and its conversion into the native state. We have also introduced a stringent validation of the approach used to calculate such structures by predicting interaction PhiIJ-values determined experimentally from a series of double-mutant cycles. The ensembles that we have obtained illustrate the heterogeneity in the nucleation-condensation process by which barnase folds. Obligatory steps of this process include the sequential formation of two folding nuclei, which correspond to the two main hydrophobic cores of the protein. Nonobligatory steps include the elongation of the strand beta1 and the formation of the helix alpha2. The results confirm that the use of experimental observables such as PhiI-values as restraints in molecular dynamics simulations is a powerful general strategy to characterize the relatively heterogeneous structural ensembles that populate nonnative regions of the energy landscapes of proteins.

Bacterial Proteins↗
PubMed2005

Prediction of "aggregation-prone" and "aggregation-susceptible" regions in proteins associated with neurodegenerative diseases.

Increasing evidence indicates that many peptides and proteins can be converted in vitro into highly organised amyloid structures, provided that the appropriate experimental conditions can be found. In this work, we define intrinsic propensities for the aggregation of individual amino acids and develop a method for identifying the regions of the sequence of an unfolded peptide or protein that are most important for promoting amyloid formation. This method is applied to the study of three polypeptides associated with neurodegenerative diseases, Abeta42, alpha-synuclein and tau. In order to validate the approach, we compare the regions of proteins that are predicted to be most important in driving aggregation, either intrinsically or as the result of mutations, with those determined experimentally. The knowledge of the location and the type of the "sensitive regions" for aggregation is important both for rationalising the effects of sequence changes on the aggregation of polypeptide chains and for the development of targeted strategies to combat diseases associated with amyloid formation.

Amino Acid Substitution↗
PubMed2005

Looking at structure, stability, and evolution of proteins through the principal eigenvector of contact matrices and hydrophobicity profiles.

We review and further develop an analytical model that describes how thermodynamic constraints on the stability of the native state influence protein evolution in a site-specific manner. To this end, we represent both protein sequences and protein structures as vectors: structures are represented by the principal eigenvector (PE) of the protein contact matrix, a quantity that resembles closely the effective connectivity of each site; sequences are represented through the "interactivity" of each amino acid type, using novel parameters that are correlated with hydropathy scales. These interactivity parameters are more strongly correlated than the other hydropathy scales that we examine with: (1) the change upon mutations of the unfolding free energy of proteins with two-states thermodynamics; (2) genomic properties as the genome-size and the genome-wide GC content; (3) the main eigenvectors of the substitution matrices. The evolutionary average of the interactivity vector correlates very strongly with the PE of a protein structure. Using this result, we derive an analytic expression for site-specific distributions of amino acids across protein families in the form of Boltzmann distributions whose "inverse temperature" is a function of the PE component. We show that our predictions are in agreement with site-specific amino acid distributions obtained from the Protein Data Bank, and we determine the mutational model that best fits the observed site-specific amino acid distributions. Interestingly, the optimal model almost minimizes the rate at which deleterious mutations are eliminated by natural selection.

Amino Acid Motifs↗