Biomedical subjects

Cristian Micheletti

Publications and source records attributed to Cristian Micheletti.

15 recordsLinked to original sources

PubMed2006

Convergent dynamics in the protease enzymatic superfamily.

Proteases regulate various aspects of the life cycle in all organisms by cleaving specific peptide bonds. Their action is so central for biochemical processes that at least 2% of any known genome encodes for proteolytic enzymes. Here we show that selected proteases pairs, despite differences in oligomeric state, catalytic residues, and fold, share a common structural organization of functionally relevant regions which are further shown to undergo similar concerted movements. The structural and dynamical similarities found pervasively across evolutionarily distant clans point to common mechanisms for peptide hydrolysis.

Computational Biology↗

PubMed2006

Efficient reconstruction of complex free energy landscapes by multiple walkers metadynamics.

Recently, we have introduced a new method, metadynamics, which is able to sample rarely occurring transitions and to reconstruct the free energy as a function of several variables with a controlled accuracy. This method has been successfully applied in many different fields, ranging from chemistry to biophysics and ligand docking and from material science to crystal structure prediction. We present an important development that speeds up metadynamics calculations by orders of magnitude and renders the algorithm much more robust. We use multiple interacting simulations, walkers, for exploring and reconstructing the same free energy surface. Each walker contributes to the history-dependent potential that, in metadynamics, is an estimate of the free energy. We show that the error on the reconstructed free energy does not depend on the number of walkers, leading to a fully linear scaling algorithm even on inexpensive loosely coupled clusters of PCs. In addition, we show that the accuracy and stability of the method are much improved by combining it with a weighted histogram analysis. We check the validity of our new method on a realistic application.

Algorithms↗

PubMed2006

Entropy-driven genome organization.

DNA and RNA polymerases active on bacterial and human genomes in the crowded environment of a cell are modeled as beads spaced along a string. Aggregation of the large polymerizing complexes increases the entropy of the system through an increase in entropy of the many small crowding molecules; this occurs despite the entropic costs of looping the intervening DNA. Results of a quantitative cost/benefit analysis are consistent with observations that active polymerases cluster into replication and transcription "factories" in both pro- and eukaryotes. We conclude that the second law of thermodynamics acts through nonspecific entropic forces between engaged polymerases to drive the self-organization of genomes into loops containing several thousands (and sometimes millions) of basepairs.

Computer Simulation↗

PubMed2006

Are structural biases at protein termini a signature of vectorial folding?

Experimental investigations of the biosynthesis of a number of proteins have pointed out that part of the native structure may be acquired already during translation. We carried out a comprehensive statistical analysis of some average structural properties of proteins that have been put forward as possible signatures of this progressive buildup process. Contrary to a widespread belief, we found that there is no major propensity of the amino acids to form contacts with residues that are closer to the N-terminus. Moreover, we found that the C-terminus is significantly more compact and locally organized than the N-terminus. This bias, though, is unlikely to be related to vectorial effects, since it correlates with subtle differences in the primary sequence. These findings indicate that even if proteins acquire their structure vectorially, no signature of this seems to be detectable in their average structural properties.

Databases, Protein↗

PubMed2005

Inferring the diameter of a biopolymer from its stretching response.

We investigate the stretching response of a thick polymer model by means of extensive stochastic simulations. The computational results are synthesized in an analytic expression that characterizes how the force versus elongation curve depends on the polymer structural parameters: its thickness and granularity (spacing of the monomers). The expression is used to analyze experimental data for the stretching of various different types of biopolymers: polypeptides, polysaccharides, and nucleic acids. Besides recovering elastic parameters (such as the persistence length) that are consistent with those obtained from standard entropic models, the approach allows us to extract viable estimates for the polymers diameter and granularity. This shows that the basic structural polymer features have such a profound impact on the elastic behavior that they can be recovered with the sole input of stretching measurements.

Biocompatible Materials↗

PubMed2005

Evolutionarily conserved functional mechanics across pepsin-like and retroviral aspartic proteases.

The biological function of the aspartic protease from HIV-1 has recently been related to the conformational flexibility of its structural scaffold. Here, we use a multistep strategy to investigate whether the same mechanism affects the functionality in the pepsin-like fold. (i) We identify the set of conserved residues by using sequence-alignment techniques. These residues cluster in three distinct regions: near the cleavage-site cavity, in the four beta-sheets cross-linking the two lobes, and in a solvent-exposed region below the long beta-hairpin in the N-terminal lobe. (ii) We elucidate the role played by the conserved residues for the enzymatic functionality of one representative member of the fold family, the human beta-secretase, by means of classical molecular dynamics (MD). The conserved regions exhibit little overall mobility and yet are involved into the most important modes of structural fluctuations. These modes influence the substrate-catalytic aspartates distance through a relative rotation of the N- and C-terminal lobes. (iii) We investigate the effects of this modulation by estimating the reaction free energy at different representative substrate/enzyme conformations. The activation free energy is strongly affected by large-scale protein motions, similarly to what has been observed in the HIV-1 enzyme. (iv) We extend our findings to all other members of the two eukaryotic and retroviral fold families by recurring to a simple, topology-based, energy functional. This analysis reveals a sophisticated mechanism of enzymatic activity modulation common to all aspartic proteases. We suggest that aspartic proteases have been evolutionarily selected to possess similar functional motions despite the observed fold variations.

Amino Acid Sequence↗

PubMed2005

Study of the Villin headpiece folding dynamics by combining coarse-grained Monte Carlo evolution and all-atom molecular dynamics.

The folding mechanism of the Villin headpiece (HP36) is studied by means of a novel approach which entails an initial coarse-grained Monte Carlo (MC) scheme followed by all-atom molecular dynamics (MD) simulations in explicit solvent. The MC evolution occurs in a simplified free-energy landscape and allows an efficient selection of marginally-compact structures which are taken as viable initial conformations for the MD. The coarse-grained MC structural representation is connected to the one with atomic resolution through a "fine-graining" reconstruction algorithm. This two-stage strategy is used to select and follow the dynamics of seven different unrelated conformations of HP36. In a notable case the MD trajectory rapidly evolves towards the folded state, yielding a typical root-mean-square deviation (RMSD) of the core region of only 2.4 A from the closest NMR model (the typical RMSD over the whole structure being 4.0 A). The analysis of the various MC-MD trajectories provides valuable insight into the details of the folding and mis-folding mechanisms and particularly about the delicate influence of local and nonlocal interactions in steering the folding process.

Algorithms↗

PubMed2004

Accurate and efficient description of protein vibrational dynamics: comparing molecular dynamics and Gaussian models.

Current all-atom potential based molecular dynamics (MD) allows the identification of a protein's functional motions on a wide-range of timescales, up to few tens of nanoseconds. However, functional, large-scale motions of proteins may occur on a timescale currently not accessible by all-atom potential based MD. To avoid the massive computational effort required by this approach, several simplified schemes have been introduced. One of the most satisfactory is the Gaussian network approach based on the energy expansion in terms of the deviation of the protein backbone from its native configuration. Here, we consider an extension of this model that captures in a more realistic way the distribution of native interactions due to the introduction of effective side-chain centroids. Since their location is entirely determined by the protein backbone, the model is amenable to the same exact and computationally efficient treatment as previous simpler models. The ability of the model to describe the correlated motion of protein residues in thermodynamic equilibrium is established through a series of successful comparisons with an extensive (14 ns) MD simulation based on the AMBER potential of HIV-1 protease in complex with a peptide substrate. Thus, the model presented here emerges as a powerful tool to provide preliminary, fast yet accurate characterizations of protein near-native motion.

Computer Simulation↗

PubMed2004

Reconstructing the density of states by history-dependent metadynamics.

We present a novel method for the calculation of the energy density of states D(E) for systems described by classical statistical mechanics. The method builds on an extension of a recently proposed strategy that allows the free-energy profile of a canonical system to be recovered within a preassigned accuracy [Proc. Natl. Acad. Sci. U.S.A. 99, 12562 (2002)]]. The method allows a good control over the error on the recovered system entropy. This fact is exploited to obtain D(E) more efficiently by combining measurements at different temperatures. The accuracy and efficiency of the method are tested for the two-dimensional Ising model (up to size 50 x 50) by comparison with both exact results and previous studies. This method is a general one and should be applicable to more realistic model systems.

Journal Article↗

PubMed2003

Thermodynamics of DNA packaging inside a viral capsid: the role of DNA intrinsic thickness.

We characterize the equilibrium thermodynamics of a thick polymer confined in a spherical region of space. This is used to gain insight into the DNA packaging process. The experimental reference system for the present study is the recent characterization of the loading process of the genome inside the phi29 bacteriophage capsid. Our emphasis is on the modelling of double-stranded DNA as a flexible thick polymer (tube) instead of a beads-and-springs chain. By using finite-size scaling to extrapolate our results to genome lengths appropriate for phi29, we find that the thickness-induced force may account for up to half the one measured experimentally at high packing densities. An analogous agreement is found for the total work that has to be spent in the packaging process. Remarkably, such agreement can be obtained in the absence of any tunable parameters and is a mere consequence of the DNA thickness. Furthermore, we provide a quantitative estimate of how the persistence length of a polymer depends on its thickness. The expression accounts for the significant difference in the persistence lengths of single and double-stranded DNA (again with the sole input of their respective sections and natural nucleotide/base-pair spacing).

Bacteriophages↗

PubMed2003

Prediction of folding rates and transition-state placement from native-state geometry.

A variety of experimental and theoretical studies have established that the folding process of monomeric proteins is strongly influenced by the topology of the native state. In particular, folding times have been shown to correlate well with the contact order, a measure of contact locality. Our investigation focuses on identifying additional topologic properties that correlate with experimentally measurable quantities, such as folding rates and transition-state placement, for both two- and three-state folders. The validation against data from 40 experiments shows that a particular topological property that measures the interdependence of contacts, termed cliquishness or clustering coefficient, can account with statistically significant accuracy both for the transition state placement and especially for folding rates. The observed correlations can be further improved by optimally combining the distinct topological information captured by cliquishness and contact order.

Kinetics↗

PubMed2002

Elastic properties of proteins: insight on the folding process and evolutionary selection of native structures.

We carry out a theoretical study of the vibrational and relaxation properties of naturally occurring proteins with the purpose of characterizing both the folding and equilibrium thermodynamics. By means of a suitable model, we provide a full characterization of the spectrum and eigenmodes of vibration at various temperatures by merely exploiting the knowledge of the protein native structure. It is shown that the rate at which perturbations decay at the folding transition correlates well with experimental folding rates. This validation is carried out on a list of about 30 two-state folders. Furthermore, the qualitative analysis of residues mean square displacements (shown to reproduce crystallographic data accurately) provides a reliable and statistically accurate method to identify crucial folding sites/contacts. This novel strategy is validated against clinical data for human immunodeficiency virus type 1 (HIV-1) protease. Finally, we compare the spectra and eigenmodes of vibration of natural proteins against randomly generated compact structures and regular random graphs. The comparison reveals a distinctive enhanced flexibility of natural structures accompanied by slow relaxation times at the folding temperature. The fact that these properties are connected intimately to the presence and assembly of secondary motifs hints at the special criteria adopted by evolution in the selection of viable folds.

Biological Evolution↗

PubMed2002

Geometry and physics of proteins.

A conceptual framework for understanding the protein folding problem has remained elusive in spite of many significant advances. We show that geometrical constraints imposed by chain connectivity, compactness, and the avoidance of steric clashes can be encompassed in a natural way using a three-body potential and lead to a selection in structure space, independent of chemical details. Strikingly, secondary motifs such as hairpins, sheets, and helices, which are the building blocks of protein folds, emerge as the chosen structures for segments of the protein backbone based just on elementary geometrical considerations.

Biophysical Phenomena↗

PubMed2002

Folding pathways of prion and doppel.

The relevance of various residue positions for the stability and the folding characteristics of the prion protein in its normal cellular form are investigated by using molecular dynamics simulations of models exploiting the topology of the native state. These models allow for reproducing the experimentally validated two-state behavior of the normal prion isoform. Highly significant correlations are found between the most topologically relevant sites in our analysis and the single point mutations known to be associated with the arousal of the genetic forms of prion disease. Insight into the conformational change is provided by comparing the folding process of cellular prion and doppel that share a similar native state topology: the folding pathways of the former can be grouped in two main classes according to which tertiary structure contacts are formed first enroute to the native state. For the latter a single class of pathways leads to the native state again through a two-state process. Our results are consistent and supportive of the recent experimental findings that doppel lacks the scrapie isoform and that such remarkably different behavior involves residues in the region containing the two beta-strands and the intervening helix.

Amino Acids↗

PubMed2002

Crucial stages of protein folding through a solvable model: predicting target sites for enzyme-inhibiting drugs.

An exactly solvable model based on the topology of a protein native state is applied to identify bottlenecks and key sites for the folding of human immunodeficiency virus type 1 (HIV-1) protease. The predicted sites are found to correlate well with clinical data on resistance to Food and Drug Administration-approved drugs. It has been observed that the effects of drug therapy are to induce multiple mutations on the protease. The sites where such mutations occur correlate well with those involved in folding bottlenecks identified through the deterministic procedure proposed in this study. The high statistical significance of the observed correlations suggests that the approach may be promisingly used in conjunction with traditional techniques to identify candidate locations for drug attacks.

Amino Acids↗