Cristian Micheletti

Scuola Internazionale Superiore di Studi Avanzati di Trieste, Trst, Friuli Venezia Giulia, Italy

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Publications (115)467.14 Total impact

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    ABSTRACT: Recent studies have shown that single-stranded viral RNAs fold into more compact structures than random RNA sequences with similar chemical composition and identical length. Based on this comparison it has been suggested that wild-type viral RNA may have evolved to be atypically compact so as to aid its encapsidation and assist the viral assembly process. In order to further explore the compactness selection hypothesis, we systematically compare the predicted sizes of more than one hundred wild-type viral sequences with those of their mutants, which are evolved in silico and subject to a number of known evolutionary constraints. In particular, we enforce mutation synonynimity, preserve the codon-bias, and leave untranslated regions intact. It is found that progressive accumulation of these restricted mutations still suffices to completely erase the characteristic compactness imprint of the viral RNA genomes, making them in this respect physically indistinguishable from randomly shuffled RNAs. This shows that maintaining the physical compactness of the genome is indeed a primary factor among ssRNA viruses evolutionary constraints, contributing also to the evidence that synonymous mutations in viral ssRNA genomes are not strictly neutral.
    10/2014;
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    ABSTRACT: The HIV-1 Tat protein and several small molecules bind to HIV-1 Trans-Activation Responsive RNA (TAR) by selecting sparsely populated but pre-existing conformations. Thus, a complete characterization of TAR conformational ensemble and dynamics is crucial to understand this paradigmatic system and could facilitate the discovery of new anti-virals targeting this essential regulatory element. We show here that molecular dynamics simulations can be effectively used towards this goal by bridging the gap between functionally-relevant timescales that are inaccessible to current experimental techniques. Specifically, we have performed several independent microsecond long molecular simulations of TAR based on one of the most advanced force fields available for RNA, the parmbsc0 AMBER . Our simulations are first validated against available experimental data, yielding an excellent agreement with measured residual dipolar couplings and order parameter S2. This contrast with previous MD simulations (Salmon et al., J. Am. Chem. Soc. 2013 135, 5457-5466) based on the CHARMM36 force field, which could achieve only modest accord with the experimental RDC values. Next, we direct the computation towards characterizing the internal dynamics of TAR over the microsecond timescale. We show that the conformational fluctuations observed over this previously-elusive timescale have a strong functionally-oriented character in that they are primed to sustain and assist ligand binding.
    Journal of the American Chemical Society 10/2014; · 10.68 Impact Factor
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    Cristian Micheletti, Marco Di Stefano, Henri Orland
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    ABSTRACT: The ongoing effort to detect and characterize physical entanglement in biopolymers has so far established that knots are present in many globular proteins and also abound in viral DNA packaged inside bacteriophages. RNA molecules, on the other hand, have not yet been systematically screened for the occurrence of physical knots. We have accordingly undertaken the systematic profiling of the ~6,000 RNA structures present in the protein data bank. The search identified no more than three deeply-knotted RNA molecules. These are ribosomal RNAs solved by cryo-em and consist of about 3,000 nucleotides. Compared to the case of proteins and viral DNA, the observed incidence of RNA knots is therefore practically negligible. This suggests that either evolutionary selection, or thermodynamic and kinetic folding mechanisms act towards minimizing the entanglement of RNA to an extent that is unparalleled by other types of biomolecules. The properties of the three observed RNA knotting patterns provide valuable clues for designing RNA sequences capable of self-tying in a twist-knot fold.
    10/2014;
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    ABSTRACT: The dynamical properties of entangled polyelectrolytes are investigated theoretically and computationally for a proposed novel micromanipulation setup. Specifically, we investigate the effects of DC and AC electric fields acting longitudinally on knotted DNA chains, modelled as semiflexible chains of charged beads, under mechanical tension. We consider various experimentally accessible values of the field amplitude and frequency as well as several of the simplest knot types. In particular, we consider both torus and twist knots because they are respectively known to be able or unable to slide along macroscopic threads and ropes. Strikingly, this qualitative distinction disappears in this microscopic context because all the considered knot types acquire a systematic drift in the direction of the electric force. Notably, the knot drift velocity and diffusion coefficient in zero field (both measurable also experimentally) can be used to define a characteristic "frictional" lengthscale for the various knot types. This previously unexplored length provides valuable information on the extent of self-interactions in the nominal knotted region. It is finally observed that the motion of a knot can effectively follow the AC field only if the driving period is larger than the knot relaxation time (for which the self-diffusion time provides an upper bound). These results suggest that salient aspects of the intrinsic dynamics of knots in DNA chains could be probed experimentally by means of external, time-dependent electric fields.
    Soft Matter 07/2014; · 4.15 Impact Factor
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    ABSTRACT: Bacteriophages initiate infection by releasing their double-stranded DNA into the cytosol of their bacterial host. However, what controls and sets the timescales of DNA ejection? Here we provide evidence from stochastic simulations which shows that the topology and organization of DNA packed inside the capsid plays a key role in determining these properties. Even with similar osmotic pressure pushing out the DNA, we find that spatially ordered DNA spools have a much lower effective friction than disordered entangled states. Such spools are only found when the tendency of nearby DNA strands to align locally is accounted for. This topological or conformational friction also depends on DNA knot type in the packing geometry and slows down or arrests the ejection of twist knots and very complex knots. We also find that the family of (2, 2k+1) torus knots unravel gradually by simplifying their topology in a stepwise fashion. Finally, an analysis of DNA trajectories inside the capsid shows that the knots formed throughout the ejection process mirror those found in gel electrophoresis experiments for viral DNA molecules extracted from the capsids.
    Proceedings of the National Academy of Sciences 11/2013; · 9.81 Impact Factor
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    ABSTRACT: Key steps in a viral life-cycle, such as self-assembly of a protective protein container or in some cases also subsequent maturation events, are governed by the interplay of physico-chemical mechanisms involving various spatial and temporal scales. These salient aspects of a viral life cycle are hence well described and rationalised from a mesoscopic perspective. Accordingly, various experimental and computational efforts have been directed towards identifying the fundamental building blocks that are instrumental for the mechanical response, or constitute the assembly units, of a few specific viral shells. Motivated by these earlier studies we introduce and apply a general and efficient computational scheme for identifying the stable domains of a given viral capsid. The method is based on elastic network models and quasi-rigid domain decomposition. It is first applied to a heterogeneous set of well-characterized viruses (CCMV, MS2, STNV, STMV) for which the known mechanical or assembly domains are correctly identified. The validated method is next applied to other viral particles such as L-A, Pariacoto and polyoma viruses, whose fundamental functional domains are still unknown or debated and for which we formulate verifiable predictions. The numerical code implementing the domain decomposition strategy is made freely available.
    PLoS Computational Biology 11/2013; 9(11):e1003331. · 4.87 Impact Factor
  • Journal of Molecular Graphics and Modelling. 08/2013; 18(s 4–5):549.
  • L. Tubiana, A. Rosa, F. Fragiacomo, C. Micheletti
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    ABSTRACT: We report on a computational study of the statics and dynamics of long flexible linear polymers that spontaneously knot and unknot. Specifically, the equilibrium self-entanglement properties, such as the knotting probability, knot length and position, are investigated with extensive Monte Carlo sampling of chains of up to 15,000 beads. Tens of such equilibrated chains of up to 4, 096 beads are next used as starting points for Langevin dynamics simulations. The complex interplay of chain dynamics and self-knotting is addressed by monitoring the time evolution of various metric and entanglement properties. In particular, the extensive duration of the simulations allows for observing the spontaneous formation and disappearance of prime and composite physical knots in linear chains. Notably, a sizeable fraction of self-knotting and unknotting events is found to involve regions that are far away from the chain termini. To the best of our knowledge this represents the first instance where spontaneous changes in knotting for linear homopolymers are systematically characterized using unbiased dynamics simulations.
    Macromolecules 05/2013; 46(9):3669-3678. · 5.93 Impact Factor
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    ABSTRACT: UreG are small GTP-binding (G) proteins that catalyse the hydrolysis of GTP necessary for the maturation of urease, a virulence factor in bacterial pathogenesis. UreG proteins are the first documented cases of intrinsically disordered enzymes. The comprehension of the dynamics of folding/unfolding events occurring in this protein could shed light on the enzymatic mechanism of UreG. Here, we used the recently developed replica exchange with solute tempering (REST2) computational methodology to explore the conformational space of UreG from Helicobacter pylori (HpUreG) and to identify its structural fluctuations. The same simulation and analysis protocol has been also applied to HypB from Methanocaldococcus jannaschii (MjHypB), which is closely related to UreG for both sequence and function, even though it is not intrinsically disordered. A comparison of the two systems reveals that both HpUreG and MjHypB feature a substantial rigidity of the protein regions involved in catalysis, justifying its residual catalytic activity. On the other hand, HpUreG tends to unfold more than MjHypB in portions involved in protein-protein interactions with metallo-chaperones necessary for the formation of multi-protein complexes known to be involved in urease activation.
    Biochemistry 04/2013; · 3.38 Impact Factor
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    ABSTRACT: We report on atomistic simulation of the folding of a natively-knotted protein, MJ0366, based on a realistic force field. To the best of our knowledge this is the first reported effort where a realistic force field is used to investigate the folding pathways of a protein with complex native topology. By using the dominant-reaction pathway scheme we collected about 30 successful folding trajectories for the 82-amino acid long trefoil-knotted protein. Despite the dissimilarity of their initial unfolded configuration, these trajectories reach the natively-knotted state through a remarkably similar succession of steps. In particular it is found that knotting occurs essentially through a threading mechanism, involving the passage of the C-terminal through an open region created by the formation of the native [Formula: see text]-sheet at an earlier stage. The dominance of the knotting by threading mechanism is not observed in MJ0366 folding simulations using simplified, native-centric models. This points to a previously underappreciated role of concerted amino acid interactions, including non-native ones, in aiding the appropriate order of contact formation to achieve knotting.
    PLoS Computational Biology 03/2013; 9(3):e1003002. · 4.87 Impact Factor
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    ABSTRACT: The connection between chromatin nuclear organization and gene activity is vividly illustrated by the observation that transcriptional coregulation of certain genes appears to be directly influenced by their spatial proximity. This fact poses the more general question of whether it is at all feasible that the numerous genes that are coregulated on a given chromosome, especially those at large genomic distances, might become proximate inside the nucleus. This problem is studied here using steered molecular dynamics simulations in order to enforce the colocalization of thousands of knowledge-based gene sequences on a model for the gene-rich human chromosome 19. Remarkably, it is found that most ([Formula: see text]) gene pairs can be brought simultaneously into contact. This is made possible by the low degree of intra-chromosome entanglement and the large number of cliques in the gene coregulatory network. A clique is a set of genes coregulated all together as a group. The constrained conformations for the model chromosome 19 are further shown to be organized in spatial macrodomains that are similar to those inferred from recent HiC measurements. The findings indicate that gene coregulation and colocalization are largely compatible and that this relationship can be exploited to draft the overall spatial organization of the chromosome in vivo. The more general validity and implications of these findings could be investigated by applying to other eukaryotic chromosomes the general and transferable computational strategy introduced here.
    PLoS Computational Biology 03/2013; 9(3):e1003019. · 4.87 Impact Factor
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    Enzo Orlandini, Cristian Micheletti
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    ABSTRACT: The amount and type of self-entanglement of DNA filaments is significantly affected by spatial confinement, which is ubiquitous in biological systems. Motivated by recent advancements in single DNA molecule experiments based on nanofluidic devices and by the introduction of algorithms capable of detecting knots in open chains, we investigate numerically the entanglement of linear, open DNA chains confined inside nano-slits. The results regard the abundance, type, and length of occurring knots and are compared with recent findings for DNA inside nano-channels. In both cases, the width of the confining region, D, spans the 30 nm-1 μm range and the confined DNA chains are 1-4 μm long. It is found that the knotting probability is maximum for slit widths in the 70-100 nm range. However, over the considered DNA contour lengths, the maximum incidence of knots remains below 20%, while for channel confinement it tops 50%. Further differences of the entanglement are seen for the average contour length of the knotted region, which drops significantly below D ~100 nm for channel-confinement, while it stays approximately constant for slit-like confinement. These properties ought to reverberate in different kinetic properties of linear DNA depending on confinement and could be detectable experimentally or exploitable in nano-technological applications.
    Journal of Biological Physics 03/2013; 39(2):267-75. · 0.95 Impact Factor
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    ABSTRACT: For several decades, the presence of knots in naturally-occurring proteins was largely ruled out a priori for its supposed incompatibility with the efficiency and robustness of folding processes. For this very same reason, the later discovery of several unrelated families of knotted proteins motivated researchers to look into the physico-chemical mechanisms governing the concerted sequence of folding steps leading to the consistent formation of the same knot type in the same protein location. Besides experiments, computational studies are providing considerable insight into these mechanisms. Here, we revisit a number of such recent investigations within a common conceptual and methodological framework. By considering studies employing protein models with different structural resolution (coarse-grained or atomistic) and various force fields (from pure native-centric to realistic atomistic ones), we focus on the role of native and non-native interactions. For various unrelated instances of knotted proteins, non-native interactions are shown to be very important for favoring the emergence of conformations primed for successful self-knotting events.
    Biomolecules. 01/2013; 4(1):1-19.
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    Michaël Bon, Cristian Micheletti, Henri Orland
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    ABSTRACT: We present McGenus, an algorithm to predict RNA secondary structures with pseudoknots. The method is based on a classification of RNA structures according to their topological genus. McGenus can treat sequences of up to 1000 bases and performs an advanced stochastic search of their minimum free energy structure allowing for non-trivial pseudoknot topologies. Specifically, McGenus uses a Monte Carlo algorithm with replica exchange for minimizing a general scoring function which includes not only free energy contributions for pair stacking, loop penalties, etc. but also a phenomenological penalty for the genus of the pairing graph. The good performance of the stochastic search strategy was successfully validated against TT2NE which uses the same free energy parametrization and performs exhaustive or partially exhaustive structure search, albeit for much shorter sequences (up to 200 bases). Next, the method was applied to other RNA sets, including an extensive tmRNA database, yielding results that are competitive with existing algorithms. Finally, it is shown that McGenus highlights possible limitations in the free energy scoring function. The algorithm is available as a web server at http://ipht.cea.fr/rna/mcgenus.php.
    Nucleic Acids Research 12/2012; · 8.81 Impact Factor
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    Cristian Micheletti
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    ABSTRACT: The growing interest for comparing protein internal dynamics owes much to the realisation that protein function can be accompanied or assisted by structural fluctuations and conformational changes. Analogously to the case of functional structural elements, those aspects of protein flexibility and dynamics that are functionally oriented should be subject to evolutionary conservation. Accordingly, dynamics-based protein comparisons or alignments could be used to detect protein relationships that are more elusive to sequence and structural alignments. Here we provide an account of the progress that has been made in recent years towards developing and applying general methods for comparing proteins in terms of their internal dynamics and advance the understanding of the structure-function relationship.
    Physics of Life Reviews 10/2012; · 6.58 Impact Factor
  • Cristian Micheletti, Enzo Orlandini
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    ABSTRACT: We report on a numerical study of the equilibrium metric scaling features and knotting properties of linear and circular model DNA chains inside cylindrical channels. The model chains are 1.2–4.8 μm long and the channels are 30–1500 nm wide. The metric scaling behaviour of confined circular chains is shown to follow the de Gennes regime down to channel widths of 85 nm, analogously to the well-studied case of open chains. A notable fact is that the same channel width (85 nm) is associated with a maximum incidence of knots in both linear and circularised chains regardless of their contour lengths. This coincidence suggests that topology-based features could aid the challenging characterization of the metric crossover behaviour. Finally, it is shown that compared to slits and cavities, channels are the confining geometry where chains have the largest probability to be tied in simple knot types. The strong width-dependent knotting probability and the abundance of simple knots suggest that nano-fluidic devices based on nano-channels could be ideally suited for producing DNA molecules that are preferentially tied in simple knots for sieving molecules based on their knotted state.
    Soft Matter 10/2012; 8(42):10959-10968. · 4.15 Impact Factor
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    A. Rosa, M. Di Ventra, C. Micheletti
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    ABSTRACT: The advent of solid state nanodevices allows for interrogating the physico-chemical properties of a polyelectrolyte chain by electrophoretically driving it through a nanopore. Salient dynamical aspects of the translocation process have been recently characterized by theoretical and computational studies of model polymer chains free from self-entanglement. However, sufficiently long equilibrated chains are necessarily knotted. The impact of such topological "defects" on the translocation process is largely unexplored, and is addressed in this study. By using Brownian dynamics simulations on a coarse-grained polyelectrolyte model we show that knots, despite being trapped at the pore entrance, do not "per se" cause the translocation process to jam. Rather, knots introduce an effective friction that increases with the applied force, and practically halts the translocation above a threshold force. The predicted dynamical crossover, which is experimentally verifiable, is of relevance in applicative contexts, such as DNA nanopore sequencing.
    Physical Review Letters 07/2012; 109(11). · 7.73 Impact Factor
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    Tatjana Skrbić, Cristian Micheletti, Pietro Faccioli
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    ABSTRACT: Stochastic simulations of coarse-grained protein models are used to investigate the propensity to form knots in early stages of protein folding. The study is carried out comparatively for two homologous carbamoyltransferases, a natively-knotted N-acetylornithine carbamoyltransferase (AOTCase) and an unknotted ornithine carbamoyltransferase (OTCase). In addition, two different sets of pairwise amino acid interactions are considered: one promoting exclusively native interactions, and the other additionally including non-native quasi-chemical and electrostatic interactions. With the former model neither protein shows a propensity to form knots. With the additional non-native interactions, knotting propensity remains negligible for the natively-unknotted OTCase while for AOTCase it is much enhanced. Analysis of the trajectories suggests that the different entanglement of the two transcarbamylases follows from the tendency of the C-terminal to point away from (for OTCase) or approach and eventually thread (for AOTCase) other regions of partly-folded protein. The analysis of the OTCase/AOTCase pair clarifies that natively-knotted proteins can spontaneously knot during early folding stages and that non-native sequence-dependent interactions are important for promoting and disfavouring early knotting events.
    PLoS Computational Biology 06/2012; 8(6):e1002504. · 4.87 Impact Factor
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    C. Micheletti, E. Orlandini
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    ABSTRACT: Advanced Monte Carlo simulations are used to study the effect of nano-slit confinement on metric and topological properties of model DNA chains. We consider both linear and circularised chains with contour lengths in the 1.2--4.8 $\mu$m range and slits widths spanning continuously the 50--1250nm range. The metric scaling predicted by de Gennes' blob model is shown to hold for both linear and circularised DNA up to the strongest levels of confinement. More notably, the topological properties of the circularised DNA molecules have two major differences compared to three-dimensional confinement. First, the overall knotting probability is non-monotonic for increasing confinement and can be largely enhanced or suppressed compared to the bulk case by simply varying the slit width. Secondly, the knot population consists of knots that are far simpler than for three-dimensional confinement. The results suggest that nano-slits could be used in nano-fluidic setups to produce DNA rings having simple topologies (including the unknot) or to separate heterogeneous ensembles of DNA rings by knot type.
    Macromolecules. 04/2012; 45(4).
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    ABSTRACT: Nanografted monolayers (NAMs) of DNA show novel physico-chemical properties that make them ideally suited for advanced biosensing applications. In comparison with alternative solid-phase techniques for diagnostic DNA detection, NAMs have the advantage of combining a small size with a high homogeneity of the DNA surface coverage. These two properties favour the extreme miniaturization and ultrasensitivity in high-throughput biosensing devices. The systematic use of NAMs for quantitative DNA (and protein) detection has so far suffered from the lack of a control on key fabrication parameters, such as the ss- or ds-DNA surface coverage. Here we report on a combined experimental-computational study that allows us to estimate the surface density of the grafted DNA by analyzing the sample mechanical response, that is the DNA patch height vs. applied tip load curves. It is shown that the same analysis scheme can be used to detect the occurrence of hybridization with complementary strands in solution and estimate its efficiency. Thanks to these quantitative relationships it is possible to use a single AFM-based setup to: (i) fabricate a DNA NAM, (ii) control the DNA surface coverage, and (iii) characterize its level of hybridization helping the design of NAMs with pre-determined fabrication parameters.
    Nanoscale 03/2012; 4(5):1734-41. · 6.73 Impact Factor

Publication Stats

2k Citations
467.14 Total Impact Points

Institutions

  • 1998–2014
    • Scuola Internazionale Superiore di Studi Avanzati di Trieste
      Trst, Friuli Venezia Giulia, Italy
  • 1997–2013
    • University of Padova
      • Department of Physics and Astronomy "Galileo Galilei"
      Padua, Veneto, Italy
  • 2009
    • The University of Edinburgh
      • School of Physics and Astronomy
      Edinburgh, Scotland, United Kingdom
    • Institut Pasteur de Montevideo
      Ciudad de Montevideo, Montevideo, Uruguay
  • 2008
    • Italian Institute of Technology (IIT)
      Genova, Liguria, Italy
  • 2005
    • The University of Warwick
      • Warwick Mathematics Institute
      Coventry, England, United Kingdom
  • 1995–2003
    • University of Oxford
      • Department of Physics
      Oxford, ENG, United Kingdom
  • 1970–2003
    • Abdus Salam International Centre for Theoretical Physics
      Trst, Friuli Venezia Giulia, Italy