Probing the quality control mechanism of theEscherichia colitwin-arginine translocase with folding variants of ade novo-designed heme protein

Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin arginine translocase (Tat). The Tat machinery exports folded and cofactor containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model Escherichia coli Tat system to recognize and translocate de novo-designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one or no heme b cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional 1H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the E. coli trimethylamine-N-oxide (TMAO) reductase (TorA) to the N-terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme b-induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system’s quality control mechanism

Modélisation Moléculaire et simulations: de system biologiques aux matériaux

I presently work at the Laboratoire de Biochimie Théorique in the Institut de Biologie et Physico- Chimique (CNRS). I have been hired as researcher (CR1) at CNRS in 2010. Previously I have been post-doc for about seven years spent in different research environments. I am a scientist with a strong background in computational biophysics and physical chemistry. My research activity involves the application and development of computational methods for tackling problems such as protein thermostability and hydration, membrane fusion and surfactant aggregation, optical and electronic properties of semiconducting polymers.The study of thermophilic protein is my principal axes of research for the next years. This research will benefit of all the expertise acquired during my past training. For example I want to unveil how the behavior of interfacial water may impact the thermodynamics stability of a protein. My past study on the structure and dynamics of water at the interface with proteins and micelles is a solid background for this future challenge. Moreover, in order to investigate the function of a protein in the high temperature regime a detailed quantum treatment of some degrees of freedom is necessary. My past activity in the field of quantum/classical as well as ab initio simulation will support this part of the research. The research on thermophilic proteins is funded by the European Research Council (ERC) via the starting grant IDEAS. A post-doc and a PhD student have joined me on this research topic. Extra-mural collaborations have been already established; I mention the collaboration with D. Madern at the Institut de Biologie Structurale (Grenoble, France), M. Maccarini at the ILL (Grenoble, France), S. Melchionna at CNR (Rome, Italy)My second line of research concerns the study of membrane fusion and in particular the role of the protein SNARE. My study will be based on a multi-scale approach using both atomistic and coarse-grain models. This research will benefit from my expertise in the field of soft-matter. My investigation will be supported by intense intra-mural collaborations, namely with P. Derreumaux and M. Baaden.Along the years I had an intense activity in code development. I am author of a code for the quantum/classical simulation of π-conjugated polymers in both ground and excited state. This code is currently used in collaboration with the group of P.J. Rossky at the university of Texas at Austin and I. Burghardt at Frankfurt University for investigating the optical and electronic properties of semiconducting plastic materials. I have also given a contribution to the development of the engine for the ab initio simulation CP2K. I have specifically implemented a sophisticated method for advanced sampling, the Temperature Accelerate Molecular Dynamics, for studying hydrogen storage and release in materials.KEYWORDS: Computer simulation, protein hydration and stability, membrane fusion, classical, quantum/classical and ab initio molecular dynamics.Je travaille actuellement au Laboratoire de Biochimie Théorique à l'Institut de Biologie et Physico-Chimique (CNRS). J'ai été recruté en tant que chercheur (CR1) au CNRS en 2010. Auparavant, j'ai été post-doc pendant environ sept ans dans différents environnements de recherche.Je suis un scientifique ayant une solide expérience en biophysique computationelle et en chimie physique. Mon activité de recherche implique l'application et le développement de méthodes de simulation pour résoudre des problèmes tels que la thermostabilité des protéines et l'hydratation, la fusion membranaire et l'agrégation des tensioactifs, les propriétés optiques et électroniques des polymères semi-conducteurs.L'étude des protéines thermophiles est mon principal axe de recherche pour les prochaines années. Cette recherche bénéficiera de toute l'expertise acquise lors de ma formation passée. Par exemple, je souhaite dévoiler comment le comportement de l'eau interfaciale peut avoir une incidence sur la stabilité thermodynamique d'une protéine. Mon étude antérieure sur la structure et la dynamique de l'eau à l'interface avec les protéines et les micelles est une base solide pour ce défi futur. En outre, afin d'étudier la fonction d'une protéine dans le régime à haute température, un traitement quantique détaillé de certains degrés de liberté est nécessaire. Mon activité passée dans le domaine de la simulation quantique / classique et ab initio appuiera cette partie de la recherche. La recherche sur les protéines thermophiles est financée par le Conseil européen de la recherche (ERC) via la subvention de départ IDEAS. Un étudiant post-doc et un doctorant m'ont rejoint sur ce sujet de recherche. Des collaborations extra-murales ont déjà été établies; Je mentionne la collaboration avec D. Madern à l'Institut de Biologie Structurale (Grenoble, France), M. Maccarini au ILL (Grenoble, France), S. Melchionna au CNR (Rome, Italie). Ma deuxième ligne de recherche concerne l'étude de fusion membranaire et en particulier le rôle de la protéine SNARE. Mon étude sera basée sur une approche multi-échelle utilisant à la fois des modèles atomiques et gros-grains. Cette recherche bénéficiera de mon expertise dans le domaine de la matière molle. Mon enquête sera soutenue par d'intenses collaborations avec de membres du laboratoire, notamment avec P. Derreumaux et M. Baaden. Au cours des années, j'ai eu une activité importante dans le développement de code. Je suis l'auteur d'un code pour la simulation quantique / classique de polymères π-conjugués à la fois à l’état fondamental et à l'état excité. Ce code est actuellement utilisé en collaboration avec le groupe de P.J. Rossky à l'université du Texas à Austin et I. Burghardt à l'Université de Francfort pour enquêter sur les propriétés optiques et électroniques des matériaux plastiques semi-conducteurs. J'ai également contribué au développement du moteur pour la simulation ab initio CP2K. J'ai spécifiquement mis en place une méthode sophistiquée pour l'échantillonnage avancé, la température Accelerate Molecular Dynamics, pour étudier le stockage et la libération d'hydrogène dans les matériaux

Computational Insights into the Unfolding of a Destabilized Superoxide Dismutase 1 Mutant

In this work, we investigate the β-barrel of superoxide dismutase 1 (SOD1) in a mutated form, the isoleucine 35 to alanine (I35A) mutant, commonly used as a model system to decipher the role of the full-length apoSOD1 protein in amyotrophic lateral sclerosis (ALS). It is known from experiments that the mutation reduces the stability of the SOD1 barrel and makes it largely unfolded in the cell at 37 degrees Celsius. We deploy state-of-the-art computational machinery to examine the thermal destabilization of the I35A mutant by comparing two widely used force fields, Amber a99SB-disp and CHARMM36m. We find that only the latter force field, when combined with the Replica Exchange with Solute Scaling (REST2) approach, reproduces semi-quantitatively the experimentally observed shift in the melting between the original and the mutated SOD1 barrel. In addition, we analyze the unfolding process and the conformational landscape of the mutant, finding these largely similar to those of the wildtype. Nevertheless, we detect an increased presence of partially misfolded states at ambient temperatures. These states, featuring conformational changes in the region of the β-strands β4−β6, might provide a pathway for nonnative aggregation

Le transfert d'électron dans le Centre Réactionnel de Rb.sphaeroides (asymétrie fonctionnelle et mutations étudiées par simulation de Dynamique moléculaire)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Are coarse-grained models apt to detect protein thermal stability? The case of \OPEP\ force field

7th IDMRCS: Relaxation in Complex SystemsInternational audienceno abstrac

Early Stage of the Dehydrogenation of NaAlH4 by Ab Initio Rare Event Simulations

We investigated the chemical species formed in the early stage of the first dehydrogenation reaction of (undoped) NaAlH4 (NaAlH4 reversible arrow 1/3Na(3)AlH(6) + 2/3Al + H-2(up arrow)). We found that the experimental barrier to dehydrogenation (120 kj/mol) is compatible with the Al-H bond breaking of an AlH4 unit. We observed the formation of AlH3, AlH5, NaH, and NaH2. We computed the free energy profiles for the process of formation of the two most frequent species, AlH5 and NaH. While the free energy barriers for creating the species are comparable, the sample containing NaH is thermodynamically much more stable than the one containing AlH5. We did not observe the formation of H-2 nor of the other products of the complete reaction. We attribute the lack of formation of H-2 at this early stage to the fact that the hydrogen released in the sample is negatively charged and cannot quickly oxidate in the absence of chemical species that can efficiently be reduced. This finding suggests a possible mechanism for the catalytic action of Ti in Ti-doped samples. Finally, we studied the concentration of the various AlHi species as a function of the distance from the surface and found that species with higher negative charge are formed far from the surface, while neutral species are formed preferably doser to the surface, in particular in the top layer

Protein thermal stability

International audienc