All-atom simulations of crowding effects on ubiquitin dynamics

It is well-known that crowded environments affect the stability of proteins, with strong biological and biotechnological implications; however, beyond this, crowding is also expected to affect the dynamic properties of proteins, an idea that is hard to probe experimentally. Here we report on a simulation study aimed at evaluating the effects of crowding on internal protein dynamics, based on fully all-atom descriptions of the protein, the solvent and the crowder. Our model system consists of ubiquitin, a protein whose dynamic features are closely related to its ability to bind to multiple partners, in a 325 g L-1 solution of glucose in water, a condition widely employed in in vitro studies of crowding effects. We observe a slight reduction in loop flexibility accompanied by a dramatic restriction of the conformational space explored in the timescale of the simulations (similar to 0.5 mu s), indicating that crowding slows down collective motions and the rate of exploration of the conformational space. This effect is attributed to the extensive and long-lasting interactions observed between protein residues and glucose molecules throughout the entire protein surface. Potential implications of the observed effects are discussed.

Multiscale simulations of protein dynamics

Enrico Spiga

Characteristic timescales associated with the function of biomolecules, like proteins, range from femtoseconds up to minutes, whereas their corresponding spatial extent ranges from few ̊A to μm when associating in large macromolecular complexes. Moreover, biomolecules are functional in a large variety of different physico-chemical conditions strongly dependent on pH, ionic strength, crowding agents, etc. This huge complexity is hard to be studied with an arbitrary level of resolution embracing all these spatial and temporal scales. Molecular simulation is a well established approach to gain mechanistic insights into the function of biomolecular systems, producing atomistically detailed models of in vitro and/or in vivo conditions. I present in this thesis two projects that aim at improving on the current limitations of multiscale molecular simulations, namely (i) the sampling of large systems, and (ii) the detailed representation and description of realistic physiological conditions. Addressing the first issue, I propose a new coarse-grained model for proteins to be used in molecular dynamics simulations. This coarse-grained model is based on a more accurate description of protein electrostatics, which accounts for dipolar contributions. The parameterization of this force field is based on force-matching methods and on the use of a particle swarm optimization heuristic algorithm. The obtained results are encouraging being structural and electrostatic properties accurately reproduced with the coarse-grained model for a variety of protein folds. Moreover, the parameterization procedure can be straightforwardly applied to any protein, and can be extended to a larger dataset to generate a fully transferable coarse-grained force field, to be applied to any protein and any large macromolecular assemblies for which long all-atom simulations are still a challenge. While the development and use of coarse-grained models are important to tackle the limited sampling of large systems, it is still important to use all-atom molecular dynamics simulation to investigate with high accuracy in physiological conditions protein dynamics. For this reason, I present here the results of state-of-art molecular dynamics simulations applied to study the influence of crowding agents on the internal dynamics of the protein ubiquitin. Their analysis allows to describe how ubiquitin dynamics is slaved by crowding agents. This work demonstrates that the description of protein dynamics should take into account its intrinsic multiscale nature. The development and applications of coarse-grained models permit to simulate proteins at low computational cost, the use of atomistic simulations allows to accurately describe proteins in absence and presence of crowding agents, and both of them permit to highlight the essence of protein dynamics.

EPFL2013

On the structural stability of the free and metal-loaded c-terminal domain of the prion protein

Maria Carola Colombo

A misfolded conformer of the cellular prion protein, denoted as scrapie prion protein, is considered responsible for a variety of fatal neurodegenerative diseases. Both, the function of the protein in its native conformation as well as the factors that trigger the protein misfolding and its mechanism remain as yet open issues. The aim of this thesis is to give a contribution to answering these questions by means of a variety of tools currently available in the computational (bio)chemistry community. The capability of the cellular prion protein isoform to bind metal is supported by controversial evidences and atomistic details of putative binding sites are missing. Based on the NMR structure of the C-terminal part of the cellular isoform, we performed a hybrid quantum/classical (QM/MM) Car-Parrinello molecular dynamics study to identify likely Cu(II) binding sites. By means of a bioinformatics approach, we localized the protein regions with the highest propensity for copper ion binding. The identified candidate structures were subsequently refined via QM/MM simulations and then probed by computing their EPR characteristics. Overall best agreement with the experimental EPR data was observed for a binding site involving H187. The previously characterized prion protein Cu(II)-binding sites were substituted for Mn(II) and Zn(II) and subsequently refined by means of the QM/MM Car-Parrinello approach. As a general conclusion, all the Cu-binding sites lead to stable arrangements upon Mn and Zn substitution with relative minor structural changes even within the ps time scale. The validity of the binding motifs obtained was assessed by means of comparison with a pool of known Mn and Zn binding proteins and common features were found and described. The Zn binding site in proximity of H177 was speculated to act as a protein interface zinc site with a possible direct involvement in the aggregation process. The EPR parameters of the Mn-containing binding sites were also computed. The influence of some environmental factors that can possibly trigger the misfolding event were investigated via classical molecular dynamics simulations. Specifically, protein solution containing a monovalent salt (NaCl) in four different concentrations (0.0, 0.05, 0.1, 0.2 M) were simulated at three different pH (pH 2, 4, 7) in order to highlight structural changes and local weaknesses in the protein native structural motifs. The effects of the ionic strength and pH on the secondary structural elements of the protein were identified and characterized whereas the main topological framework was preserved in all simulations. The energy landscape of the cellular to scrapie isoform misfolding pathway is currently under investigation by means of a parallel tempering (replica exchange) enhanced sampling technique in combination with classical molecular dynamics. The screening of the data collected led to the identification of a pool of prion protein scrapie candidates. A set of new folding motifs were characterized. Preliminary results and plans for future investigations are presented. With the goal of providing a structural model for the minimal misfolded aggregate based on the most likely prion protein scrapie candidates, a protein-protein docking approach was used to generate protofibril models and their structural stability and transformations investigated by means of classical molecular dynamics simulations.

EPFL2006

Solute energy based REMD

Molecular dynamics (MD) simulations have increasingly contributed to the understanding of biomolecular processes, allowing for predictions of thermodynamic and structural properties. Unfortunately, the holy grail of protein structure prediction was soon found to be severely hampered by the very rugged free energy surface of proteins, with small relative free energies separating native, folded protein conformations from unfolded states. These multiple minima frequently trap present-day protein MD simulations permanently. In order to allow the simulation to escape minima and explore wider portions of conformational space, enhanced sampling techniques were developed. One of the most popular ones, replica exchange molecular dynamics (REMD), is based on multiple parallel MD simulations that are performed with replicas of a system at increasing temperatures T1, T2, etc. Periodic Monte Carlo exchange moves are attempted, aiming to allow conformations to exchange temperature ensembles with a probability that depends on their potential energy and temperature difference. Thus, conformations are simulated at all temperatures and escape local minima with the kinetic energy provided at higher temperatures, while Boltzmann distributions are generated at all temperatures. REMD has been successful in ab-initio folding of a variety of small peptides and proteins (up to 20-30 residues). However, with larger proteins, the overlap of potential energy distributions diminishes, since the potential energy and its fluctuation scale with fkBT, respectively with √fkBT, where f is the number of degrees of freedom of the system, kB Boltzmann's constant and T the temperature. Consequently, the related Monte Carlo exchange probability and number of exchanges in a simulation are also diminished. This is generally compensated by choosing smaller temperature intervals between replicas and thereby increasing the number of necessary replicas (as well as the computational cost of the simulation) to cover a given temperature range. An additional problem relates to explicit solvent simulations, in which solvent to solvent interactions account for the largest part of the total potential energy. Consequently, explicit solvent REMD simulations almost exclusively sample solvent degrees of freedom. These two limitations have lead to the development of REMD protocols for large explicit solvent systems that are based on exchange probabilities computed with subsystem (e.g. protein only) potential energy functions, allowing for a targeted sampling of protein degrees of freedom and a reduction of the computational effort. In this thesis, this approximation is tested by implementing its simplest variation that entirely neglects solvent-solvent interaction as a new REMD protocol termed REM Dpe (Chapter 2). Possible REMD limitations for large explicit solvent systems are tested (Chapter 3) with REM Dpe, which is further applied to perform a thorough and comparative investigation of prion (Chapter 4) and doppel (Chapter 5) protein misfolding. In Chapter 2, the practical validity of the REM Dpe approximation is assessed with simulations of the prion protein, a system that is too large (i.e. 103 residues) to allow for efficient simulations using traditional REMD over the necessary temperature range. A first validation consists in testing whether protein and total potential energy distributions are consistent with their analogs from straightforward reference MD simulations. Second, the overlap pattern of the total potential energy distributions are characterized at different temperatures and show that the exchanges in the REM Dpe simulations are performed according to a Boltzmann weight. Native structures are found to have the lowest protein and total potential energies, as compared to higher energies found for various unfolded structures. Although no obvious bias is detected in the three validations, the conformational landscapes of the REM Dpe simulation at low temperatures progressively shift to non-native regions of the free energy surface. In Chapter 3, this phenomenon is quantified, and its origin identified in insufficient low temperature residence times required for refolding native-like structures. REMD is based on the assumption that systems have to be decorrelated between exchange attempts. Increasing inter-exchange times accordingly would allow for decorrelation and sufficient low temperature residence times but is practically impossible to achieve for large protein simulations, highlighting a major limitation and possible source of bias for present-day REMD simulations. We have chosen the prion as a test case because of its link to transmissible spongiform encephalopathies. Diseases of this category are believed to be caused by a rare prion protein (PrP) misfold leading from the cellular, monomeric, soluble, α-helical PrPC isoform to a pathogenic, aggregated, insoluble, β-rich PrPSc isoform of unknown structure. Gaining experimental knowledge of the PrPSc structure has remained elusive, and aroused interest in predictions supplied by computer simulations. REMD provides a powerful tool allowing to explore a diversity of misfolds and select stable ones that accumulate at lower temperatures. In Chapter 4, we describe a PrP REM Dpe simulation in which rare new β-strands are formed and arrange into a multitude of different β-sheets, reproducing the α-helix → β-sheet conversion observed with circular dichroism spectra. The α-helical and β-sheet propensities along the sequence can thus be computed. We develop and apply the β contact map clustering (bcmc) protocol to identify the most frequent β-sheet pattern defining β-rich folds. 10 new β-rich folds are found and compared to recent experimental data characterizing PrPSc, providing atomistically detailed models for putative monomeric precursors of PrPSc or β-oligomeric conformations. In Chapter 5, an analogous simulation is performed with doppel, a structural homolog of prion (with an identical three α-helix, two β-strand fold) originating from the same gene family, but characterized by a different sequence (only 25% sequence homology), expression pattern and physiological function. Unrelated to amyloid neurodegenerative diseases, doppel supplies the perfect test system to investigate the misfolding of a non-amyloidogenic protein. Prion and doppel misfolding are compared in their monomeric form in the quest to identify prion-specific features that might reveal the mechanism of conversion to PrPSc. In agreement with experiments, we find a lower thermal stability for doppel. Surprisingly, we also observe β-rich forms for doppel. However, the β-rich folds of the two proteins are very different. Moreover, a major difference is found in the free energy barriers leading from the native structure to such conformations as well as to non-native conformations in general: These barriers are low for prion and can already be crossed at 300K, while for doppel they are at least 3 times higher. This difference suggests an intrinsic misfolding and β-enrichment propensity for the monomeric form of prion as compared to doppel.

EPFL2008