Solute energy based REMD | EPFL Graph Search

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.

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

CO2 capture in amine solutions: modelling and simulations with non-empirical methods

Wanda Andreoni, Fabio Pietrucci

Absorption in aqueous amine solutions is the most advanced technology for the capture of CO2, although suffering from drawbacks that do not allow exploitation on large scale. The search for optimum solvents has been pursued with empirical methods and has also motivated a number of computational approaches over the last decade. However, a deeper level of understanding of the relevant chemical reactions in solution is required so as to contribute to this effort. We present here a brief critical overview of the most recent applications of computer simulations using ab initio methods. Comparison of their outcome shows a strong dependence on the structural models employed to represent the molecular systems in solution and on the strategy used to simulate the reactions. In particular, the results of very recent ab initio molecular dynamics augmented with metadynamics are summarized, showing the crucial role of water, which has been so far strongly underestimated both in the calculations and in the interpretation of experimental data. Indications are given for advances in computational approaches that are necessary if meant to contribute to the rational design of new solvents.

Iop Publishing Ltd2016

Computational Investigation of Intracellular Signalling Cascades

Siri Camee van Keulen

In order to adjust to changes in the environment, cells can communicate via signalling cascades. A common pathway for intercellular communication is the G-protein-coupled- receptor (GPCR) signal transduction cascade which is triggered by a (chemical or physical) extracellular signal, such as light or small molecules. This external interaction induces conformational changes that can lead either to GPCR activation or deactivation. In the active form, GPCRs can induce the activation of trimeric G proteins, which results in the dissociation of the Galpha from the beta and gamma subunits. Subsequently, active Galpha can interact with other cytosolic proteins, such as adenylyl cyclase (AC), which converts adenosine triphosphate to cyclic adenosine monophosphate (cAMP) upon stimulation. AC activation can be induced via stimulatory Galpha subunit (Galpha_s) interactions, while inhibition is related to inhibitory Galpha (Galpha_i) association. X-ray crystallography is crucial for the computational study of the GPCR signal transduction pathway. However, sometimes important molecules or moieties can become lost or cannot be incorporated during crystallisation procedures, which can have a significant effect on a protein’s conformation and function. In this thesis, three steps in the GPCR signal transduction cascade have been studied, rhodopsin activation (i), the conformation of active Galpha_i (ii) and the interaction between Galpha_i and AC (iii). In all three cases some groups or molecules that are absent in the X-ray structures play a vital role in the function of the proteins. In the first project, rhodopsin’s early intermediates after photon exposure are investigated together with a deprotonation reaction that takes place in the later stages of the activation pathway. The classical molecular dynamics (MD) and mixed quantum mechanics/molecular mechanics MD results suggest that photon exposure induces active site rearrangements which increase the stability of rhodopsin’s deprotonated Schiff base state. A bridging water molecule in the active site that is absent in the X-ray structure appears to play an important role during the deprotonation mechanism, which demonstrates the dependence of the protein’s function on its environment. The second study investigates the conformation of the active soluble form of Galpha_i, which has not yet been fully resolved via experimental studies due to crystallisation difficulties of the lipidated N-terminus. To investigate the effect of lipidation via classical MD simulations, a model of soluble lipid-bound Galpha_i was constructed. The simulations show that Galpha_i can form a hydrophobic pocket for the lipid on the protein surface. Other regions of the protein, important for protein-protein interaction, are adjusted as well. Hence, the post-translational modification appears to have a significant impact on Galpha_i’s active conformation and function, which was not captured via the unlipidated X-ray structures. The last project is devoted to a complex of active soluble Galpha_i and AC (Galpha_i :AC). While the interaction site for Galpha_s on AC is known, the interaction site for Galpha_i has not yet been identified. Classical MD simulations were performed on a docked Galpha_i:AC complex to understand the inhibition mechanisms as well as the differentiation between AC stimulation/inhibition. The results show that Galpha_i binding not only leads to AC inhibition via impeding ATP binding but also by preventing re-activation via Galpha_s through closure of the subunit’s interaction site on AC. Keywords: GPCR signal transduction pathway, G-protein-coupled recepter, rhodopsin, G protein, complex formation, cis-trans isomerisation, adenylyl cyclase.