Fitting Force Field Parameters to NMR Relaxation Data.

We present an approach to optimize force field parameters using time-dependent data from NMR relaxation experiments. To do so, we scan parameters in the dihedral angle potential energy terms describing the rotation of the methyl groups in proteins and compare NMR relaxation rates calculated from molecular dynamics simulations with the modified force fields to deuterium relaxation measurements of T4 lysozyme. We find that a small modification of Cγ methyl groups improves the agreement with experiments both for the protein used to optimize the force field and when validating using simulations of CI2 and ubiquitin. We also show that these improvements enable a more effective a posteriori reweighting of the MD trajectories. The resulting force field thus enables more direct comparison between simulations and side-chain NMR relaxation data and makes it possible to construct ensembles that better represent the dynamics of proteins in solution.

Double Mutant of Chymotrypsin Inhibitor 2 Stabilized through Increased Conformational Entropy.

The conformational heterogeneity of a folded protein can affect not only its function but also stability and folding. We recently discovered and characterized a stabilized double mutant (L49I/I57V) of the protein CI2 and showed that state-of-the-art prediction methods could not predict the increased stability relative to the wild-type protein. Here, we have examined whether changed native-state dynamics, and resulting entropy changes, can explain the stability changes in the double mutant protein, as well as the two single mutant forms. We have combined NMR relaxation measurements of the ps-ns dynamics of amide groups in the backbone and the methyl groups in the side chains with molecular dynamics simulations to quantify the native-state dynamics. The NMR experiments reveal that the mutations have different effects on the conformational flexibility of CI2: a reduction in conformational dynamics (and entropy estimated from this) of the native state of the L49I variant correlates with its decreased stability, while increased dynamics of the I57V and L49I/I57V variants correlates with their increased stability. These findings suggest that explicitly accounting for changes in native-state entropy might be needed to improve the predictions of the effect of mutations on protein stability.

Transient exposure of a buried phosphorylation site in an autoinhibited protein.

Autoinhibition is a mechanism used to regulate protein function, often by making functional sites inaccessible through the interaction with a cis-acting inhibitory domain. Such autoinhibitory domains often display a substantial degree of structural disorder when unbound, and only become structured in the inhibited state. These conformational dynamics make it difficult to study the structural origin of regulation, including effects of regulatory post-translational modifications. Here, we study the autoinhibition of the Dbl Homology domain in the protein Vav1 by the so-called acidic inhibitory domain. We use molecular simulations to study the process by which a mostly unstructured inhibitory domain folds upon binding and how transient exposure of a key buried tyrosine residue makes it accessible for phosphorylation. We show that the inhibitory domain, which forms a helix in the bound and inhibited stated, samples helical structures already before binding and that binding occurs via a molten-globule-like intermediate state. Together, our results shed light on key interactions that enable the inhibitory domain to sample a finely tuned equilibrium between an inhibited and a kinase-accessible state.

Fitting Side-Chain NMR Relaxation Data Using Molecular Simulations.

Proteins display a wealth of dynamical motions that can be probed using both experiments and simulations. We present an approach to integrate side-chain NMR relaxation measurements with molecular dynamics simulations to study the structure and dynamics of these motions. The approach, which we term ABSURDer (average block selection using relaxation data with entropy restraints), can be used to find a set of trajectories that are in agreement with relaxation measurements. We apply the method to deuterium relaxation measurements in T4 lysozyme and show how it can be used to integrate the accuracy of the NMR measurements with the molecular models of protein dynamics afforded by the simulations. We show how fitting of dynamic quantities leads to improved agreement with static properties and highlight areas needed for further improvements of the approach.

Structural and Biophysical Properties of Supercharged and Circularized Nanodiscs.

Nanodiscs based on membrane scaffold proteins (MSPs) and phospholipids are used as membrane mimics to stabilize membrane proteins in solution for structural and functional studies. Combining small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and time-resolved small-angle neutron scattering (TR-SANS), we characterized the structure and lipid bilayer properties of five different nanodiscs made with dimyristoylphosphatidylcholine and different MSPs varying in size, charge, and circularization. Our SAXS modeling showed that the structural parameters of the embedded lipids are all similar, irrespective of the MSP properties. DSC showed that the lipid packing is not homogeneous in the nanodiscs and that a 20 Å wide boundary layer of lipids with perturbed packing is located close to the MSP, while the packing of central lipids is tighter than in large unilamellar vesicles. Finally, TR-SANS showed that lipid exchange rates in nanodiscs decrease with increasing nanodisc size and are lower for the nanodiscs made with supercharged MSPs compared to conventional nanodiscs. Altogether, the results provide a thorough biophysical understanding of the nanodisc as a model membrane system, which is important in order to carry out and interpret experiments on membrane proteins embedded in such systems.

Ab initio determination of the shape of membrane proteins in a nanodisc.

New software, called Marbles, is introduced that employs SAXS intensities to predict the shape of membrane proteins embedded into membrane nanodiscs. To gain computational speed and efficient convergence, the strategy is based on a hybrid approach that allows one to account for the contribution of the nanodisc to the SAXS intensity through a semi-analytical model, while the embedded membrane protein is treated as a set of beads, similarly to as in well known ab initio methods. The reliability and flexibility of this approach is proved by benchmarking the code, implemented in C++ with a Python interface, on a toy model and two proteins with very different geometry and size.

Pharmacological inactivation of the prion protein by targeting a folding intermediate.

Recent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. These techniques allowed us to design a drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein levels by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP and identified four different small molecule ligands for this conformer, all capable of selectively lowering the load of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression.

How to learn from inconsistencies: Integrating molecular simulations with experimental data.

Molecular simulations and biophysical experiments can be used to provide independent and complementary insights into the molecular origin of biological processes. A particularly useful strategy is to use molecular simulations as a modeling tool to interpret experimental measurements, and to use experimental data to refine our biophysical models. Thus, explicit integration and synergy between molecular simulations and experiments is fundamental for furthering our understanding of biological processes. This is especially true in the case where discrepancies between measured and simulated observables emerge. In this chapter, we provide an overview of some of the core ideas behind methods that were developed to improve the consistency between experimental information and numerical predictions. We distinguish between situations where experiments are used to refine our understanding and models of specific systems, and situations where experiments are used more generally to refine transferable models. We discuss different philosophies and attempt to unify them in a single framework. Until now, such integration between experiments and simulations have mostly been applied to equilibrium data, and we discuss more recent developments aimed to analyze time-dependent or time-resolved data.

Full atomistic model of prion structure and conversion.

Prions are unusual protein assemblies that propagate their conformationally-encoded information in absence of nucleic acids. The first prion identified, the scrapie isoform (PrPSc) of the cellular prion protein (PrPC), caused epidemic and epizootic episodes [1]. Most aggregates of other misfolding-prone proteins are amyloids, often arranged in a Parallel-In-Register-ß-Sheet (PIRIBS) [2] or ß-solenoid conformations [3]. Similar folding models have also been proposed for PrPSc, although none of these have been confirmed experimentally. Recent cryo-electron microscopy (cryo-EM) and X-ray fiber-diffraction studies provided evidence that PrPSc is structured as a 4-rung ß-solenoid (4RßS) [4, 5]. Here, we combined different experimental data and computational techniques to build the first physically-plausible, atomic resolution model of mouse PrPSc, based on the 4RßS architecture. The stability of this new PrPSc model, as assessed by Molecular Dynamics (MD) simulations, was found to be comparable to that of the prion forming domain of Het-s, a naturally-occurring ß-solenoid. Importantly, the 4RßS arrangement allowed the first simulation of the sequence of events underlying PrPC conversion into PrPSc. This study provides the most updated, experimentally-driven and physically-coherent model of PrPSc, together with an unprecedented reconstruction of the mechanism underlying the self-catalytic propagation of prions.

Assessment of groundwater geochemistry and diffusion of hexavalent chromium contamination in an industrial town of Italy.

Groundwater contamination by hexavalent chromium (Cr (VI)) is currently a very serious and challenging issue. Therefore, in the present study, 108 shallow groundwater samples were collected during the years 2012 to 2015 (n = 27 samples each year) from an industrial town (Aosta) to assess the contamination status of the area. To evaluate the Cr (VI) concentration level, sources of pollutants and groundwater geochemistry of the study area, a combined approach of the multi-statistical and hydrogeochemical techniques were used. Furthermore, a geographic information system (GIS) was applied for the spatial distribution of Cr (VI) so that the most contaminated sites can be identified for a quick decision by policymakers. Results show that the groundwater chemistry was dominated by HCO3- and SO42- in the anionic chemistry and Ca2+ and Na+ in the cationic chemistry, while Ca-Mg-HCO3 and Ca-Mg-Cl-SO4 were the main water types during the years 2012-2015 in the study area. The central part and some north-eastern parts of the Aosta town groundwater was contaminated with Cr (VI) and are unsafe for drinking use. Concentration levels of Cr (VI) in the groundwater were 0.09 µg/L (lowest value) and 165 µg/L (highest value) in the years studied, and the spatial analysis shows the diffusion of Cr (VI) to north-east direction from the source. Contamination of Cr (VI) in the groundwater was due to the superficial slag deposits from a steel company within the study area. This study would be helpful for current and future water resource management in the area concerned.

Allostery in Its Many Disguises: From Theory to Applications.

Allosteric regulation plays an important role in many biological processes, such as signal transduction, transcriptional regulation, and metabolism. Allostery is rooted in the fundamental physical properties of macromolecular systems, but its underlying mechanisms are still poorly understood. A collection of contributions to a recent interdisciplinary CECAM (Center Européen de Calcul Atomique et Moléculaire) workshop is used here to provide an overview of the progress and remaining limitations in the understanding of the mechanistic foundations of allostery gained from computational and experimental analyses of real protein systems and model systems. The main conceptual frameworks instrumental in driving the field are discussed. We illustrate the role of these frameworks in illuminating molecular mechanisms and explaining cellular processes, and describe some of their promising practical applications in engineering molecular sensors and informing drug design efforts.

All-Atom Simulations Reveal How Single-Point Mutations Promote Serpin Misfolding.

Protein misfolding is implicated in many diseases, including serpinopathies. For the canonical inhibitory serpin α1-antitrypsin, mutations can result in protein deficiencies leading to lung disease, and misfolded mutants can accumulate in hepatocytes, leading to liver disease. Using all-atom simulations based on the recently developed bias functional algorithm, we elucidate how wild-type α1-antitrypsin folds and how the disease-associated S (Glu264Val) and Z (Glu342Lys) mutations lead to misfolding. The deleterious Z mutation disrupts folding at an early stage, whereas the relatively benign S mutant shows late-stage minor misfolding. A number of suppressor mutations ameliorate the effects of the Z mutation, and simulations on these mutants help to elucidate the relative roles of steric clashes and electrostatic interactions in Z misfolding. These results demonstrate a striking correlation between atomistic events and disease severity and shine light on the mechanisms driving chains away from their correct folding routes.

Atomic Detail of Protein Folding Revealed by an Ab Initio Reappraisal of Circular Dichroism.

Circular dichroism (CD) is known to be an excellent tool for the determination of protein secondary structure due to fingerprint signatures of α and ß domains. However, CD spectra are also sensitive to the 3D arrangement of the chain as a result of the excitonic nature of additional signals due to the aromatic residues. This double sensitivity, when extended to time-resolved experiments, should allow protein folding to be monitored with high spatial resolution. To date, the exploitation of this very appealing idea has been limited, due to the difficulty in relating the observed spectral evolution to specific configurations of the chain. Here, we demonstrate that the combination of atomistic molecular dynamics simulations of the folding pathways with a quantum chemical evaluation of the excitonic spectra provides the missing key. This is exemplified for the folding of canine milk lysozyme protein.