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Biomolecular condensates play a key role in cytoplasmic compartmentalization and cell functioning. Despite extensive research on the physico-chemical, thermodynamic, or crowding aspects of the formation and stabilization of the condensates, one less studied feature is the role of external perturbative fluid flow. In fact, in living cells, shear stress may arise from streaming or active transport processes. Here, we investigate how biomolecular condensates are deformed under different types of shear flows. We first model Couette flow perturbations via two-way coupling between the condensate dynamics and fluid flow by deploying Lattice Boltzmann Molecular Dynamics. We then show that a simplified approach where the shear flow acts as a static perturbation (one-way coupling) reproduces the main features of the condensate deformation and dynamics as a function of the shear rate. With this approach, which can be easily implemented in molecular dynamics simulations, we analyze the behavior of biomolecular condensates described through residue-based coarse-grained models, including intrinsically disordered proteins and protein/RNA mixtures. At lower shear rates, the fluid triggers the deformation of the condensate (spherical to oblated object), while at higher shear rates, it becomes extremely deformed (oblated or elongated object). At very high shear rates, the condensates are fragmented. We also compare how condensates of different sizes and composition respond to shear perturbation, and how their internal structure is altered by external flow. Finally, we consider the Poiseuille flow that realistically models the behavior in microfluidic devices in order to suggest potential experimental designs for investigating fluid perturbations in vitro., (© 2024 Author(s). Published under an exclusive license by AIP Publishing.)

In this work, we investigate how fluid flows impact the aggregation mechanisms of Aβ 40 proteins and Aβ 16-22 peptides and mechanically perturb their (pre)fibrillar aggregates. We exploit the OPEP coarse-grained model for proteins and the Lattice Boltzmann Molecular Dynamics technique. We show that beyond a critical shear rate, amyloid aggregation speeds up in Couette flow because of the shorter collisions times between aggregates, following a transition from diffusion limited to advection dominated dynamics. We also characterize the mechanical deformation of (pre)fibrillar states due to the fluid flows (Couette and Poiseuille), confirming the capability of (pre)fibrils to form pathological loop-like structures as detected in experiments. Our findings can be of relevance for microfluidic applications and for understanding aggregation in the interstitial brain space.

Molecular mechanisms underlying the thermal response of cells remain elusive. On the basis of the recent result that the short-time diffusive dynamics of the Escherichia coli proteome is an excellent indicator of temperature-dependent bacterial metabolism and death, we used neutron scattering (NS) spectroscopy and molecular dynamics (MD) simulations to investigate the sub-nanosecond proteome mobility in psychro-, meso-, and hyperthermophilic bacteria over a wide temperature range. The magnitude of thermal fluctuations, measured by atomic mean square displacements, is similar among all studied bacteria at their respective thermal cell death. Global roto-translational motions turn out to be the main factor distinguishing the bacterial dynamical properties. We ascribe this behavior to the difference in the average proteome net charge, which becomes less negative for increasing bacterial thermal stability. We propose that the chemical-physical properties of the cytoplasm and the global dynamics of the resulting proteome are fine-tuned by evolution to uphold optimal thermal stability conditions.

In this work, we investigated the lactate dehydrogenase (LDH) from Selenomonas ruminantium (S. rum), an enzyme that differs at key amino acid positions from canonical allosteric LDHs. The wild type (Wt) of this enzyme recognises pyuvate as all LDHs. However, introducing a single point mutation in the active site loop (I85R) allows S. Rum LDH to recognize the oxaloacetate substrate as a typical malate dehydrogenase (MalDH), whilst maintaining homotropic activation as an LDH. We report the tertiary structure of the Wt and I85RLDH mutant. The Wt S. rum enzyme structure binds NADH and malonate, whilst also resembling the typical compact R-active state of canonical LDHs. The structure of the mutant with I85R was solved in the Apo State (without ligand), and shows no large conformational reorganization such as that observed with canonical allosteric LDHs in Apo state. This is due to a local structural feature typical of S. rum LDH that prevents large-scale conformational reorganization. The S. rum LDH was also studied using Molecular Dynamics simulations, probing specific local deformations of the active site that allow the S. rum LDH to sample the T-inactive state. We propose that, with respect to the LDH/MalDH superfamily, the S. rum enzyme possesses a specificstructural and dynamical way to ensure homotropic activation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Atomic characterization of large nonfibrillar aggregates of amyloid polypeptides cannot be determined by experimental means. Starting from β-rich aggregates of Y and elongated topologies predicted by coarse-grained simulations and consisting of more than 100 Aβ16-22 peptides, we performed atomistic molecular dynamics (MD), replica exchange with solute scaling (REST2), and umbrella sampling simulations using the CHARMM36m force field in explicit solvent. Here, we explored the dynamics within 3 μs, the free energy landscape, and the potential of mean force associated with either the unbinding of one single peptide in different configurations within the aggregate or fragmentation events of a large number of peptides. Within the time scale of MD and REST2, we find that the aggregates experience slow global conformational plasticity, and remain essentially random coil though we observe slow beta-strand structuring with a dominance of antiparallel beta-sheets over parallel beta-sheets. Enhanced REST2 simulation is able to capture fragmentation events, and the free energy of fragmentation of a large block of peptides is found to be similar to the free energy associated with fibril depolymerization by one chain for longer Aβ sequences., (© 2023 Wiley Periodicals LLC.)

The bacterial fimbrial adhesin FimH is a remarkable and well-studied catch-bond protein found at the tip of E. coli type 1 pili, which allows pathogenic strains involved in urinary tract infections to bind high-mannose glycans exposed on human epithelia. The catch-bond behavior of FimH, where the strength of the interaction increases when a force is applied to separate the two partners, enables the bacteria to resist clearance when they are subjected to shear forces induced by urine flow. Two decades of experimental studies performed at the single-molecule level, as well as x-ray crystallography and modeling studies, have led to a consensus picture whereby force separates the binding domain from an inhibitor domain, effectively triggering an allosteric conformational change in the former. This force-induced allostery is thought to be responsible for an increased binding affinity at the core of the catch-bond mechanism. However, some important questions remain, the most challenging one being that the crystal structures corresponding to these two allosteric states show almost superimposable binding site geometries, which questions the molecular origin for the large difference in affinity. Using molecular dynamics with a combination of enhanced-sampling techniques, we demonstrate that the static picture provided by the crystal structures conceals a variety of binding site conformations that have a key impact on the apparent affinity. Crucially, the respective populations in each of these conformations are very different between the two allosteric states of the binding domain, which can then be related to experimental affinity measurements. We also evidence a previously unappreciated but important effect: in addition to the well-established role of the force as an allosteric regulator via domain separation, application of force tends to directly favor the high-affinity binding site conformations. We hypothesize that this additional "local" catch-bond effect could delay unbinding between the bacteria and the host cell before the "global" allosteric transition occurs, as well as stabilizing the complex even more once in the high-affinity allosteric state., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Macromolecular crowding has profound effects on the mobility of proteins, with strong implications on the rates of intracellular processes. To describe the dynamics of crowded environments, detailed molecular models are needed, capturing the structures and interactions arising in the crowded system. In this work, we present OPEPv7, which is a coarse-grained force field at amino-acid resolution, suited for rigid-body simulations of the structure and dynamics of crowded solutions formed by globular proteins. Using the OPEP protein model as a starting point, we have refined the intermolecular interactions to match the experimentally observed dynamical slowdown caused by crowding. The resulting force field successfully reproduces the diffusion slowdown in homogeneous and heterogeneous protein solutions at different crowding conditions. Coupled with the lattice Boltzmann technique, it allows the study of dynamical phenomena in protein assemblies and opens the way for the in silico rheology of protein solutions.

Probing the structures of amyloid-β (Aβ) peptides in the early steps of aggregation is extremely difficult experimentally and computationally. Yet, this knowledge is extremely important as small oligomers are the most toxic species. Experiments and simulations on Aβ42 monomer point to random coil conformations with either transient helical or β-strand content. Our current conformational description of small Aβ42 oligomers is funneled toward amorphous aggregates with some β-sheet content and rare high energy states with well-ordered assemblies of β-sheets. In this study, we emphasize another view based on metastable α-helix bundle oligomers spanning the C-terminal residues, which are predicted by the machine-learning AlphaFold2 method and supported indirectly by low-resolution experimental data on many amyloid polypeptides. This finding has consequences in developing novel chemical tools and to design potential therapies to reduce aggregation and toxicity., (© 2023 Wiley Periodicals LLC.)

Mucociliary clearance is the first defense mechanism of the respiratory tract against inhaled particles. This mechanism is based on the collective beating motion of cilia at the surface of epithelial cells. Impaired clearance, either caused by malfunctioning or absent cilia, or mucus defects, is a symptom of many respiratory diseases. Here, by exploiting the lattice Boltzmann particle dynamics technique, we develop a model to simulate the dynamics of multiciliated cells in a two-layer fluid. First, we tuned our model to reproduce the characteristic length- and time-scales of the cilia beating. We then check for the emergence of the metachronal wave as a consequence of hydrodynamic mediated correlations between beating cilia. Finally, we tune the viscosity of the top fluid layer to simulate the mucus flow upon cilia beating, and evaluate the pushing efficiency of a carpet of cilia. With this work, we build a realistic framework that can be used to explore several important physiological aspects of mucociliary clearance.

Temperature variations have a big impact on bacterial metabolism and death, yet an exhaustive molecular picture of these processes is still missing. For instance, whether thermal death is determined by the deterioration of the whole or a specific part of the proteome is hotly debated. Here, by monitoring the proteome dynamics of E. coli , we clearly show that only a minor fraction of the proteome unfolds at the cell death. First, we prove that the dynamical state of the E. coli proteome is an excellent proxy for temperature-dependent bacterial metabolism and death. The proteome diffusive dynamics peaks at about the bacterial optimal growth temperature, then a dramatic dynamical slowdown is observed that starts just below the cell's death temperature. Next, we show that this slowdown is caused by the unfolding of just a small fraction of proteins that establish an entangling interprotein network, dominated by hydrophobic interactions, across the cytoplasm. Finally, the deduced progress of the proteome unfolding and its diffusive dynamics are both key to correctly reproduce the E. coli growth rate., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Understanding the atomistic resolution changes during the self-assembly of amyloid peptides or proteins is important to develop compounds or conditions to alter the aggregation pathways and suppress the toxicity and potentially aid in the development of drugs. However, the complexity of protein aggregation and the transient order/disorder of oligomers along the pathways to fibril are very challenging. In this Perspective, we discuss computational studies of amyloid polypeptides carried out under various conditions, including conditions closely mimicking in vivo and point out the challenges in obtaining physiologically relevant results, focusing mainly on the amyloid-beta Aβ peptides.

Nuclear magnetic relaxation is widely used to probe protein dynamics. For decades, most analyses of relaxation in proteins have relied successfully on the model-free approach, forgoing mechanistic descriptions of motion. Model-free types of correlation functions cannot describe a large carbon-13 relaxation dataset in protein side chains. Here, we use molecular dynamics simulations to design explicit models of motion and solve Fokker-Planck diffusion equations. These models of motion provide better agreement with relaxation data, mechanistic insight, and a direct link to configuration entropy.

Self-assembled artificial water channels (AWCs) are reshaping current water desalination technologies. Recently, the improvements achieved by incorporating hydrophilic compounds into polyamide membranes (PA) at the interface were confirmed experimentally. However, the determination of the nanoscale structures of AWCs remains unclear. An important step in the preparation of PA membranes is the solubilization of a colloidal suspension of the solid phase in a water-ethanol mixture. We perform molecular dynamics simulations to study the nanoscale structures of AWC aggregates. We characterize the size and shape of the aggregates at several key locations in the ternary phase diagram. The role of ethanol in the formation of the interface between the solvent and the solute phase is highlighted. We found that the structure of the aggregates formed in the ternary solution resembled the disordered sponge-like structures observed when AWCs were inserted into lipid membranes. Such permeable sponge architectures allow the passage of water despite their noncrystalline organization and were previously shown to be consistent with AWC permeation measurements in membrane environments.

We unveil the intimate relationship between protein dynamics and allostery by following the trajectories of model proteins in their conformational and sequence spaces. Starting from a nonallosteric hyperthermophilic malate dehydrogenase, we have tracked the role of protein dynamics in the evolution of the allosteric capacity. Based on a large phylogenetic analysis of the malate (MalDH) and lactate dehydrogenase (LDH) superfamily, we identified two amino acid positions that could have had a major role for the emergence of allostery in LDHs, which we targeted for investigation by site-directed mutagenesis. Wild-type MalDH and the single and double mutants were tested with respect to their substrate recognition profiles. The double mutant displayed a sigmoid-shaped profile typical of homotropic activation in LDH. By using molecular dynamics simulations, we showed that the mutations induce a drastic change in the protein sampling of its conformational landscape, making transiently T-like (inactive) conformers, typical of allosteric LDHs, accessible. Our data fit well with the seminal key concept linking protein dynamics and evolvability. We showed that the selection of a new phenotype can be achieved by a few key dynamics-enhancing mutations causing the enrichment of low-populated conformational substates., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Aggregation of amyloid-β (Aβ42) protein is one hallmark of Alzheimer's disease, and the conformations of the smallest Aβ42 oligomers are largely unknown. Here, we explore the application of the deep learning AlphaFold2 method to the structure determination of Aβ42 monomers up to hexamers. The results shed light on the early Aβ42 aggregation steps in the bulk solution.

Stress granules (SGs) are among the most studied membraneless organelles that form upon heat stress (HS) to sequester unfolded, misfolded, or aggregated protein, supporting protein quality control (PQC) clearance. The folding states that are primarily associated with SGs, as well as the function of the phase separated environment in adjusting the energy landscapes, remain unknown. Here, we investigate the association of superoxide dismutase 1 (SOD1) proteins with different folding stabilities and aggregation propensities with condensates in cells, in vitro and by simulation. We find that irrespective of aggregation the folding stability determines the association of SOD1 with SGs in cells. In vitro and in silico experiments however suggest that the increased flexibility of the unfolded state constitutes only a minor driving force to associate with the dynamic biomolecular network of the condensate. Specific protein-protein interactions in the cytoplasm in comparison to SGs determine the partitioning of folding states between the respective phases during HS.

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.

Von Willebrand Factor (vWf) is a giant multimeric extracellular blood plasma involved in hemostasis. In this work we present multi-scale simulations of its three-domains fragment A1A2A3. These three domains are essential for the functional regulation of vWf. Namely the A2 domain hosts the site where the protease ADAMTS13 cleavages the multimeric vWf allowing for its length control that prevents thrombotic conditions. The exposure of the cleavage site follows the elongation/unfolding of the domain that is caused by an increased shear stress in blood. By deploying Lattice Boltzmann molecular dynamics simulations based on the OPEP coarse-grained model for proteins, we investigated at molecular level the unfolding of the A2 domain under the action of a perturbing shear flow. We described the structural steps of this unfolding that mainly concerns the β-strand structures of the domain, and we compared the process occurring under shear with that produced by the action of a directional pulling force, a typical condition of single molecule experiments. We observe, that under the action of shear flow, the competition among the elongational and rotational components of the fluid field leads to a complex behaviour of the domain, where elongated structures can be followed by partially collapsed melted globule structures with a very different degree of exposure of the cleavage site. Our simulations pose the base for the development of a multi-scale in-silico description of vWf dynamics and functionality in physiological conditions, including high resolution details for molecular relevant events, e.g., the binding to platelets and collagen during coagulation or thrombosis.

In this work, we combined biochemical and structural investigations with molecular dynamics (MD) simulations to analyze the very different thermal-dependent allosteric behavior of two lactate dehydrogenases (LDH) from thermophilic bacteria. We found that the enzyme from Petrotoga mobilis (P. mob) necessitates an absolute requirement of the allosteric effector (fructose 1, 6-bisphosphate) to ensure functionality. In contrast, even without allosteric effector, the LDH from Thermus thermophilus (T. the) is functional when the temperature is raised. We report the crystal structure of P. mob LDH in the Apo state solved at 1.9 Å resolution. We used this structure and the one from T. the, obtained previously, as a starting point for MD simulations at various temperatures. We found clear differences between the thermal dynamics, which accounts for the behavior of the two enzymes. Our work demonstrates that, within an allosteric enzyme, some areas act as local gatekeepers of signal transmission, allowing the enzyme to populate either the T-inactive or the R-active states with different degrees of stringency., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Macromolecular crowding influences protein mobility and stability in vivo . A precise description of the crowding effect on protein thermal stability requires the estimate of the combined effects of excluded volume, specific protein-environment interactions, as well as the thermal response of the crowders. Here, we explore an ideal model system, the lysozyme protein in powder state, to dissect the factors controlling the melting of the protein under extreme crowding. By deploying state-of-the art molecular simulations, supported by calorimetric experiments, we assess the role of the environment flexibility and of intermolecular electrostatic interactions. In particular, we show that the temperature-dependent flexibility of the macromolecular crowders, along with specific interactions, significantly alleviates the stabilizing contributions of the static volume effect.

Understanding water transport mechanisms at the nanoscale level remains a challenge for theoretical chemical physics. Major advances in chemical synthesis have allowed us to discover new artificial water channels, rivaling with or even surpassing water conductance and selectivity of natural protein channels. In order to interpret experimental features and understand microscopic determinants for performance improvements, numerical approaches based on all-atom molecular dynamics simulations and enhanced sampling methods have been proposed. In this study, we quantify the influence of microscopic observables, such as channel radius and hydrogen bond connectivity, and of meso-scale features, such as the size of self-assembly blocks, on the permeation rate of a self-assembled nanocrystal-like artificial water channel. Although the absolute permeation rate extrapolated from these simulations is overestimated by one order of magnitude compared to the experimental measurement, the detailed analysis of several observed conductive patterns in large assemblies opens new pathways to scalable membranes with enhanced water conductance for the future design.

Artificial water channels (AWCs) are known to selectively transport water, with ion exclusion. Similarly to natural porins, AWCs encapsulate water wires or clusters, offering continuous and iterative H-bonding that plays a vital role in their stabilization. Herein, we report octyl-ureido-polyol AWCs capable of self-assembly into hydrophilic hydroxy channels. Variants of ethanol, propanediol, and trimethanol are used as head groups to modulate the water transport permeabilities, with rejection of ions. The hydroxy channels achieve a single-channel permeability of 2.33 × 10 8 water molecules per second, which is within the same order of magnitude as the transport rates for aquaporins. Depending on their concentration in the membrane, adaptive channels are observed in the membrane. Over increased concentrations, a significant shift occurs, initiating unexpected higher water permeation. Molecular simulations probe that spongelike or cylindrical aggregates can form to generate transient cluster water pathways through the bilayer. Altogether, the adaptive self-assembly is a key feature influencing channel efficiency. The adaptive channels described here may be considered an important milestone contributing to the systematic discovery of artificial water channels for water desalination.

Protein misfolding and aggregation is observed in many amyloidogenic diseases affecting either the central nervous system or a variety of peripheral tissues. Structural and dynamic characterization of all species along the pathways from monomers to fibrils is challenging by experimental and computational means because they involve intrinsically disordered proteins in most diseases. Yet understanding how amyloid species become toxic is the challenge in developing a treatment for these diseases. Here we review what computer, in vitro , in vivo , and pharmacological experiments tell us about the accumulation and deposition of the oligomers of the (Aβ, tau), α-synuclein, IAPP, and superoxide dismutase 1 proteins, which have been the mainstream concept underlying Alzheimer's disease (AD), Parkinson's disease (PD), type II diabetes (T2D), and amyotrophic lateral sclerosis (ALS) research, respectively, for many years.

The effect of macromolecular crowding on the stability of proteins can change with temperature. This dependence might reveal a delicate balance between two factors: the entropic excluded volume and the stability-modulating quinary interactions. Here we computationally investigate the thermal stability of the native state of chymotrypsin inhibitor 2 (CI2), which was previously shown by experiments to be destabilized by protein crowders at room temperature. Mimicking experimental conditions, our enhanced-sampling atomistic simulations of CI2 surrounded by lysozyme and bovine serum albumin reproduce this destabilization but also provide evidence of a crossover temperature above which lysozyme is found to become stabilizing, as previously predicted by analysis of thermodynamic data. We relate this crossover to the different CI2-crowder interactions and the local packing experienced by CI2. In fact, we clearly show that the pronounced stabilization induced by lysozyme at high temperatures stems from the tight local packing created around CI2 by this smaller crowder.

A key aspect of life's evolution on Earth is the adaptation of proteins to be stable and work in a very wide range of temperature conditions. A detailed understanding of the associated molecular mechanisms would also help to design enzymes optimized for biotechnological processes. Despite important advances, a comprehensive picture of how thermophilic enzymes succeed in functioning under extreme temperatures remains incomplete. Here, we examine the temperature dependence of stability and of flexibility in the mesophilic monomeric Escherichia coli (Ec) and thermophilic dimeric Thermotoga maritima (Tm) homologs of the paradigm dihydrofolate reductase (DHFR) enzyme. We use all-atom molecular dynamics simulations and a replica-exchange scheme that allows to enhance the conformational sampling while providing at the same time a detailed understanding of the enzymes' behavior at increasing temperatures. We show that this approach reproduces the stability shift between the two homologs, and provides a molecular description of the denaturation mechanism by identifying the sequence of secondary structure elements melting as temperature increases, which is not straightforwardly obtained in the experiments. By repeating our approach on the hypothetical TmDHFR monomer, we further determine the respective effects of sequence and oligomerization in the exceptional stability of TmDFHR. We show that the intuitive expectation that protein flexibility and thermal stability are correlated is not verified. Finally, our simulations reveal that significant conformational fluctuations already take place much below the melting temperature. While the difference between the TmDHFR and EcDHFR catalytic activities is often interpreted via a simplified two-state picture involving the open and closed conformations of the key M20 loop, our simulations suggest that the two homologs' markedly different activity temperature dependences are caused by changes in the ligand-cofactor distance distributions in response to these conformational changes.

Thermal adaptation of enzymes is essential for both living organism development in extreme conditions and efficient biocatalytic applications. However, the molecular mechanisms leading to a shift in catalytic activity optimum temperatures remain unclear, and there is increasing experimental evidence that thermal adaptation involves complex changes in both structural and reactive properties. Here, a combination of enhanced protein conformational sampling with an explicit chemical reaction description was applied to mesophilic and thermophilic homologues of the dihydrofolate reductase enzyme, and a quantitative description of the stability and catalytic activity shifts between homologues was obtained. The key role played by temperature-induced shifts in protein conformational distributions is revealed. In contrast with pictures focusing on protein flexibility and dynamics, it is shown that while the homologues' reaction free energies are similar, the striking discrepancy between their activation energies is caused by their different conformational changes with temperature. An analytic model is proposed that combines catalytic activity and structural stability, and which quantitatively predicts the shift in homologue optimum temperatures. It is shown that this general model provides a molecular explanation of changes in optimum temperatures for several other enzymes., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The thermal stability of the superoxide dismutase 1 protein in a crowded solution is investigated by performing enhanced sampling molecular simulations. By complementing thermal unfolding experiments done close to physiological conditions (200 mg/mL), we provide evidence that the presence of the protein crowder bovine serum albumin in different packing states has only a minor, and essentially destabilizing, effect. The finding that quinary interactions counteract the pure stabilization contribution stemming from excluded volume is rationalized here by exploring the SOD1 unfolding mechanism in microscopic detail. In agreement with recent experiments, we unveil the importance of intermediate unfolded states as well as the correlation between protein conformations and local packing with the crowders. This link helps us to elucidate why certain SOD1 mutations involved in the ALS disease reverse the stability effect of the intracellular environment.

Protein misfolding and aggregation of amyloid proteins is the fundamental cause of more than 20 diseases. Molecular mechanisms of the self-assembly and the formation of the toxic aggregates are still elusive. Computer simulations have been intensively used to study the aggregation of amyloid peptides of various amino acid lengths related to neurodegenerative diseases. We review atomistic and coarse-grained simulations of short amyloid peptides aimed at determining their transient oligomeric structures and the early and late aggregation steps., Competing Interests: Conflict of interest The authors declare no competing financial interest., (© 2020 Elsevier Inc. All rights reserved.)

Proteins, in general, fold to a well-organized three-dimensional structure in order to function. The stability of this functional shape can be perturbed by external environmental conditions, such as temperature. Understanding the molecular factors underlying the resistance of proteins to the thermal stress has important consequences. First of all, it can aid the design of thermostable enzymes able to perform efficient catalysis in the high-temperature regime. Second, it is an essential brick of knowledge required to decipher the evolutionary pathways of life adaptation on Earth. Thanks to the development of atomistic simulations and ad hoc enhanced sampling techniques, it is now possible to investigate this problem in silico, and therefore provide support to experiments. After having described the methodological aspects, the chapter proposes an extended discussion on two problems. First, we focus on thermophilic proteins, a perfect model to address the issue of thermal stability and molecular evolution. Second, we discuss the issue of how protein thermal stability is affected by crowded in vivo-like conditions., (© 2020 Elsevier Inc. All rights reserved.)

In this work we present the coupling between Dry Martini, an efficient implicit solvent coarse-grained model for lipids, and the Lattice Boltzmann Molecular Dynamics (LBMD) simulation technique in order to include naturally hydrodynamic interactions in implicit solvent simulations of lipid systems. After validating the implementation of the model, we explored several systems where the action of a perturbing fluid plays an important role. Namely, we investigated the role of an external shear flow on the dynamics of a vesicle, the dynamics of substrate release under shear, and inquired the dynamics of proteins and substrates confined inside the core of a vesicle. Our methodology enables future exploration of a large variety of biological entities and processes involving lipid systems at the mesoscopic scale where hydrodynamics plays an essential role, e.g. by modulating the migration of proteins in the proximity of membranes, the dynamics of vesicle-based drug delivery systems, or, more generally, the behaviour of proteins in cellular compartments.

The great importance of pH for molecular processes has motivated the continuous development of numerical methods to improve the physical description of molecular mechanisms in computer simulations. Although rigid titration models are able to provide several pieces of useful information, the coupling between the molecular conformational changes and the acid-base equilibrium is necessary to more completely model the pH effects in biomolecules. Previously reported convergence issues with atomistic simulations indicated that a promising approach would require coarse-grained models. By means of the coupling between the successful OPEP force field for proteins with the fast proton titration scheme, we proposed a new protocol for constant-pH molecular dynamics simulations that takes advantage of both coarse-grained approaches to circumvent sampling difficulties faced by other numerical schemes and also to be able to properly describe electrostatic and structural properties at lower CPU costs. Here, we introduce this new protocol that defines now OPEP6 and its p K a 's benchmark for a set of representative proteins (HP36, BBL, HEWL, NTL9, and a protein G variant). In comparison with experimental measurements, our calculated p K a values have the average, maximum absolute, and root-mean-square deviations of [0.3-1.1], [0.6-2.5], and [0.4-1.3] pH units, respectively, for these five studied proteins. These numbers are within the ones commonly observed when similar comparisons are done among different theoretical models and are slightly better than the accuracy obtained by a rigid model using the same titration engine. For BBL, the predicted p K a are closer to experimental results than other analyzed theoretical data. Structural properties were tested for insulin where separation distances between the groups were compared and found in agreement with experimental crystallographic data obtained at different pH conditions. These indicate the ability of the new OPEP to properly describe the system physics and open up more possibilities to study pH-mediated biological processes.

In this work we investigate the multiscale dynamics of the aggregation process of an amyloid peptide, Aβ 16-22 . By performing massive coarse-grained simulations at the quasi-atomistic resolution and including hydrodynamic effects, we followed the formation and growth of a large elongated aggregate and its slow structuring. The elongation proceeds via a two-step nucleation mechanism with disordered aggregates formed initially and subsequently fusing to elongate the amorphous prefibril. A variety of coagulation events coexist, including lateral growth. The latter mechanism, sustained by long-range hydrodynamics correlations, actually can create a large branched structure spanning a few tens of nanometers. Our findings confirm the experimental hypothesis of a critical contribution of lateral growth to the amyloid aggregation kinetics and the capability of our model to sample critical structures like prefibril hosting annular pores.

The omega-3 and omega-6 polyunsaturated fatty acids are two important components of cell membranes in human brains. When incorporated into phospholipids, omega-3 slows the progression of Alzheimer's disease (AD), whereas omega-6 is linked to increased risk of AD. Little is known on the amyloid-β (Aβ) conformations in membranes rich in omega-3 and omega-6 phospholipids. Herein, the structural properties of the Aβ 29-42 dimer embedded in both fatty acid membranes were comparatively studied to a 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) bilayer using all-atom molecular dynamics (MD) simulations. Starting from α-helix, both omega-6 and omega-3 membranes promote new orientations and conformations of the dimer, in agreement with the observed dependence of Aβ production upon addition of these two fatty acids. This conformational result is corroborated by atomistic MD simulations of the dimer of the 99 amino acid C-terminal fragment of amyloid precursor protein spanning the residues 15-55. Starting from β-sheet, omega-6 membrane promotes helical and disordered structures of Aβ 29-42 dimer, whereas omega-3 membrane preserves the β-sheet structures differing however from those observed in POPC. Remarkably, the mixture of the two fatty acids and POPC depicts another conformational ensemble of the Aβ 29-42 dimer. This finding demonstrates that variation in the abundance of the molecular phospholipids, which changes with age, modulates membrane-embedded Aβ oligomerization.

In cells, proteins are embedded in a crowded environment that controls their properties via manifold avenues including weak protein-macromolecule interactions. A molecular level understanding of these quinary interactions and their contribution to protein stability, function, and localization in the cell is central to modern structural biology. Using a mutational analysis to quantify the energetic contributions of single amino acids to the stability of the ALS related protein superoxide dismutase I (SOD1) in mammalian cells, we show that quinary interactions destabilize SOD1 by a similar energetic offset for most of the mutants, but there are notable exceptions: Mutants that alter its surface properties can even lead to a stabilization of the protein in the cell as compared to the test tube. In conclusion, quinary interactions can amplify and even reverse the mutational response of proteins, being a key aspect in pathogenic protein misfolding and aggregation.