Molecular mechanisms of chaperone-directed protein folding: Insights from atomistic simulations.

Molecular chaperones, a family of proteins of which Hsp90 and Hsp70 are integral members, form an essential machinery to maintain healthy proteomes by controlling the folding and activation of a plethora of substrate client proteins. This is achieved through cycles in which Hsp90 and Hsp70, regulated by task-specific co-chaperones, process ATP and become part of a complex network that undergoes extensive compositional and conformational variations. Despite impressive advances in structural knowledge, the mechanisms that regulate the dynamics of functional assemblies, their response to nucleotides, and their relevance for client remodeling are still elusive. Here, we focus on the glucocorticoid receptor (GR):Hsp90:Hsp70:co-chaperone Hop client-loading and the GR:Hsp90:co-chaperone p23 client-maturation complexes, key assemblies in the folding cycle of glucocorticoid receptor (GR), a client strictly dependent upon Hsp90/Hsp70 for activity. Using a combination of molecular dynamics simulation approaches, we unveil with unprecedented detail the mechanisms that underpin function in these chaperone machineries. Specifically, we dissect the processes by which the nucleotide-encoded message is relayed to the client and how the distinct partners of the assemblies cooperate to (pre)organize partially folded GR during Loading and Maturation. We show how different ligand states determine distinct dynamic profiles for the functional interfaces defining the interactions in the complexes and modulate their overall flexibility to facilitate progress along the chaperone cycle. Finally, we also show that the GR regions engaged by the chaperone machinery display peculiar energetic signatures in the folded state, which enhance the probability of partial unfolding fluctuations. From these results, we propose a model where a dynamic cross-talk emerges between the chaperone dynamics states and remodeling of client-interacting regions. This factor, coupled to the highly dynamic nature of the assemblies and the conformational heterogeneity of their interactions, provides the basis for regulating the functions of distinct assemblies during the chaperoning cycle.

Conformational Behavior of SARS-Cov-2 Spike Protein Variants: Evolutionary Jumps in Sequence Reverberate in Structural Dynamic Differences.

SARS-CoV-2 has evolved rapidly in the first 3 years of pandemic diffusion. The initial evolution of the virus appeared to proceed through big jumps in sequence changes rather than through the stepwise accumulation of point mutations on already established variants. Here, we examine whether this nonlinear mutational process reverberates in variations of the conformational dynamics of the SARS-CoV-2 Spike protein (S-protein), the first point of contact between the virus and the human host. We run extensive microsecond-scale molecular dynamics simulations of seven distinct variants of the protein in their fully glycosylated state and set out to elucidate possible links between the mutational spectrum of the S-protein and the structural dynamics of the respective variant, at global and local levels. The results reveal that mutation-dependent structural and dynamic modulations mostly consist of increased coordinated motions in variants that acquire stability and in an increased internal flexibility in variants that are less stable. Importantly, a limited number of functionally important substructures (the receptor binding domain, in particular) share the same time of movements in all variants, indicating efficient preorganization for functional regions dedicated to host interactions. Our results support a model in which the internal dynamics of the S-proteins from different strains varies in a way that reflects the observed random and non-stepwise jumps in sequence evolution, while conserving the functionally oriented traits of conformational dynamics necessary to support productive interactions with host receptors.

SARS-CoV-2 Spike Protein Mutations and Escape from Antibodies: A Computational Model of Epitope Loss in Variants of Concern.

The SARS-CoV-2 spike (S) protein is exposed on the viral surface and is the first point of contact between the virus and the host. For these reasons it represents the prime target for Covid-19 vaccines. In recent months, variants of this protein have started to emerge. Their ability to reduce or evade recognition by S-targeting antibodies poses a threat to immunological treatments and raises concerns for their consequences on vaccine efficacy. To develop a model able to predict the potential impact of S-protein mutations on antibody binding sites, we performed unbiased multi-microsecond molecular dynamics of several glycosylated S-protein variants and applied a straightforward structure-dynamics-energy based strategy to predict potential changes in immunogenic regions on each variant. We recover known epitopes on the reference D614G sequence. By comparing our results, obtained on isolated S-proteins in solution, to recently published data on antibody binding and reactivity in new S variants, we directly show that modifications in the S-protein consistently translate into the loss of potentially immunoreactive regions. Our findings can thus be qualitatively reconnected to the experimentally characterized decreased ability of some of the Abs elicited against the dominant S-sequence to recognize variants. While based on the study of SARS-CoV-2 spike variants, our computational epitope-prediction strategy is portable and could be applied to study immunoreactivity in mutants of proteins of interest whose structures have been characterized, helping the development/selection of vaccines and antibodies able to control emerging variants.

Effect of Antioxidants on High-Temperature Stability of Renewable Bio-Oils Revealed by an Innovative Method for the Determination of Kinetic Parameters of Oxidative Reactions.

Bio-oils employed for various industrial purposes, such as biodiesel production, undergo extensive oxidation and degradation during transformation processes. Therefore, it is extremely important to predict their stability at high temperature. We report herein a new procedure based on the optically detected profile of headspace O2 concentration during isotherms at 130 °C for evaluating the oxidation kinetic parameters of several bio-oil feedstocks. The slope of O2 consumption and the induction period duration were related to the oil characteristics (molecular structure, acidity, and presence of intrinsic antioxidants or metals). The increase of the induction time caused by a standardized propyl gallate addition yielded a semiquantitative value of radical generation rate. Investigated oils included used cooking oils; mono-, di-, and triglycerides from natural sources; free fatty acids; transesterified oils; and their blends. With respect to other methods, this characterization presents the advantage of disentangling and evaluating the role of both fatty acids composition and naturally occurring antioxidants, and allows the development of rational strategies for antioxidant protection of oils and of their blends.