Insights into the Mechanism of Tryptophan Fluorescence Quenching due to Synthetic Crowding Agents: A Combined Experimental and Computational Study.

Intrinsic tryptophan fluorescence spectroscopy is an important tool for examining the effects of molecular crowding and confinement on the structure, dynamics, and function of proteins. Synthetic crowders such as dextran, ficoll, polyethylene glycols, polyvinylpyrrolidone, and their respective monomers are used to mimic crowded intracellular environments. Interactions of these synthetic crowders with tryptophan and the subsequent impact on its fluorescence properties are therefore critically important for understanding the possible interference created by these crowders. In the present study, the effects of polymer and monomer crowders on tryptophan fluorescence were assessed by using experimental and computational approaches. The results of this study demonstrated that both polymer and monomer crowders have an impact on the tryptophan fluorescence intensity; however, the molecular mechanisms of quenching were different. Using Stern-Volmer plots and a temperature variation study, a physical basis for the quenching mechanism of commonly used synthetic crowders was established. The quenching of free tryptophan was found to involve static, dynamic, and sphere-of-action mechanisms. In parallel, computational studies employing Kohn-Sham density functional theory provided a deeper insight into the effects of intermolecular interactions and solvation, resulting in differing quenching modes for these crowders. Taken together, the study offers new physical insights into the quenching mechanisms of some commonly used monomer and polymer synthetic crowders.

Polyethylene Glycol 20k. Does It Fluoresce?

Polyethylene glycol (PEG) is a polyether compound commonly used in biological research and medicine because it is biologically inert. This simple polymer exists in variable chain lengths (and molecular weights). As they are devoid of any contiguous π-system, PEGs are expected to lack fluorescence properties. However, recent studies suggested the occurrence of fluorescence properties in non-traditional fluorophores like PEGs. Herein, a thorough investigation has been conducted to explore if PEG 20k fluoresces. Results of this combined experimental and computational study suggested that although PEG 20k could exhibit "through-space" delocalization of lone pairs of electrons in aggregates/clusters, formed via intermolecular and intramolecular interactions, the actual contributor of fluorescence between 300 and 400 nm is the stabilizer molecule, i.e., 3-tert-butyl-4-hydroxyanisole present in the commercially available PEG 20k. Therefore, the reported fluorescence properties of PEG should be taken with a grain of salt, warranting further investigation.

Pre-Existing Oxidative Stress Creates a Docking-Ready Conformation of the SARS-CoV-2 Receptor-Binding Domain.

The redox-dependent changes on the binding between the receptor-binding domain of the severe acute respiratory syndrome-coronavirus-2 spike protein and the peptidase domain of the human cell surface receptor angiotensin-converting enzyme II were investigated by performing molecular dynamics simulations. The reduced states of the protein partners were generated in silico by converting the disulfides to thiols. The role of redox transformation on the protein-protein binding affinity was assessed from the time-evolved structures after 200 ns simulations using electrostatic field calculations and implicit solvation. The present simulations revealed that the bending motion at the protein-protein interface is significantly altered when the disulfides are reduced to thiols. In the native complex, the presence of disulfide bonds preserves the structural complementarity of the protein partners and maintains the intrinsic conformational dynamics. Also, the study demonstrates that when already bound, the disulfide-to-thiol conversion of the receptor-binding domain has a limited impact on the binding of the spike protein to the receptor. However, if the reduction occurs before binding to the receptor, a spectacular conformational change of the receptor-binding domain occurs that fully impairs the binding. In other words, the formation of disulfide bonds, prevalent during oxidative stress, creates a conformation ready to bind to the receptor. Taken together, the present study demonstrates the role of pre-existing oxidative stress in elevating the binding affinity of the spike protein for the human receptor, offering future clues for alternate therapeutic possibilities.

Role of Oxidative Stress on SARS-CoV (SARS) and SARS-CoV-2 (COVID-19) Infection: A Review.

Novel coronavirus disease 2019 (COVID-19) has resulted in a global pandemic and is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Several studies have suggested that a precise disulfide-thiol balance is crucial for viral entry and fusion into the host cell and that oxidative stress generated from free radicals can affect this balance. Here, we reviewed the current knowledge about the role of oxidative stress on SARS-CoV and SARS-CoV-2 infections. We focused on the impact of antioxidants, like NADPH and glutathione, and redox proteins, such as thioredoxin and protein disulfide isomerase, that maintain the disulfide-thiol balance in the cell. The possible influence of these biomolecules on the binding of viral protein with the host cell angiotensin-converting enzyme II receptor protein as well as on the severity of COVID-19 infection was discussed.

Editing Domain Motions Preorganize the Synthetic Active Site of Prolyl-tRNA Synthetase.

Prolyl-tRNA synthetases (ProRSs) catalyze the covalent attachment of proline onto cognate tRNAs, an indispensable step for protein synthesis in all living organisms. ProRSs are modular enzymes and the "prokaryotic-like" ProRSs are distinguished from "eukaryotic-like" ProRSs by the presence of an editing domain (INS) inserted between motifs 2 and 3 of the main catalytic domain. Earlier studies suggested the presence of coupled-domain dynamics could contribute to catalysis; however, the role that the distal, highly mobile INS domain plays in catalysis at the synthetic active site is not completely understood. In the present study, a combination of theoretical and experimental approaches has been used to elucidate the precise role of INS domain dynamics. Quantum mechanical/molecular mechanical simulations were carried out to model catalytic Pro-AMP formation by Enterococcus faecalis ProRS. The energetics of the adenylate formation by the wild-type enzyme was computed and contrasted with variants containing active site mutations, as well as a deletion mutant lacking the INS domain. The combined results revealed that two distinct types of dynamics contribute to the enzyme's catalytic power. One set of motions is intrinsic to the INS domain and leads to conformational preorganization that is essential for catalysis. A second type of motion, stemming from the electrostatic reorganization of active site residues, impacts the height and width of the energy profile and has a critical role in fine tuning the substrate orientation to facilitate reactive collisions. Thus, motions in a distal domain can preorganize the active site of an enzyme to optimize catalysis.