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Understanding which aspects contribute to the thermostability of proteins is a challenge that has persisted for decades, and it is of great relevance for protein engineering. Several types of interactions can influence the thermostability of a protein. Among them, the electrostatic interactions have been a target of particular attention. Aiming to explore how this type of interaction can affect protein thermostability, this paper investigated four homologous cold shock proteins from psychrophilic, mesophilic, thermophilic, and hyperthermophilic organisms using a set of theoretical methodologies. It is well-known that electrostatics as well as hydrophobicity are key-elements for the stabilization of these proteins. Therefore, both interactions were initially analyzed in the native structure of each protein. Electrostatic interactions present in the native structures were calculated with the Tanford–Kirkwood model with solvent accessibility, and the amount of hydrophobic surface area buried upon folding was estimated by measuring both folded and extended structures. On the basis of Energy Landscape Theory, the local frustration and the simplified alpha-carbon structure-based model were modeled with a Debye–Hückel potential to take into account the electrostatics and the effects of an implicit solvent. Thermodynamic data for the structure-based model simulations were collected and analyzed using the Weighted Histogram Analysis and Stochastic Diffusion methods. Kinetic quantities including folding times, transition path times, folding routes, and Φ values were also obtained. As a result, we found that the methods are able to qualitatively infer that electrostatic interactions play an important role on the stabilization of the most stable thermophilic cold shock proteins, showing agreement with the experimental data.

Angiotensin-converting enzyme 2 (ACE2) is the host cellular receptor that locks onto the surface spike protein of the 2002 SARS coronavirus (SARS-CoV-1) and of the novel, highly transmissible and deadly 2019 SARS-CoV-2, responsible for the COVID-19 pandemic. One strategy to avoid the virus infection is to design peptides by extracting the human ACE2 peptidase domain α1-helix, which would bind to the coronavirus surface protein, preventing the virus entry into the host cells. The natural α1-helix peptide has a stronger affinity to SARS-CoV-2 than to SARS-CoV-1. Another peptide was designed by joining α1with the second portion of ACE2 that is far in the peptidase sequence yet grafted in the spike protein interface with ACE2. Previous studies have shown that, among several α1-based peptides, the hybrid peptidic scaffold is the one with the highest/strongest affinity for SARS-CoV-1, which is comparable to the full-length ACE2 affinity. In this work, binding and folding dynamics of the natural and designed ACE2-based peptides were simulated by the well-known coarse-grained structure-based model, with the computed thermodynamic quantities correlating with the experimental binding affinity data. Furthermore, theoretical kinetic analysis of native contact formation revealed the distinction between these processes in the presence of the different binding partners SARS-CoV-1 and SARS-CoV-2 spike domains. Additionally, our results indicate the existence of a two-state folding mechanism for the designed peptide en route to bind to the spike proteins, in contrast to a downhill mechanism for the natural α1-helix peptides. The presented low-cost simulation protocol demonstrated its efficiency in evaluating binding affinities and identifying the mechanisms involved in the neutralization of spike-ACE2 interaction by designed peptides. Finally, the protocol can be used as a computer-based screening of more potent designed peptides by experimentalists searching for new therapeutics against COVID-19.