Electrolyte-amino acid interplay in the stability mechanisms of halophilic proteins.

Proteins of halophilic microorganisms thrive in high-salt environments. Compared to mesophilic proteins, they are enriched in acidic residues and small polar/apolar amino acids while being depleted in large hydrophobic residues, features that strongly influence their structure and stability. Here we critically examine experimental and computational studies investigating the mechanistic connection between the halophilic proteome and thermodynamic stability of halophilic proteins as a function of salt concentration. A defining feature of halophilic proteins is their highly negative surface charge, arising from abundant acidic residues. Some studies suggest this property is essential to ensure proteins remain folded at high salt concentrations, while others argue that a net negative protein charge might always be destabilizing. Alternative views propose that reducing solvent-exposed hydrophobic surface area is more critical than charge for stability at high salt concentrations. Advancing our understanding on this topic will require addressing multiple knowledge gaps. The unfolded states of both protein classes remain poorly characterized, leaving differences in local and non-local entropy contributions between the folded and the unfolded states to salt-dependent protein stability largely unexplored. Packing, cation-carbonyl and hydrophobic SASA differences between both protein classes are also insufficiently quantified. Atomistic molecular dynamics simulations with explicit solvent can advantageously be used to investigate these issues. Simultaneously, theoretical frameworks to understand how small perturbations in protein composition impact its stability as a function of salt concentration need to be expanded to include these contributions, which to date have been neglected, to fully understand how a halophilic proteome impacts salt-dependent stability.