Mechanical Aspects of Protein Thermostability

Abstract

Single-molecule force-spectroscopies are remarkable tools for studying protein folding and unfolding. However it is legitimate to wonder whether the explored pathways differ from that traditionally explored via chemical or thermal denaturation, especially because the experimental reaction coordinates are very different. Here we address the following questions: how well do proteins that are very stable upon thermal denaturation resist under force? Are the same mechanisms involved for both types of stability? Our paradigm system is a ∼70-residue protein, cold-shock, that is present in thermophilic organisms and that has a very high melting temperature. We compare it to one of its mesophilic counterparts. Both thermal and mechanical stabilities are investigated with all-atom molecular dynamics simulations in explicit solvent. To verify the ability of our models to reproduce the experimental shift in the melting temperature of both protein homologues, we have implemented an enhanced-sampling algorithm (REST2) based on an Hamiltonian exchange scheme, into a publicly available simulation code (NAMD2). This allows us to explore the proteins’ free-energy landscape over a wide temperature range much faster than would be obtained using unperturbed simulations. The proteins mechanical stability is investigated using constant-force steered molecular-dynamics. In agreement with experimental data, we find that the typical unfolding force of the both variants are non-negligible but significantly lower than that of well-characterized mechanically stable proteins that are thermally stable as well. Our results suggest that thermal stability does not guarantee a mechanical one and that the associated mechanisms are very different in nature. Our simulations also aim at identifying the weak-points of the protein mechanical stability and how more stable mutants can be designed; such mutants are then expected to be more stable against thermal denaturation and could prove useful in numerous applications.