Role of topology in protein thermal stability

For several decades, experimental and computational studies have been used to investigate the potential functional role of knots in protein structures. A property that has attracted considerable attention is thermal stability, i.e., the extent to which a protein retains its native conformation and biological activity at high temperatures, without undergoing denaturation or aggregation. Thermal stability is quantified by the melting temperature T m , an equilibrium property that corresponds to the peak of heat capacity in differential scanning calorimetry (DSC) experiments. Experimental and computational studies report conflicting effects of knotting on protein thermal stability [M. A. Soler and P. F. N. Faísca, ;Y.-C. Chuang, I.-C. Hu, P.-C. Lyu, and S.-T. D. Hsu, ]. Here, we use extensive Monte Carlo simulations of a simple C-alpha model of protein YibK, with energetics modeled by the Go potential, to show that T m does not depend on the topological state of the protein. Our simulations further support the view that the discrepancy between the experimental and computational results stems from a pronounced separation of timescales for unknotting and unfolding that is inherent to deeply knotted proteins like YibK. In particular, the timescale separation implies that the complete unfolding-untying transition may not be accessible within the duration of a DSC experiment, whose apparent T m measurements likely reflect a nonequilibrium distribution lacking unfolded states that are also unknotted.