Increasing temperature accelerates protein unfolding without changing the pathway of unfolding

We have traditionally relied on extremely elevated temperatures (498K, 225 degrees C) to investigate the unfolding process of proteins within the timescale available to molecular dynamics simulations with explicit solvent. However, recent advances in computer hardware have allowed us to extend our thermal denaturation studies to much lower temperatures. Here we describe the results of simulations of chymotrypsin inhibitor 2 at seven temperatures, ranging from 298K to 498K. The simulation lengths vary from 94ns to 20ns, for a total simulation time of 344ns, or 0.34 micros. At 298K, the protein is very stable over the full 50ns simulation. At 348K, corresponding to the experimentally observed melting temperature of CI2, the protein unfolds over the first 25ns, explores partially unfolded conformations for 20ns, and then refolds over the last 35ns. Above its melting temperature, complete thermal denaturation occurs in an activated process. Early unfolding is characterized by sliding or breathing motions in the protein core, leading to an unfolding transition state with a weakened core and some loss of secondary structure. After the unfolding transition, the core contacts are rapidly lost as the protein passes on to the fully denatured ensemble. While the overall character and order of events in the unfolding process are well conserved across temperatures, there are substantial differences in the timescales over which these events take place. We conclude that 498K simulations are suitable for elucidating the details of protein unfolding at a minimum of computational expense.