Abstract
Free energy calculations were carried out to understand the effect of the I56V mutation of human lysozyme on its thermal stability. In the simulation of the denatured state, a short peptide including the mutation site in the middle is employed. To study the dependence of the stability on the denatured-state structure, five different initial conformations, native-like, extended, and three random-coil-like conformations, were examined. We found that the calculated free energy difference, DeltaDeltaGcal, depends significantly on the structure of the denatured state. When native-like structure is employed, DeltaDeltaGcal is in good agreement with the experimental free energy difference, DeltaDeltaGexp, whereas in the other four models, DeltaDeltaGcal differs sharply from DeltaDeltaGexp. It is therefore strongly suggested that the structure around the mutation site takes a native-like conformation rather than an extended or random-coil conformation. From the free energy component analysis, it has been shown that free energy components originating from Lennard-Jones and covalent interactions dominantly determine DeltaDeltaGcal. The contribution of protein-protein interactions to the nonbonded component of DeltaDeltaGcal is about the same as that from protein-water interactions. The residues that are located in a hydrophobic core (F3, L8, Y38, N39, T40, and I89) contribute significantly to the nonbonded free energy component of DeltaDeltaGcal. We also propose a general computational strategy for the study of protein stability that is equally conscious of the denatured and native states.