Dependence of Protein Stability on the Structure of the Denatured State: Free Energy Calculations of I56V Mutation in Human Lysozyme

AbstractFree 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, ΔΔGcal, depends significantly on the structure of the denatured state. When native-like structure is employed, ΔΔGcal is in good agreement with the experimental free energy difference, ΔΔGexp, whereas in the other four models, ΔΔGcal differs sharply from ΔΔGexp. 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 ΔΔGcal. The contribution of protein-protein interactions to the nonbonded component of ΔΔGcal 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 ΔΔGcal. We also propose a general computational strategy for the study of protein stability that is equally conscious of the denatured and native states