Free energy calculations of the mutation of Ile96 → Ala in barnase: Contributions to the difference in stability

Free energy calculations were carried out to determine the relative unfolding free energy of the Ile96 wild type and Ala96 mutant barnases. The total calculated free energies suggest that substitution of Ile96 with Ala destabilizes barnase by 3.9 kcal/mol, which is in good agreement with the independently determined experimental values of 4.0 and 3.3 kcal/mol and a previous simulation. However, a decomposition of the free energy finds the dominant contributions to this free energy arising from the non-covalent interactions between the perturbed group and distant residues of barnase in the sequence and water molecules and only a very small contribution from covalent interactions. This is in contrast to the previous simulation, using the dual topology methodology, which produced a decomposition with an ~ 60% free energy contribution from changes in covalent interactions. The use of the single topology employed in the present calculations and the dual topology employed in the previous study are analyzed in order to understand the contrast between the present results and the results of the previous study.