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 relativeunfolding free energy of the Ile96 wild type and Ala96 mutantbarnases. The total calculated free energies suggest that substitutionof Ile96 with Ala destabilizes barnase by 3.9 kcal/mol, whichis in good agreement with the independently determined experimentalvalues of 4.0 and 3.3 kcal/mol and a previous simulation. However,a decomposition of the free energy finds the dominant contributionsto this free energy arising from the noncovalent Interactionsbetween the perturbed group and distant residues of barnasein the sequence and water molecules and only a very small contributionfrom covalent interactions. This is in contrast to the previoussimulation, using the dual topology methodology, which produceda decomposition with an {small tilde}60% free energy contributionfrom changes in covalent interactions. The use of the singletopology employed in the present calculations and the dual topologyemployed in the previous study are analyzed in order to understandthe contrast between the present results and the results ofthe previous study.