ESTIMATION OF STABILITIES OF STAPHYLOCOCCAL NUCLEASE MUTANTS (MET(32)-]ALA AND MET(32)-]LEU) USING MOLECULAR-DYNAMICS FREE-ENERGY PERTURBATION

We performed molecular dynamics (MD)/free energy perturbation (FEP) ca lculations to reproduce the experimental free energy difference of den aturation for staphylococcal nuclease mutant Met(32) --> Ala (M32A) an d to predict the stability of the mutant Met(32) --> Leu (M32L). The c alculated free energy difference of denaturation for the M32A of -1.9 kcal/mol was in good agreement with the experimental value. In the M32 A, a small hydrophobic core formed by three aromatic rings (Tyr-27, Ph e-34, Phe-76) in a wild-type crumbled as a result of exposure to water . The van der Waals interactions in the native state of the M32A were weaker than those of the wild-type, which strongly suggests that the M et-32 is important for the stability of the enzyme. The M32L has not b een available yet, but is expected to retain the small hydrophobic cor e. The free energy difference of denaturation for the M32L was calcula ted to be 1.6 kcal/mol. The MD/FEP simulation showed that the native s tate structure of the M32L was only slightly changed when compared wit h that of the wild-type. It was suggested that the M32L is more stable than the wild-type because the electrostatic interactions in the dena tured state are more disadvantageous than those in the native state.