Estimation of stabilities of staphylococcal nuclease mutants (Met32-->Ala and Met32-->Leu) using molecular dynamics/free energy perturbation.
We performed molecular dynamics (MD)/free energy perturbation (FEP) calculations to reproduce the experimental free energy difference of denaturation for staphylococcal nuclease mutant Met32-->Ala (M32A) and to predict the stability of the mutant Met32-->Leu (M32L). The calculated free energy difference of denaturation for the M32A of -1.9 kcal/mol was in good agreement with the experimental value. In the M32A, a small hydrophobic core formed by three aromatic rings (Tyr-27, Phe-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 Met-32 is important for the stability of the enzyme. The M32L has not been available yet, but is expected to retain the small hydrophobic core. The free energy difference of denaturation for the M32L was calculated to be 1.6 kcal/mol. The MD/FEP simulation showed that the native state structure of the M32L was only slightly changed when compared with that of the wild-type. It was suggested that the M32L is more stable than the wild-type because the electrostatic interactions in the denatured state are more disadvantageous than those in the native state.
Yamaotsu
N
N
School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan.
Moriguchi
I
I
Hirono
S
S
D004795:Enzyme Stability; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics; D015394:Molecular Structure; D017354:Point Mutation; D011489:Protein Denaturation; D013816:Thermodynamics
D008836:Micrococcal Nuclease