| Abstract
| - The stabilization of proteins due to cavity-filling mutations are thought to be attributable to removal ofhydrophobic residues from solvent exposure in denatured (D) state and formation of close packing in native(N) state. However, it is still unclear which contribution is dominant to stabilize proteins, because experimentscan probe only the free energy difference between the two states (N and D). To address this question, wecarried out molecular dynamics simulations, circular dichroism (CD) measurements, and X-ray crystallographicexperiments on the cavity-filling mutations of the DNA-binding domain of the Myb transcriptional regulator.The cavity size was altered by systematic natural and nonnatural amino acid substitutions at a fixed site. Thestability free energy change (ΔΔG(N→D;W→M)) and the cavity-size change (ΔV) calculated for the mutationsagreed with the experimental data observed by urea-titration/CD measurements and crystallographic structureanalysis, respectively. We found that the experimental ΔΔG values correlate well with the calculated native-state free energy change due to mutations ΔG(N;W→M) and with ΔV (their correlation coefficients are largerthan 0.9) but not with the denatured-state ΔG(D;W→M). These results demonstrated that the decrease incavity size increases the protein stability by lowering the free energy of native state for this protein. Wediscussed physicochemical meanings of our calculation results for ΔG(N;W→M) and ΔG(D;W→M).
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