The stabilization of proteins due to cavity-filling mutations are thought to be attributable to removal of hydrophobic 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 experiments can probe only the free energy difference between the two states (N and D). To address this question, we carried out molecular dynamics simulations, circular dichroism (CD) measurements, and X-ray crystallographic experiments 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. The stability free energy change (ΔΔG(N→D;W→M)) and the cavity-size change (ΔV) calculated for the mutations agreed with the experimental data observed by urea-titration/CD measurements and crystallographic structure analysis, respectively. We found that the expe...