Cavity-filling mutations enhance protein stability by lowering the free energy of native state

The stabilization of proteins due to cavity-filling mutations are thought t o 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 pr oteins, because experiments can probe only the free energy difference betwe en the two states (N and D). To address this question, we carried out molec ular dynamics simulations, circular dichroism (CD) measurements, and X-ray crystallographic experiments on the cavity-filling mutations of the DNA-bin ding domain of the Myb transcriptional regulator. The cavity size was alter ed by systematic natural and nonnatural amino acid substitutions at a fixed site. The stability free energy change (Delta Delta G(N-->D;W-->M)) and th e cavity-size change (Delta V) calculated for the mutations agreed with the experimental data observed by urea-titration/CD measurements and crystallo graphic structure analysis, respectively. We found that the experimental De lta Delta G values correlate well with the calculated native-state free ene rgy change due to mutations Delta G(N;W-->M) and with Delta V(their correla tion coefficients are larger than 0.9) but not with the denatured-state Del ta G(D;W-->M). These results demonstrated that the decrease in cavity size increases the protein stability by lowering the free energy of native state for this protein. We discussed physicochemical meanings of our calculation results for Delta G(N;W-->M) and Delta G(D;W-->M).