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1 Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812–8582, Japan
2 Faculty of Science and Technology, Hirosaki University, Hirosaki, Aomori, Japan
Reprint requests to: Taiji Imoto, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan; e-mail: imoto{at}phar.kyushu-u.ac.jp; fax: 81-92-642-6667.
Stabilization of a protein using cavity-filling strategy has hardly been successful because of unfavorable van der Waals contacts. We succeeded in stabilizing lysozymes by cavity-filling mutations. The mutations were checked by a simple energy minimization in advance. It was shown clearly that the sum of free energy change caused by the hydrophobicity and the cavity size was correlated very well with protein stability. We also considered the aromatic–aromatic interaction. It is reconfirmed that the cavity-filling mutation in a hydrophobic core is a very useful method to stabilize a protein when the mutation candidate is selected carefully.
Keywords: Cavity-filling mutation; lysozyme; stability; aromatic-aromatic interaction
Keywords: HEL, hen egg white lysozyme; vdW, van der Waals; DSC, differential scanning calorimetry; WT, the wild-type lysozyme; LL mutant, a mutant lysozyme where Met12 is mutated to Leu; FL mutant, a mutant lysozyme where Met12 is mutated to Phe; LF mutant, a double mutant lysozyme where Met12 is mutated to Leu and Leu56 is mutated to Phe; FF mutant, a double mutant lysozyme where Met12 is mutated to Phe and Leu56 is mutated to Phe; GC, glycol chitin; (NAG)3, trimer of N-acetyl-glucosamine; MD, molecular dynamics; Gdn-HCl, Guanidine-hydrochloride; RMSD, root-mean-square deviation; RMSF, root-mean-square fluctuation calculated by molecular dynamics simulation; B-RMSF, root-mean-square fluctuation calculated by B-factor of crystallographic data; Vc, the volume of the cavity was defined to be the volume contained within the cavity surface which was the area swept out by a sphere of radius 1.2 Å as it rolls over the cavity surface; CSA, the cavity surface area, that is, the area swept out by a sphere of radius 1.2 Å as it rolls over the cavity surface; Gtm, Gibbs free energy calculated from 
Tm; Gvc, the differences in free energy change with changes of cavity volume; GCSA, the differences in free energy change with changes of cavity surface; Gtr, the virtual free energy of transfer of residues from the exterior to the interior of globular protein.
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