Stabilization of hen egg white lysozyme by a cavity-filling mutation

Abstract

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.

Fig. 1.

Ribbon model of secondary and tertiary structures of HEL, showing the side chain of the hydrophobic core residues mutated in this study. The side chain is shown as a ball-and-stick model. This figure was made with the MOLSCRIPT program (Kraulis 1991).

Fig. 2.

(A) Stereo view of space-filling model of cavities in the hydrophobic core of WT. (B) Comparisons of the core region in the refined structures of the mutants and WT. The alignments are based on the least-squares superposition of the main chain atoms. FL mutant (thick line) and LF mutant (thin line) versus WT (dotted line).

Fig. 2.

(A) Stereo view of space-filling model of cavities in the hydrophobic core of WT. (B) Comparisons of the core region in the refined structures of the mutants and WT. The alignments are based on the least-squares superposition of the main chain atoms. FL mutant (thick line) and LF mutant (thin line) versus WT (dotted line).

Fig. 3.

Correlation between glycol chitin (GC) activity and ΔVc (A) and ΔΔGtm value (B).

Fig. 4.

Correlation between ΔΔGtm value and the sum of ΔGtr and ΔΔGvc (A) and the sum of ΔGtr and ΔΔG_CSA (B). LF′ and FF′ are the corrected points for the aromatic–aromatic interaction in the LF mutant and the FF mutant, respectively.