Stabilization of lysozyme by the introduction of gly-pro sequence

Three mutant lysozymes where the Asp¹⁰¹ - Gly¹⁰² sequence of lysozyme was converted to Asp¹⁰¹-Pro¹⁰², Gly¹⁰¹-Pro102 and Pro¹⁰¹-Gly¹⁰² were prepared to investigate the effect of proline residues on the stabilization of proteins. The free energy changes of lysozymes for the unfolding in aqueous solution at pH 5.5 and 35°C were 10.0, 10.1, 11.0 and 7.7 kcal/mol for wild type, Asp¹⁰¹Pro¹⁰², Gly¹⁰¹Pro¹⁰² and Pro¹⁰¹Gly¹⁰² lysozyme respectively. When the energy level in the unfolded state of wild type lysozyme was fixed at a standard level, the energy levels in the folded state of Asp¹⁰¹Pro¹⁰² and Pro¹⁰¹Gly¹⁰² lysozymes were found to be higher than that of wild type lysozyme on the basis of ΔG_D(H₂O) and entropy losses of their polypeptide chains in the unfolded state. The presence of some strain in the folded state of these lysozymes was supported by both the calculation of conformational energy for a trans-L-prolyl residue [Schimmel, P.R. and Flory,P.J. (1968) J. Mol. Biol, 34, 105 -120] and the analysis of structures of energy-minimized mutant lysozymes. Therefore, it is concluded that the formation of the Gly-Pro sequence is effective in avoiding possible strain in the folded state of a protein caused by the introduction of proline residue(s).