Role of amino acid residues at turns in the conformational stability and folding of human lysozyme

TO clarify the role of amino acid residues at turns in the conformational s tability and folding of a globular protein, six mutant human lysozymes dele ted or substituted at turn structures were investigated by calorimetry, GuH Cl denaturation experiments, and X-ray crystal analysis. The thermodynamic properties of the mutant and wild-type human lysozymes were compared and di scussed on the basis of their three-dimensional structures. For the deletio n mutants, Delta 47-48 and Delta 101, the deleted residues are in turns on the surface and are absent in human alpha-lactalbumin, which is homologous to human lysozyme in amino acid sequence and tertiary structure. The stabil ity of both mutants would be expected to increase due to a decrease in conf ormational entropy in the denatured state; however, both proteins were dest abilized. The destabilizations were mainly caused by the disappearance of i ntramolecular hydrogen bonds. Each part deleted was recovered by the turn r egion like the alpha-lactalbumin structure, but there were differences in t he main-chain conformation of the turn between each deletion mutant and or- lactalbumin even if the loop length was the same. For the point mutants, R5 0G, Q58G, H78G, and G37Q, the main-chain conformations of these substitutio n residues located in turns adopt a left-handed helical region in the wild- type structure. It is thought that the left-handed non-Gly residue has unfa vorable conformational energy compared to the left-handed Gly residue. Q58G was stabilized, but the others had little effect on the stability. The str uctural. analysis revealed that the turns could rearrange the main-chain co nformation to accommodate the left-handed non-Gly residues. The present res ults indicate that turn structures are able to change their main-chain conf ormations, depending upon the side-chain features of amino acid residues on the turns. Furthermore, stopped-flow GuHCl denaturation experiments on the six mutants were performed. The effects of mutations on unfolding-refoldin g kinetics were significantly different among the mutant proteins. The dele tion/substitutions in turns located in the alpha-domain of human lysozyme a ffected the refolding rate, indicating the contribution of turn structures to the folding of a globular protein.