title: Analysis of the transition state in the unfolding of hen lysozyme by introduction of Gly-Pro and Pro-Gly sequences at the same site.
abstract: We developed a sensitive method for analyzing the conformation of the transition state in the unfolding of hen lysozyme. The activation free energy changes of mutant lysozymes with Gly-Pro and Pro-Gly sequences at the same sites (Gly47Pro47', Pro47Gly47', Gly101Pro102, Pro101Gly102, Gly117Pro118, Pro117Gly118, Gly121Pro122, and Pro121Gly122 lysozymes) were obtained for the unfolding in aqueous solution at pH 5.5 and 35 degrees C. Since we had shown that the difference of energies of the unfolded state in lysozymes having an introduced Gly-Pro or Pro-Gly sequence at the same site was much smaller than the difference of energies of the folded states [Motoshima, H., Ueda, T., Hashimoto, Y., Tsutsumi, M., and Imoto, T. (1995) J. Biochem. 118, 1138-1144], we could estimate the difference of energies of the folded and the transition states unequivocally. We defined the phi-value as the ratio of the difference in the free energy change in the transition state to that in the free energy change in the folded state between lysozymes with Gly-Pro and Pro-Gly sequences at the same site. The phi-values gave information on how much the mutated sites retained the folded structure in the transition state. These values were 0.45 around position 47, which is located in the beta-sheet structure, 0.12 at position 101-102, which is located in the loop at the upper part of the active site, 0.17 at position 117-118, which is located in the beta-turn and 0.64 at position 121-122, which is located in the 3(10)-helix. Therefore, in the transition state in the unfolding of lysozyme, it was found that the 3(10)-helical region had a similar structure to the intact region, while both the beta-turn and the loop at the upper part of the active site were considerably unfolded. The beta-sheet structure was also moderately disrupted in the transition state.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka.
authors.forename: H
authors.initials: H
authors.lastname: Motoshima
authors.forename: T
authors.initials: T
authors.lastname: Ueda
authors.forename: T
authors.initials: T
authors.lastname: Imoto
mesh_terms: D000818:Animals; D002645:Chickens; D005998:Glycine / Q000737:chemistry; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics
chemical_list: D011392:Proline; D009113:Muramidase; D005998:Glycine