caa a22 4500 397556276 CHVBK 20180308164803.0 cr unu---uuuuu 161202e199606 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021342 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021342 Analysis of the Transition State in the Unfolding of Hen Lysozyme by Introduction of Gly-Pro and Pro-Gly Sequences at the Same Site [Elektronische Daten] [Hiroyuki Motoshima, Tadashi Ueda, Taiji Imoto] 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′, Gly1O1-Prol02, Pro101Gly102, Gly117Pro118, Pro117Gly118, Gly121Pro122, and Pro121Gly122 lysozymes) were obtained for the unfolding in aqueous solution at pH 5.5 and 35°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 denned the φ- 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 φ-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 β-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 β-turn and 0.64 at position 121-122, which is located in the 310-helix. Therefore, in the transition state in the unfolding of lysozyme, it was found that the 310-helical region had a similar structure to the intact region, while both the β-turn and the loop at the upper part of the active site were considerably unfolded. The β-sheet structure was also moderately disrupted in the transition state. © by the Journal of Biochemistry Regular Papers nationallicence Gly-Pro sequence nationallicence lysozyme nationallicence Pro-Gly sequence nationallicence stability nationallicence transition state nationallicence Motoshima Hiroyuki Graduate School of Pharmaceutical Sciences, Kyushu University 62, Maidashi, Higashi-ku, Fukuoka 812 aut Ueda Tadashi Graduate School of Pharmaceutical Sciences, Kyushu University 62, Maidashi, Higashi-ku, Fukuoka 812 aut Imoto Taiji Graduate School of Pharmaceutical Sciences, Kyushu University 62, Maidashi, Higashi-ku, Fukuoka 812 aut The Journal of Biochemistry Oxford University Press 119/6(1996-06), 1019-1023 0021-924X 119:6<1019 1996 119 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021342 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021342 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Motoshima Hiroyuki Graduate School of Pharmaceutical Sciences, Kyushu University 62, Maidashi, Higashi-ku, Fukuoka 812 aut NATIONALLICENCE 700 1- Ueda Tadashi Graduate School of Pharmaceutical Sciences, Kyushu University 62, Maidashi, Higashi-ku, Fukuoka 812 aut NATIONALLICENCE 700 1- Imoto Taiji Graduate School of Pharmaceutical Sciences, Kyushu University 62, Maidashi, Higashi-ku, Fukuoka 812 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 119/6(1996-06), 1019-1023 0021-924X 119:6<1019 1996 119 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford