Evidence for intermediates during unfolding and refolding of a two-domain protein, phage T4 lysozyme: equilibrium and kinetic studies.
Evidence for intermediates during unfolding and refolding of a two-domain protein, phage T4 lysozyme: equilibrium and kinetic studies.
复制标题
双结构域蛋白（噬菌体 T4 溶菌酶）展开和重折叠过程中中间体的证据：平衡和动力学研究。
DOI：
10.1021/bi00296a003
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发表时间：
1984
期刊：
影响因子：
2.9
通讯作者：
J. Yon
中科院分区：
文献类型：
--
作者：
M. Desmadril;J. Yon
Equilibrium and kinetic studies of the unfolding-refolding of phage T4 lysozyme induced by guanidine hydrochloride are reported. Tryptophan fluorescence and circular dichroism (CD) were used as observables. Several results indicated the existence of intermediates in the unfolded-folded transition, including (1) the noncoincidence of the transition observed by fluorescence and CD, (2) the asymmetry of the transition recorded by CD, and (3) triphasic kinetics, followed by both observables, accounting for the forward and reverse processes. Our data were inconsistent with an independent unfolding or refolding of each domain. They indicated the existence of residual structured regions particularly resistant to denaturant, which involved one or more of the three tryptophans located in the C-terminal domain. Kinetic analysis has made it possible to propose a minimum pathway corresponding to a sequential refolding, with dead-end species arising from an intermediate. We compared our data with the experimental results of Elwell and Schellman [Elwell, M., & Schellman, J.A. (1975) Biochim. Biophys. Acta 386, 309-313] and the theoretical analysis of B. Maigret (unpublished results) and proposed that the C-terminal domain refolds first, after which the overall structure can be formed and stabilized.
