Probing intradomain and interdomain conformational changes during equilibrium unfolding of phosphoglycerate kinase: fluorescence and circular dichroism study of tryptophan mutants.
Probing intradomain and interdomain conformational changes during equilibrium unfolding of phosphoglycerate kinase: fluorescence and circular dichroism study of tryptophan mutants.
复制标题
探测磷酸甘油酸激酶平衡展开过程中的域内和域间构象变化：色氨酸突变体的荧光和圆二色性研究。
DOI：
10.1021/bi00042a027
复制
发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Mas,MT
中科院分区：
文献类型：
--
作者：
Sherman,MA;Beechem,JM;Mas,MT
Revised Manuscript Received August 29, 1995® abstract: Phosphoglycerate kinase is a monomeric protein composed of two globular domains of the a1/3 type. Extensive domain—domain interactionsinvolve three segments of the polypeptide chain that are distant from one another in the primary sequence: the N-terminus, the C-terminus, and a centrally located a-helix. In order to monitor spectroscopically the conformational changes that occur in the individual domains andat the interdomain interface during the unfolding process, we have constructed a series of single-tryptophan mutants. In addition to two previously described mutants, each with single tryptophans in the C-terminal domain (W308 and W333)[Szpikowska, B. K., Beechem, J. M., Sherman, MA, & Mas, M. T.(1994) Biochemistry 33, 2217—2225], four new single-tryptophan mutants have been constructed: two with tryptophans located in the interdomain region (W194 and W399) and two with tryptophans in the N-terminal domain (W48 and W122). The equilibrium unfolding transitions induced by guanidine hydrochloride were monitored using far-UV CD, near-UV CD, steady-state, and time-resolved fluorescence. These studies reveal two unfolding transitions and suggest a sequential unfolding process for the mutants described in this paper. During the first transition (Cm~ 0.5 M) the interdomain region and C-terminal domain unfold; the N-terminal domain remains relatively compact butlacks much of the tertiary structure that characterizes the native state. A hyperfluorescent intermediate is detected during this transition by tryptophan probes placed within the N-terminal domain. Complete unfolding of the N-terminal domain occurs during the second transition (Cm~
