Real-time measurement of multiple intramolecular distances during protein folding reactions: a multisite stopped-flow fluorescence energy-transfer study of yeast phosphoglycerate kinase.
Real-time measurement of multiple intramolecular distances during protein folding reactions: a multisite stopped-flow fluorescence energy-transfer study of yeast phosphoglycerate kinase.
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蛋白质折叠反应过程中多个分子内距离的实时测量：酵母磷酸甘油酸激酶的多位点停流荧光能量转移研究。
DOI：
10.1021/bi970789z
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Beechem,JM
中科院分区：
文献类型：
--
作者：
Lillo,MP;Szpikowska,BK;Mas,MT;Sutin,JD;Beechem,JM
Understanding the set of rules which dictate how the primary amino acid sequence determines tertiary structure is an unsolved problem in biophysics. If it were possible to simultaneously measure all of the intramolecular distances in a protein (in real time) during a folding reaction, the “second” genetic code problem would be solved. Regrettably, no such technique currently exists. As a first step toward this goal, an optical distance assay system has been developed for a two-domain protein, yeast phosphoglycerate kinase (PGK), using Förster resonance energy transfer [Lillo, M. P., et al. (1997)Biochemistry36, 11261−11272]. In this study, real-time stopped-flow distance changes are measured using six unique pairs of donor/acceptor fluorescent labels strategically placed throughout the tertiary structure of PGK. These multiple donor/acceptor sites were genetically engineered into PGK by cysteine substitution mutagenesis followed by extrinsic labeling with fluorescent probes, 5-[[[(2-iodoacetyl)amino]ethyl]amino]naphthalenesulfonic acid (as a donor) and 5-iodoacetamidofluorescein (acceptor). The unfolding of PGK is found to be a sequential multistep process (native → I1→ I2→ unfolded) with rate constants of 0.30, 0.16, and 0.052 s-1, respectively (from native to unfolded). Unique to this unfolding study, six intramolecular distance vectors have been resolved for both the I1and I2states. With this distance information, it is shown that the transition from the native to I1state can be modeled as a large hinge-bending motion, in which both domains “swing away” from each other by about 15 Å. As the domains move apart, the carboxyl-terminal domain rotates almost 90° about the hinge region connecting the two domains. It is also shown that the amino-terminal domain remains intact during the native → I1transition, consistent with our previous site-specific tryptophan fluorescence anisotropy stopped-flow study [Beechem, J. M., et al. (1995)Biochemistry 34, 13943−13948]. Future experiments are proposed which will attempt to resolve in detail the unfolding/refolding transitions in this protein with a resolution of approximately 5−10 Å.
