Lillo M P, Szpikowska B K, Mas M T, Sutin J D, Beechem J M
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, USA.
Biochemistry. 1997 Sep 16;36(37):11273-81. doi: 10.1021/bi970789z.
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) Biochemistry 36, 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 I1 and I2 states. With this distance information, it is shown that the transition from the native to I1 state can be modeled as a large hinge-bending motion, in which both domains "swing away" from each other by about 15 A. As the domains move apart, the carboxyl-terminal domain rotates almost 90 degrees about the hinge region connecting the two domains. It is also shown that the amino-terminal domain remains intact during the native --> I1 transition, 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 A.
理解决定一级氨基酸序列如何形成三级结构的那套规则，是生物物理学中一个尚未解决的问题。如果在折叠反应过程中能够（实时）同时测量蛋白质中所有的分子内距离，那么“第二”遗传密码问题就能得到解决。遗憾的是，目前还不存在这样的技术。作为朝着这个目标迈出的第一步，已利用福斯特共振能量转移为一种双结构域蛋白——酵母磷酸甘油酸激酶（PGK）开发了一种光学距离测定系统[利洛，M. P. 等人（1997年）《生物化学》36卷，第11261 - 11272页]。在本研究中，使用六对独特的供体/受体荧光标记物来测量实时停流距离变化，这些标记物被巧妙地放置在PGK三级结构的各处。通过半胱氨酸替代诱变，随后用荧光探针5 - [[[(2 - 碘乙酰基)氨基]乙基]氨基]萘磺酸（作为供体）和5 - 碘乙酰氨基荧光素（受体）进行外部标记，将这些多个供体/受体位点基因工程导入PGK。发现PGK的去折叠是一个连续的多步过程（天然态→I1→I2→去折叠态），其速率常数分别为0.30、0.16和0.052 s⁻¹（从天然态到去折叠态）。在这项去折叠研究中独特的是，已解析出I1和I2状态下的六个分子内距离向量。有了这些距离信息，结果表明从天然态到I1态的转变可以被模拟为一种大的铰链弯曲运动，其中两个结构域彼此“摆动分开”约15埃。随着结构域分开移动，羧基末端结构域围绕连接两个结构域的铰链区域旋转近90度。还表明在天然态→I1转变过程中氨基末端结构域保持完整，这与我们之前的位点特异性色氨酸荧光各向异性停流研究结果一致[比切姆，J. M. 等人（1995年）《生物化学》34卷，第13943 - 13948页]。提议开展未来的实验，尝试以大约5 - 10埃的分辨率详细解析该蛋白质的去折叠/再折叠转变过程。
