Conformation of T4 lysozyme in solution. Hinge-bending motion and the substrate-induced conformational transition studied by site-directed spin labeling
Conformation of T4 lysozyme in solution. Hinge-bending motion and the substrate-induced conformational transition studied by site-directed spin labeling
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DOI：
10.1021/bi962114m
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发表时间：
1997-01-14
期刊：
影响因子：
2.9
通讯作者：
Hubbell, WL
中科院分区：
文献类型：
--
作者：
Mchaourab, HS;Oh, KJ;Hubbell, WL
T4溶菌酶及其突变体以不同的构象结晶，这些构象通过围绕分子中铰链的弯曲而彼此相关。这一观察结果表明，野生型蛋白质在溶液中可能经历铰链弯曲运动，以允许底物接近否则封闭的活性位点裂缝[Faber，H. R.，&马修斯，B. W.（1990）Nature 348，263-266]。为了检验这一假设，无论是单一或对氮氧侧链被引入到蛋白质中，以监测三级接触相互作用和残基间的距离，分别在溶液中。这些结构参数的一组约束条件来自参考状态，共价酶-底物加合物，其中酶被锁定在封闭状态。在不存在底物的情况下，相对于该参考状态，残基间距离和三级接触相互作用的差异与打开活性位点裂隙的铰链弯曲运动一致。氮氧自由基对之间的自旋-自旋相互作用的定量分析揭示了8埃的相对域运动后，基板结合。此外，它表明，I3 P突变，这产生了一个大的铰链弯曲角的晶体，没有影响的解决方案的构象。因此，铰链运动不是突变的结果，而是溶液中T4溶菌酶催化的组成部分，如最近提出的[Zhang，X. J.，沃兹尼亚克，J. A.，&马修斯，B. W.等人（1995）J. Mol. Biol. 250，527-552]。本文所采用的基于定点自旋标记的方法，可普遍应用于蛋白质构象及其在溶液中构象变化的研究。
T4 lysozyme and mutants thereof crystallize in different conformations that are related to each other by a bend about a hinge in the molecule. This observation suggests that the wild type protein may undergo a hinge-bending motion in solution to allow substrate access to an otherwise closed active site cleft [Faber, H. R., & Matthews, B. W. (1990) Nature 348, 263-266]. To test this hypothesis, either single or pairs of nitroxide side chains were introduced into the protein to monitor tertiary contact interactions and inter-residue distances, respectively, in solution. A set of constraints for these structural parameters was derived from a reference state, a covalent enzyme-substrate adduct where the enzyme is locked in the closed state. In the absence of substrate, differences in both inter-residue distances and tertiary contact interactions relative to this reference state are consistent with a hinge-bending motion that opens the active site cleft. Quantitative analysis of spin-spin interactions between nitroxide pairs reveals an 8 Angstrom relative domain movement upon substrate binding. In addition, it is demonstrated that the I3P mutation, which produces a large hinge-bending angle in the crystal, has no effect on the solution conformation. Thus, the hinge motion is not the result of the mutation but is an integral part of T4 lysozyme catalysis in solution, as suggested recently [Zhang, X. J., Wozniak, J. A., & Matthews, B. W. (1995) J. Mol. Biol. 250, 527-552]. The strategy employed here, based on site-directed spin labeling, should be generally applicable to the study of protein conformation and conformational changes in solution.