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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- 作者：Hassane S. Mchaourab ; Kyoung Joon Oh ; Celia J. Fang ; and Wayne L. Hubbell
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：January 14, 1997
- 年：1997
- 卷：36
- 期：2
- 页码：307 - 316
- 全文大小：329K
- 年卷期：v.36,no.2(January 14, 1997)
- ISSN：1520-4995
文摘
T4 lysozyme and mutants thereof crystallize in differentconformations that are related toeach other by a bend about a hinge in the molecule. Thisobservation suggests that the wild type proteinmay undergo a hinge-bending motion in solution to allow substrateaccess to an otherwise closed activesite cleft [Faber, H. R., & Matthews, B. W. (1990) Nature348, 263-266]. To test this hypothesis,eithersingle or pairs of nitroxide side chains were introduced into theprotein to monitor tertiary contactinteractions and inter-residue distances, respectively, in solution.A set of constraints for these structuralparameters was derived from a reference state, a covalentenzyme-substrate adduct where the enzyme islocked in the closed state. In the absence of substrate,differences in both inter-residue distances andtertiary contact interactions relative to this reference state areconsistent with a hinge-bending motion thatopens the active site cleft. Quantitative analysis of spin-spininteractions between nitroxide pairs revealsan 8 Å relative domain movement upon substrate binding. Inaddition, it is demonstrated that the I3Pmutation, which produces a large hinge-bending angle in the crystal,has no effect on the solutionconformation. Thus, the hinge motion is not the result of themutation but is an integral part of T4lysozyme catalysis in solution, as suggested recently [Zhang, X. J.,Wozniak, J. A., & Matthews, B. W.(1995) J. Mol. Biol. 250, 527-552]. The strategyemployed here, based on site-directed spin labeling,should be generally applicable to the study of protein conformation andconformational changes in solution.