Department of Integrative Structural and Computational Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
Biochemistry. 2013 May 21;52(20):3464-77. doi: 10.1021/bi400322e. Epub 2013 May 7.
Escherichia coli dihydrofolate reductase (DHFR) provides a paradigm for the integrated study of the role of protein dynamics in enzyme function. Previous studies of backbone and side chain dynamics have yielded unprecedented insights into the mechanism by which DHFR progresses through the structural changes that occur during its catalytic cycle. Here we report a comprehensive study of the χ1 rotamer populations of the aromatic and γ-methyl containing residues for complexes of the catalytic cycle, based on NMR measurement of (3)JCγCO and (3)JCγN coupling constants. We report conformational and dynamic information for eight distinct complexes, where transitions between rotamer wells may occur on a broad picosecond to millisecond time scale. This large volume of (3)J data has allowed us to fit new Karplus parameterizations for aromatic side chains and to select the best available of previously determined parameters for Ile, Thr, and Val. The (3)JCγCO and (3)JCγN coupling constants are found to be extremely sensitive measures of side chain χ1 rotamers and to give important insights into the extent of conformational averaging. For a subset of residues in DHFR, the extent of rotamer averaging is invariant to the nature of the bound ligand, while for other residues the rotamer averaging differs in one or more complexes of the enzymatic cycle. These variable-averaging residues are generally located near the active site, but the phenomenon extends into the adenosine binding domain. For several residues, the rotamer populations in different DHFR complexes appear to depend on whether the complex is in the closed or occluded state, and some residues are exquisitely sensitive to small changes in the nature of the bound ligand.
大肠杆菌二氢叶酸还原酶（DHFR）为研究蛋白质动力学在酶功能中的作用提供了一个范例。先前对骨架和侧链动力学的研究为 DHFR 在其催化循环中发生的结构变化过程中如何发挥作用的机制提供了前所未有的见解。在这里，我们根据（3）JCγCO 和（3）JCγN 偶合常数的 NMR 测量，报告了催化循环中芳香族和含γ-甲基残基的 χ1 构象体群体的综合研究。我们报告了 8 种不同复合物的构象和动态信息，其中构象体阱之间的转变可能发生在宽皮秒到毫秒的时间尺度上。大量的（3）J 数据使我们能够为芳香族侧链拟合新的 Karplus 参数化，并为 Ile、Thr 和 Val 选择最佳的现有参数。（3）JCγCO 和（3）JCγN 偶合常数被发现是侧链 χ1 构象体的极其敏感的测量方法，并为构象平均程度提供了重要的见解。对于 DHFR 中的一小部分残基，构象体平均程度与结合配体的性质无关，而对于其他残基，构象体平均程度在酶循环的一个或多个复合物中不同。这些可变量平均残基通常位于活性部位附近，但这种现象延伸到腺苷结合域。对于几个残基，不同 DHFR 复合物中的构象体群体似乎取决于复合物是处于闭合还是闭塞状态，并且一些残基对结合配体性质的微小变化非常敏感。
