Department of Integrative Structural and Computational Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037.
Proc Natl Acad Sci U S A. 2014 Jan 28;111(4):E445-54. doi: 10.1073/pnas.1323440111. Epub 2014 Jan 13.
Detailed descriptions of atomic coordinates and motions are required for an understanding of protein dynamics and their relation to molecular recognition, catalytic function, and allostery. Historically, NMR relaxation measurements have played a dominant role in the determination of the amplitudes and timescales (picosecond-nanosecond) of bond vector fluctuations, whereas high-resolution X-ray diffraction experiments can reveal the presence of and provide atomic coordinates for multiple, weakly populated substates in the protein conformational ensemble. Here we report a hybrid NMR and X-ray crystallography analysis that provides a more complete dynamic picture and a more quantitative description of the timescale and amplitude of fluctuations in atomic coordinates than is obtainable from the individual methods alone. Order parameters (S(2)) were calculated from single-conformer and multiconformer models fitted to room temperature and cryogenic X-ray diffraction data for dihydrofolate reductase. Backbone and side-chain order parameters derived from NMR relaxation experiments are in excellent agreement with those calculated from the room-temperature single-conformer and multiconformer models, showing that the picosecond timescale motions observed in solution occur also in the crystalline state. These motions are quenched in the crystal at cryogenic temperatures. The combination of NMR and X-ray crystallography in iterative refinement promises to provide an atomic resolution description of the alternate conformational substates that are sampled through picosecond to nanosecond timescale fluctuations of the protein structure. The method also provides insights into the structural heterogeneity of nonmethyl side chains, aromatic residues, and ligands, which are less commonly analyzed by NMR relaxation measurements.
详细的原子坐标和运动描述对于理解蛋白质动力学及其与分子识别、催化功能和变构的关系是必要的。历史上，NMR 弛豫测量在确定键矢量波动的幅度和时间尺度（皮秒-纳秒）方面发挥了主导作用，而高分辨率 X 射线衍射实验可以揭示蛋白质构象集合中多个弱占据亚态的存在，并提供原子坐标。在这里，我们报告了一种 NMR 和 X 射线晶体学的混合分析，它提供了一个更完整的动态图像，以及对原子坐标波动的时间尺度和幅度的更定量描述，比单独使用单个方法所能获得的更完整、更定量。序参数（S(2)）是根据室温和低温 X 射线衍射数据为二氢叶酸还原酶拟合的单构象和多构象模型计算的。从 NMR 弛豫实验得出的骨架和侧链序参数与从室温单构象和多构象模型计算出的序参数非常吻合，表明在溶液中观察到的皮秒时间尺度运动也发生在晶体状态。这些运动在低温下被晶体淬火。NMR 和 X 射线晶体学的迭代 refinement 相结合，有望提供对通过蛋白质结构的皮秒到纳秒时间尺度波动采样的交替构象亚态的原子分辨率描述。该方法还深入了解了非甲基侧链、芳香族残基和配体的结构异质性，这些在 NMR 弛豫测量中较少被分析。
