Evaluation of backbone proton positions and dynamics in a small protein by liquid crystal NMR spectroscopy
Evaluation of backbone proton positions and dynamics in a small protein by liquid crystal NMR spectroscopy
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DOI：
10.1021/ja0350684
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发表时间：
2003-07-30
影响因子：
15
通讯作者：
Bax, A
中科院分区：
文献类型：
--
作者：
Ulmer, TS;Ramirez, BE;Bax, A
对大量蛋白质骨架单键偶极偶联进行了 NMR 测量，以细化蛋白质 G (GB3) 的第三个 IgG 结合域的结构，该结构之前通过 X 射线晶体学以 1.1 埃的分辨率解决。除了常用的 bicelle、聚乙二醇和丝状噬菌体液晶介质外，当蛋白质在带正电或带负电的拉伸丙烯酰胺凝胶内排列时，也会测量偶极耦合。针对 C-13(α)-C-13' 和 C-13'-N-15 偶极耦合的 GB3 晶体结构的细化提高了实验和预测的 N-15-H-1(N) 以及 C-13(α)-H-1(α) 偶极耦合之间的一致性。肽键 N-H 方向的评估显示肽键扭转角 omega 与 180 度的偏差与 N-H 向量与 C'-N-C-α 平面之间的角度之间存在弱反相关性。该相关性的斜率为 -1，表明平均而言，肽 N 的金字塔化会导致肽键平面性的小偏差（= 179.3 +/- 3.1 度），其程度与围绕 C'-N 键的真实扭曲相同。虽然氢通常构建在晶体结构上，假设 N-H 矢量方向落在平分 C'-N-C-α 角的线上，但更好的近似将 C-α-C'-N-H 扭转角调整为 -2 度。 N-15-H-1(N)偶极数据与普遍接受的运动模型并不矛盾，其中正交于肽平面的N-H键的角波动大于面内运动，但正交于C-i-1(α)-N-i-C-i(α)平面的角波动幅度最多超过面内运动的幅度10-15度。偶极耦合分析表明，对于大多数 GB3 主链，酰胺有序参数 S 高度均匀，变化小于 +/-7%。 H-α 质子位置的评估表明，平均 C-α-H-α 向量方向与与 C-α-C-β 和 C-α-N 向量形成理想四面体角的方向偏离不到 1 度。
NMR measurements of a large set of protein backbone one-bond dipolar couplings have been carried out to refine the structure of the third IgG-binding domain of Protein G (GB3), previously solved by X-ray crystallography at a resolution of 1.1 Angstrom. Besides the commonly used bicelle, poly(ethylene glycol), and filamentous phage liquid crystalline media, dipolar couplings were also measured when the protein was aligned inside either positively or negatively charged stretched acrylamide gels. Refinement of the GB3 crystal structure against the C-13(alpha)-C-13' and C-13'-N-15 dipolar couplings improves the agreement between experimental and predicted N-15-H-1(N) as well as C-13(alpha)-H-1(alpha) dipolar couplings. Evaluation of the peptide bond N-H orientations shows a weak anticorrelation between the deviation of the peptide bond torsion angle omega from 180degrees and the angle between the N-H vector and the C'-N-C-alpha, plane. The slope of this correlation is -1, indicating that, on average, pyramidalization of the peptide N contributes to small deviations from peptide bond planarity ( = 179.3 +/- 3.1degrees) to the same degree as true twisting around the C'-N bond. Although hydrogens are commonly built onto crystal structures assuming the N-H vector orientation falls on the line bisecting the C'-N-C-alpha, angle, a better approximation adjusts the C-alpha-C'-N-H torsion angle to -2degrees. The N-15-H-1(N) dipolar data do not contradict the commonly accepted motional model where angular fluctuations of the N-H bond orthogonal to the peptide plane are larger than in-plane motions, but the amplitude of angular fluctuations orthogonal the C-i-1(alpha)-N-i-C-i(alpha) plane exceeds that of in-plane motions by at most 10-15degrees. Dipolar coupling analysis indicates that for most of the GB3 backbone, the amide order parameters, S, are highly homogeneous and vary by less than +/-7%. Evaluation of the H-alpha proton positions indicates that the average C-alpha-H-alpha vector orientation deviates by less than 1degrees from the direction that makes ideal tetrahedral angles with the C-alpha-C-beta and C-alpha-N vectors.