Skrynnikov N R, Goto N K, Yang D, Choy W Y, Tolman J R, Mueller G A, Kay L E
Contribution Protein Engineering Network Centres of Excellence and Department of Medical Genetics, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
J Mol Biol. 2000 Feb 4;295(5):1265-73. doi: 10.1006/jmbi.1999.3430.
Protein function is often regulated by conformational changes that occur in response to ligand binding or covalent modification such as phosphorylation. In many multidomain proteins these conformational changes involve reorientation of domains within the protein. Although X-ray crystallography can be used to determine the relative orientation of domains, the crystal-state conformation can reflect the effect of crystal packing forces and therefore may differ from the physiologically relevant form existing in solution. Here we demonstrate that the solution-state conformation of a multidomain protein can be obtained from its X-ray structure using an extensive set of dipolar couplings measured by triple-resonance multidimensional NMR spectroscopy in weakly aligning solvent. The solution-state conformation of the 370-residue maltodextrin-binding protein (MBP) loaded with beta-cyclodextrin has been determined on the basis of one-bond (15)N-H(N), (15)N-(13)C', (13)C(alpha)-(13)C', two-bond (13)C'-H(N), and three-bond (13)C(alpha)-H(N) dipolar couplings measured for 280, 262, 276, 262, and 276 residues, respectively. This conformation was generated by applying hinge rotations to various X-ray structures of MBP seeking to minimize the difference between the experimentally measured and calculated dipolar couplings. Consistent structures have been derived in this manner starting from four different crystal forms of MBP. The analysis has revealed substantial differences between the resulting solution-state conformation and its crystal-state counterpart (Protein Data Bank accession code 1DMB) with the solution structure characterized by an 11(+/-1) degrees domain closure. We have demonstrated that the precision achieved in these analyses is most likely limited by small uncertainties in the intradomain structure of the protein (ca 5 degrees uncertainty in orientation of internuclear vectors within domains). In addition, potential effects of interdomain motion have been considered using a number of different models and it was found that the structures derived on the basis of dipolar couplings accurately represent the effective average conformation of the protein.
蛋白质功能通常受构象变化调控，这种构象变化是对配体结合或共价修饰（如磷酸化）的响应。在许多多结构域蛋白质中，这些构象变化涉及蛋白质内结构域的重新定向。虽然X射线晶体学可用于确定结构域的相对取向，但晶体状态构象可能反映晶体堆积力的影响，因此可能与溶液中存在的生理相关形式不同。在此我们证明，使用在弱排列溶剂中通过三共振多维核磁共振光谱测量的大量偶极耦合，可以从其X射线结构获得多结构域蛋白质的溶液状态构象。基于分别针对280、262、276、262和276个残基测量的一键（¹⁵N-H(N)）、（¹⁵N-¹³C'）、（¹³Cα-¹³C'）、二键（¹³C'-H(N)）和三键（¹³Cα-H(N)）偶极耦合，已确定了负载β-环糊精的370个残基的麦芽糖糊精结合蛋白（MBP）的溶液状态构象。通过对MBP的各种X射线结构应用铰链旋转来生成这种构象，以尽量减少实验测量和计算的偶极耦合之间的差异。从MBP的四种不同晶体形式开始，以这种方式获得了一致的结构。分析揭示了所得溶液状态构象与其晶体状态对应物（蛋白质数据库登录号1DMB）之间的显著差异，溶液结构的特征是结构域闭合11（±1）度。我们已经证明，这些分析中实现的精度很可能受蛋白质结构域内结构的小不确定性（结构域内核间矢量取向的约5度不确定性）限制。此外，使用多种不同模型考虑了结构域间运动的潜在影响，发现基于偶极耦合推导的结构准确地代表了蛋白质的有效平均构象。
