Protein structure and dynamics from residual dipolar couplings

描述蛋白质在液晶介质中的排列的Saupe矩阵包含五个独立的元素，使得能够产生多达五个线性独立的排列条件。 在这些条件下，在五维排列空间中正交的NMR光谱法测量核间残余偶极耦合（RDC），提供了对核间矢量运动的幅度、不对称性和方向的访问。它表明，对于小的蛋白质结构域GB 3（56个残基），适当的正交对齐条件可以产生在一个单一的液晶介质中的Pf 1噬菌体，通过产生一系列的保守突变体，具有可忽略不计的时间平均的骨干结构域的影响。 突变涉及几个溶剂暴露侧链的电荷变化，以及通过N-或C-末端His-标签肽（通常用于蛋白质纯化）的蛋白质延伸。 这些蛋白质突变体绘制了五维对齐空间，提供了对结构和动力学的独特见解，并提供了对各向异性参数的访问，如13 C，15 N和1H化学屏蔽张量。 位点特异性的15 N化学位移各向异性（CSA）张量已被推导出的有序的骨干酰胺15 N核蛋白G（GB 3）的B3域从残留的化学位移各向异性（RCSA）测量在6个不同的突变体，保留了天然结构，但不同的对齐相对于静态磁场时，溶解在液晶Pf 1悬浮液。 这些信息通过测量15 N CSA张量与15 N-1H或15 N-13 C '偶极相互作用之间的交叉相关弛豫速率来补充。 与最近的固态NMR测量结果一致，15 N CSA张量仅表现出与平均值的中等程度的变化，但在α-螺旋（~ 173 7 ppm）中具有比β-折叠（~ 162 6 ppm）残基更大的幅度，这一发现也通过量子计算得到证实。 屏蔽最少的张量分量的取向紧密地聚集在与N-H键成19.62.5 °角的肽平面内矢量周围，15 N CSA张量的不对称性在α-螺旋（eta=0.230.17）中比在β-折叠（eta=0.310.11）中略小。 残留物特定的15 N CSA值进行了验证，通过改进的协议计算和实验15 N R1 rho弛豫速率测量的15 N-2 H网站在GB 3中，这是占主导地位的CSA机制。 使用特定于残基的15 N CSA值还导致更均匀的广义序参数S2，并预测TROSY线变窄最有效的磁场强度中的相当大的残基变化。 还通过液晶NMR研究了蛋白质GB 3的骨架肽基团中的N-H键长度，使用该蛋白质的上述结构保守的突变体中的五种。 在没有额外信息的情况下，N-H矢量取向的动态波动对15 N-1H偶极相互作用的影响不能与N-H键长的变化分开。然而，在N-H键长的变化直接影响的取向的C '-H载体的肽组，和13 C'-HN和15 N-HN残留偶极耦合，在五个不同的对齐方向下测量的同时分析，允许无模型测定的平均平衡N-H键长GB 3，产生rNHeq = 1.008 - 0.006。键拉伸的非谐性导致稍微更长的时间平均键长<rNH>= 1.015 0.006，并且有效键长reff = <rNH-3>-1/3 = 1.023 0.006与NMR弛豫分析相关，不包括N-H取向中零点或其他角度波动的影响。 使用由蛋白质的骨架C-C '载体定义的参考系，发现二级结构元件中的N-H载体的角波动对于平面外波动比肽平面内的运动大约1.5倍，并且比基于其零点振动的量子力学分析所预期的大不了多少。

The Saupe matrix describing protein alignment in a liquid crystalline medium contains five independent elements, enabling the generation of up to five linearly independent alignment conditions. Measurement of internuclear residual dipolar couplings (RDCs) by NMR spectroscopy under these conditions, orthogonal in five-dimensional alignment space, provides access to the amplitude, asymmetry, and direction of motions of the internuclear vector. It is demonstrated for the small protein domain GB3 (56 residues) that suitably orthogonal alignment conditions can be generated in a single liquid crystalline medium of Pf1 phage, by generating a series of conservative mutants that have negligible impact on the time-averaged backbone structure of the domain. Mutations involve changes in the charge of several solvent-exposed sidechains, as well as extension of the protein by either an N- or C-terminal His-tag peptide, commonly used for protein purification. These protein mutants map out the five-dimensional alignment space, providing unique insights into the structure and dynamics, and providing access to anisotropic parameters such as the 13C, 15N and 1H chemical shielding tensors. Site-specific 15N chemical shift anisotropy (CSA) tensors have been derived for the well-ordered backbone amide 15N nuclei in the B3 domain of protein G (GB3) from residual chemical shift anisotropy (RCSA) measured in six different mutants that retain the native structure but align differently relative to the static magnetic field when dissolved in a liquid crystalline Pf1 suspension. This information is complemented by measurement of cross-correlated relaxation rates between the 15N CSA tensor and either the 15N-1H or 15N-13C' dipolar interaction. In agreement with recent solid state NMR measurements, the 15N CSA tensors exhibit only a moderate degree of variation from averaged values, but have larger magnitudes in alpha-helical (-173 7 ppm) than in beta-sheet (-162 6 ppm) residues, a finding also confirmed by quantum computations. The orientations of the least shielded tensor component cluster tightly around an in-peptide-plane vector that makes an angle of 19.62.5 with the N-H bond, with the asymmetry of the 15N CSA tensor being slightly smaller in alpha-helix (eta=0.230.17) than in beta-sheet (eta=0.310.11). The residue-specific 15N CSA values are validated by improved agreement between computed and experimental 15N R1rho relaxation rates measured for 15N-2H sites in GB3, which are dominated by the CSA mechanism. Use of residue-specific 15N CSA values also results in more uniform generalized order parameters, S2, and predicts considerable residue-by-residue variations in the magnetic field strengths where TROSY line narrowing is most effective. The N-H bond length in backbone peptide groups of the protein GB3 has also been studied by liquid crystal NMR, using five of the above mentioned structurally conserved mutants of this protein. In the absence of additional information, the impact of dynamic fluctuations of the N-H vector orientation on the 15N-1H dipolar interaction cannot be separated from a change in N-H bond length. However, a change in N-H bond length directly impacts the orientation of C'-H vectors in the peptide group, and simultaneous analysis of 13C'-HN and 15N-HN residual dipolar couplings, measured under five different alignment orientations, permitted modelfree determination of the average equilibrium N-H bond length in GB3, yielding rNHeq = 1.008 0.006 . Anharmonicity of the bond stretching resulted in a slightly longer time-averaged bond length <rNH> = 1.015 0.006 , and an effective bond length reff = <rNH-3>-1/3 = 1.023 0.006 pertinent for NMR relaxation analysis, not including the impact of zero-point or other angular fluctuations in N-H orientation. Using a reference frame defined by the backbone C&#61537;-C' vectors of the protein, angular fluctuations for N-H vectors in elements of secondary structure were found to be approximately 1.5 fold larger for out-of-plane fluctuations than motions within the peptide plane and not much larger than anticipated on the basis of quantum mechanical analysis of their zero-point librations.