Protein structure and dynamics from residual dipolar couplings

描述液晶介质中蛋白质比对的索普矩阵包含五个独立的元素，从而能够产生最多五个线性独立的比对条件。在这些条件下，用核磁共振波谱测量核间残留偶极耦合(RDC)，在五维排列空间中正交，提供了核间矢量的幅度、不对称性和运动方向的途径。研究表明，对于小的蛋白质结构域GB3(56个残基)，通过产生一系列对结构域的时间平均骨架结构影响可忽略不计的保守突变体，可以在PF1噬菌体的单一液晶介质中产生合适的正交比对条件。突变包括几个暴露在溶剂中的侧链电荷的变化，以及蛋白质的N-末端或C-末端组氨酸标记肽的延伸，通常用于蛋白质纯化。这些蛋白质突变体绘制了五维排列空间，提供了对结构和动力学的独特见解，并提供了访问各向异性参数的途径，如13C，15N和1H化学屏蔽张量。 根据在六个不同突变体中测量的残余化学位移各向异性(RCSA)，推导出了蛋白质G(GB3)B3结构域中有序的主干酰胺15N核的位点特定的15N化学位移各向异性(CSA)张量，这些突变体保留了天然结构，但在溶解在液晶PF1悬浮液中时，相对于静态磁场的排列方式不同。这一信息通过测量15N CSA张量和15N-1H或15N-13C‘偶极相互作用之间的互相关弛豫速率来补充。与最近的固体核磁共振测量结果一致，15N CSA张量与平均值只有中等程度的变化，但α-螺旋(-173 7ppm)比β-折叠(-162 6ppm)有更大的幅度，这一发现也得到了量子计算的证实。屏蔽最少的张量组分的取向紧密地聚集在与N-H键成19.62.5角的肽平面矢量周围，15N CSA张量的不对称性在α-螺旋中(ETA=0.230.17)略小于在β-折叠中(ETA=0.310.11)。通过对GB3中15N-2H位的15N-2H位的15N-2H驰豫速率的计算与实验结果的一致性，验证了残基特定的15N CSA值，这些驰豫速率由CSA机制主导。使用残基特定的15N CSA值还会产生更均匀的广义有序参数S2，并预测在TROSY线收窄最有效的地方，每个残基的磁场强度会有相当大的变化。 利用上述5个结构保守的突变体，用液晶核磁共振方法研究了GB3蛋白主链多肽基团的N-H键长度。在没有附加信息的情况下，N-H矢量取向的动态波动对15N-1H偶极相互作用的影响与N-H键长的变化是分不开的。然而，N-H键长的变化直接影响多肽中C‘-H载体的取向，并且同时分析在五种不同排列方向下测量的13C’-HN和15N-HN残余偶极耦合，允许无模型地确定GB3中平均平衡N-H键长，得到rNHeq=1.008 0.006。键拉伸的非谐性导致时间平均键长&lt；rnh&gt；=1.015 0.006，有效键长reff=&lt；rnh-3&gt；-1/3=1.023 0.006，不包括零点或N-H取向上其他角波动的影响。使用由蛋白质主干C&#61537；-C‘矢量定义的参照系，二级结构元素中N-H矢量的角波动被发现对于平面外的波动比在肽平面内的运动大约1.5倍，并且根据对其零点振动的量子力学分析并不比预期的大很多。

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.