Protein structure and dynamics from residual dipolar couplings

残余偶极耦合的蛋白质结构和动力学

• 批准号: 8148713
• 负责人: Ad Bax
• 金额: $ 38.84万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8148713
• 关键词: Residual state; protein structure

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.

描述液晶介质中蛋白质比对的SAUPE基质包含五个独立元素，从而可以产生多达五个线性独立的比对条件。 在这些条件下，NMR光谱法测量了核内残留偶性耦合（RDC），在五维对齐空间中正交，可访问幅度，不对称和核心内部载体运动的方向。对于小蛋白质结构域GB3（56个残基）证明，可以通过产生一系列对域域的反向主链结构的保守性突变体来生成一系列保守的突变体，从而在PF1噬菌体的单个液体晶体介质中适当地进行正交对准条件。 突变涉及几个暴露溶剂的侧技术的电荷，以及通常用于蛋白质纯化的N-或C末端HIS-TAG肽对蛋白质的延伸。 这些蛋白质突变体绘制了五维对齐空间，为结构和动力学提供了独特的见解，并提供了对各向异性参数的访问，例如13C，15N和1H化学屏蔽量张量。 位点特异性15N各向异性（CSA）张量已为良好的骨干酰胺酰胺15N核从蛋白G（GB3）的B3域（GB3）中的残留化学移动各向异性（RCSA）得出，在六个不同的突变体中测量的六个不同突变体中测量的液体结构不同，但在液体中保持液位相对于液态静止1个液体，但在液体中均不同。 通过测量15N CSA张量与15n-1h或15n-13c'偶极相互作用之间的交叉相关弛豫率，可以补充此信息。 与最近的固态NMR测量值一致，15N CSA张量仅显示平均值的变化程度中等，但在α-螺旋（-173 7 ppm）中的幅度大于beta-sheet（-162 6 ppm）残基（-173 7 ppm），这一发现还通过量子计算证实了这一发现。 最低屏蔽量张量的组件的方向紧密地围绕肽平面矢量，该载体与N-H键的角度为19.62.5，而15N CSA张量的不对称性在α-螺旋体中略小（ETA = 0.230.17）（ETA = 0.230.17）。 特定于残基的15N CSA值通过改进的计算和实验性15N R1RHO弛豫率在GB3中测得的15N-2H位点（由CSA机制支配）的一致性得到了验证。 使用残留特异性15N CSA值还会导致更均匀的概括性阶参数S2，并预测磁场强度缩小最有效的磁场强度中的相当大的残基变化。 液晶NMR还使用了上述蛋白质的五个结构保守的突变体研究了蛋白质GB3的骨架肽组的N-H键长。 在没有其他信息的情况下，N-H矢量取向的动态波动对15N-1H偶极相互作用的影响不能与N-H键长度的变化分开。但是，N-H键长的变化直接影响肽组中C'-H载体的方向，并对13C'-HN和15N-HN残留偶性偶联的同时分析，在五个不同的对齐方向下测量，允许在平均n-h-h-h-e键长度的平均平均键长度的模量上测量Gb3，gb3，gb3，gb3，fust rnheq。键拉伸的非谐性导致较长的时间平均键长<rnh> = 1.015 0.006，有效的键长reff = <rnh-3> -1/3 = 1.023 0.006 0.006 0.006涉及NMR松弛分析，不包括在N-H-H-H-H-H-H-H-H-H-H-H-H-HERERISSATION中的零点的影响。 使用由蛋白质的骨架C-C'向量定义的参考框，发现二级结构元素中N-H矢量的角波动的角度波动比肽平面内的运动外运动大约1.5倍，并且比肽平面内的运动的动作高，并且根据其量子机械分析的量子机械分析，而不是预期的。