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: 7967277
• 负责人: Ad Bax
• 金额: $ 28.11万
• 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/7967277
• 关键词: Agreement; Anisotropy; Antibodies; Bacteriophage Pf1; C-terminal; Charge; Chemicals; Elements; Generations; Hour; Hydrogen Bonding; Indium; Liquid substance; Maps; Measurement; Measures; Methods; Motion; Mutation; NMR Spectroscopy; Peptides; Procedures; Process; Proteins; Relaxation; Reporting; Residual state; Series; Solutions; Solvents; Structure; Surface; Suspension substance; Suspensions; Tertiary Protein Structure; Time; Torsion; Variant; Vertebral column; insight; mutant; novel; protein purification; protein structure; solid state nuclear magnetic resonance; vector

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. A novel iterative procedure has been developed which allows both the orientation and dynamics of internuclear bond vectors to be determined from direct interpretation of NMR dipolar couplings, measured under at least three orthogonal alignment conditions. With the five orthogonal alignments available from the above mentioned mutation procedure, the approach also yields information on the degree of motional anisotropy and the direction in which the largest amplitude internal motion of each bond vector takes place. The method is demonstrated for the backbone 15N-1H, 13Ca-1Ha, and 13Ca-13C' interactions in the previously well studied protein domain GB3, dissolved in a liquid crystalline suspension of filamentous phage Pf1. Alignment variation is achieved by using conservative mutations of charged surface residues. Results indicate remarkably uniform backbone dynamics, with amplitudes that agree well with those of previous 15N relaxation studies for most residues involved in elements of secondary structure, but larger amplitude dynamics than found by 15N relaxation for residues in loop and turn regions. In agreement with a previous analysis of dipolar couplings, the N-H bonds in the second beta-strand, which is involved in antibody recognition, show elevated dynamics with largest amplitudes orthogonal to the chain direction. The same set of mutants also provides access to residue-specific determination of the 15N chemical shift anisotropy (CSA) tensor. Very extensive prior experimental has resulted in contradictory findings regarding the uniformity of the CSA tensor, which is a key parameter underlying NMR relaxation studies of protein backbone dynamics. Our results on GB3 indicate that recent solid-state NMR results are clearly superior to most other prior studies, and indicate a moderate degree of residue by residue variation, which is mostly dominated by the backbone torsion angles phi and psi, and by the type and orientation of the intraresidue and preceding residue sidechains.

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。 已经开发了一种新型的迭代程序，该程序允许通过直接解释NMR偶性耦合来确定核键矢量的方向和动力学，并在至少三个正交对齐条件下测量。通过上述突变程序可获得的五个正交比对，该方法还产生有关运动各向异性程度的信息以及每个键矢量发生的最大振幅内部运动的方向。在先前研究良好的蛋白质结构域GB3中，骨架15N-1H，13CA-1HA和13CA-13C的相互作用证明了该方法，该方法溶解在丝状噬菌体PF1的液晶悬浮液中。通过使用带电表面残基的保守突变来实现比对变化。结果表明，主链动力学非常均匀，幅度与先前15N弛豫研究的幅度非常吻合，大多数与二级结构元件相关的残留物相比，但幅度动力学比15N弛豫在循环和转弯区域中发现的幅度更大。 与先前对偶极耦合的分析一致，第二个β链中涉及抗体识别的N-H键表现出升高的动力学，其幅度最大，幅度最大。 相同的一组突变体还提供了15N化学偏移各向异性（CSA）张量的残基特异性测定。 非常广泛的先验实验导致有关CSA张量的均匀性的矛盾发现，这是蛋白质骨干动力学的NMR弛豫研究基础的关键参数。我们对GB3的结果表明，最近的固态NMR结果显然优于大多数其他先前的研究，并表明残基变化的残留度中等，这主要由主链扭转角PHI和PSI以及内内的类型和方向的内置和方向而主导。