Nuclear Magnetic Resonance--new Methods And Molecular St

核磁共振--新方法与分子研究

• 批准号: 6673405
• 负责人: Ad - Bax
• 金额: --
• 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/6673405
• 关键词: biomagnetism measurement; calcium ion; calmodulin; chemical binding; intermolecular interaction; liquid crystal; magnetism; method development; micelles; nuclear magnetic resonance spectroscopy; nucleic acid structure; protein structure; structural biology

A new NMR approach has been developed to measure ultraprecise distance and orientational information in proteins. The so-called cross-correlated magnetization transfer, carried out in a quantitative fashion, relies on interference between 1H-1H and 1H-13C dipolar interactions. The magnitude of the effect scales with the inverse sixth power of the interproton distance, and in contrast to NOE distance information, it is essentially insensitive to indirect effects. Comparison of rates measured in the third Igg-binding domain of protein G, for which a 1.1-A resolution X-ray structure is available, shows quite good agreement, which nevertheless remains limited by the coordinate accuracy of the crystal structure. Comparison of rates measured for HIV protease, for which multiple 1.8-A X-ray structures are available, shows considerably poorer agreement, but agreement among predicted rates for the crystal structures is even poorer, indicating that the coordinate uncertainty in these structures is high, and that inclusion of the cross correlation rates in refinement of structures should yield considerable improvements in coordinate accuracy. Measurement of anisotropic interactions in weakly aligned proteins remains a main focus of our work. Improvements in alignment methodology have been developed, including the introduction of anisotropically compressed charged acrylamide gels. These gels are compatible with detergent, and comparison of the structure of an HIV gp41 peptide solubilized either in detergent or by disk-shaped micelles shows that in both cases the peptide is alpha-helical, but solubilization by detergent micelles yields strong curvature of the helix axis, whereas the use of flat micelles yields essentially straight helices. More effective methods for measurement of 1H-1H dipolar interactions in aligned proteins have also been developed, and interactions over distances exceeding 7 A have been demonstrated. It is anticipated that this approach will be useful for the study of larger, perdeuterated systems.

一种新的核磁共振方法已经发展到测量蛋白质的超精确距离和取向信息。以定量方式进行的所谓交叉相关磁化转移依赖于1H-1H和1H-13 C偶极相互作用之间的干扰。效应的大小与质子间距离的六次方成反比，与NOE距离信息相反，它对间接效应基本上不敏感。在蛋白G的第三个IgG结合结构域，其中1.1-A分辨率的X-射线结构是可用的速率测量的比较，显示出相当好的协议，但仍然受到晶体结构的坐标精度的限制。HIV蛋白酶，其中多个1.8-A的X-射线结构的测量速率的比较，显示出相当差的协议，但预测的晶体结构的速率之间的协议是更差，这表明在这些结构中的坐标不确定性是高的，并列入交叉相关率在细化的结构应该产生相当大的改善坐标精度。弱排列蛋白质中各向异性相互作用的测量仍然是我们工作的主要焦点。已经开发了对准方法的改进，包括引入各向异性压缩的带电丙烯酰胺凝胶。这些凝胶与洗涤剂是相容的，并且在洗涤剂中或通过盘形胶束增溶的HIV gp 41肽的结构的比较显示，在这两种情况下，肽是α-螺旋的，但是通过洗涤剂胶束增溶产生螺旋轴的强曲率，而使用平胶束产生基本上直的螺旋。更有效的方法来测量1H-1H偶极相互作用在对齐的蛋白质也已经开发出来，和相互作用的距离超过7A已被证明。预计这种方法将有助于研究更大的全氘系统。