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.

已经开发了一种新的NMR方法来测量蛋白质中的超频距离和定向信息。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-13C偶极相互作用之间的干扰。效应的大小与脉冲距离距离的第六功率相反，与NOE距离信息相反，它对间接效应根本不敏感。比较蛋白质G的第三个IgG结合结构域中测量的速率，为此，可以使用1.1-A分辨率X射线结构，显示出很好的一致性，但仍受到晶体结构的坐标精度的限制。可用的多个1.8-A X射线结构可用的比较艾滋病毒蛋白酶的速率比较表明一致性较差，但是晶体结构的预测速率之间的一致性甚至较差，这表明这些结构中的坐标不确定性很高，并且在结构中的相关速率应大大提高，均应提高了坐标精度。弱对准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重点。已经开发了对齐方法的改进，包括引入各向异性压缩带电的丙烯酰胺凝胶。这些凝胶与洗涤剂兼容，并比较在洗涤剂或盘状胶束中溶解的HIV GP41肽的结构表明，在这两种情况下，肽都是α-螺旋，但是用污水胶束溶解的溶剂化，但降解型螺旋轴的螺旋轴均均具有固定型均方根的产量。还已经开发了对排列蛋白中1H-1H偶极相互作用的更有效方法，并且已经证明了超过7 A的距离的相互作用。预计这种方法对于研究较大，perdeuterate的系统的研究将很有用。