Nuclear Magnetic Resonance--new Methods And Molecular St

发展了一种新的核磁共振方法来测量蛋白质中的超精密距离和方向信息。所谓的相互关联磁化转移是以定量的方式进行的，依赖于1H-1H和1H-13C偶极相互作用之间的干扰。这种效应的大小与质子间距离的倒数六次方成正比，与NOE距离信息相比，它基本上不受间接影响的影响。在蛋白质G的第三个免疫球蛋白结合区域测得的速率的比较表明，相当好的一致性，但仍然受到晶体结构的坐标精度的限制。对HIV蛋白酶(有多个1.8-A X射线结构可用)测得的速率的比较显示，一致性要差得多，但晶体结构的预测速率之间的一致性甚至更差，这表明这些结构中的坐标不确定性很高，在结构精化中纳入互相关速率应该会在坐标精度方面产生相当大的改进。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重点。已开发了对齐方法的改进，包括引入各向异性压缩的带电丙烯酰胺凝胶。这些凝胶与洗涤剂相容，对HIV gp41肽在洗涤剂中溶解或被盘状胶束增溶的结构比较表明，在这两种情况下，该肽都是α螺旋的，但被洗涤剂胶束增溶产生强烈的螺旋轴弯曲，而使用平面胶束产生基本上直的螺旋。还开发了更有效的方法来测量比对蛋白质中的1H-1H偶极相互作用，并且已经证明了超过7A的距离的相互作用。预计这种方法将对研究更大的过氚系统有用。

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.