NUCLEAR MAGNETIC RESONANCE--NEW METHODS AND MOLECULAR STRUCTURE DETERMINATION

蛋白质的净磁化率各向异性导致其非 当各向同性解被放置在一个强 磁场。已经开发出新的方法来测量 由小度偶极耦合引起的小偶极耦合 与磁场对齐。即使是小的抗磁性的 蛋白质偶极偶联可能产生有用的限制 用于结构计算和/或验证。其他重要的 与大分子结构测定相关的研究进展 核磁共振，包括第一次实验确定一个完整的 非对称旋转扩散张量，针对HIV 蛋白酶同源二聚体与氢气之间的实验相关性 键长与主链上的主链--氚四极偶合。这 后一项实验也代表了主链酰胺第一次 已经在蛋白质中测量到了重离子四极耦合。 HIV-a辅助蛋白Nef的结构已确定 通过多维异核核磁共振。结构确定 涉及许多非标准元素，包括使用 不可交换的氢的过氢化反应，产生一种蛋白质 折叠和建议的突变以减少蛋白质聚集。 然后对蛋白质的较少聚集形式进行标准实验 产生了一个低分辨率的三维结构。NEF与 与HCK SH3的亚微摩尔亲和力，相互作用表面为 根据化学位移微扰确定的。这 交互表示SH3域交互的第一个示例 具有不止一个(富含脯氨酸的)表位。类似的实验 用于鉴定特定的、尽管亲和力低(Kd)的 约1 mM)，胞质N-的多肽片段的结合位置 CD4的末端结构域。Nef与CD4多肽和HCK的结合 Sh#是弱合作事件。

Net magnetic susceptibility anisotropy of a protein causes its non- random distribution when an isotropic solution is placed in a strong magnetic field. New methods have been developed for measurement of the small dipolar couplings which result from the small degree of alignment with the magnetic field. Even for small diamagnetic proteins the dipolar couplings potentially yield useful constraints for structure calculation and/or validation. Other significant developments, related to macromolecular structure determination by NMR, include the first experimental determination of a fully asymmetric rotational diffusion tensor, carried out for the HIV protease homodimer, and an experimental correlation between hydrogen bond length and the backbone amide deuteron quadrupole coupling. This latter experiment also represents the first time that backbone amide deuteron quadrupole couplings have been measured in a protein. The structure of the HIV-a accessory protein Nef has been determined by multi-dimensional heteronuclear NMR. Structure determination involved a number of non-standard elements, including the use of perdeuteration of non-exchangeable hydrogens, which yielded a protein fold and suggested mutations for reducing protein aggregation. Standard experiments on the less-aggregating form of the protein then yielded a low-resolution three-dimensional structure. Nef binds with submicromolar affinity to Hck SH3, and the interaction surface was determined on the basis of chemical shift perturbation. This interaction represents the first example of an SH3 domain interacting with more than a single (proline-rich) epitope. Similar experiments were used for identification of a specific, albeit low affinity (Kd about 1mM), binding site for a peptide fragment of the cytosolic N- terminal domain of CD4. Binding of Nef to the CD4 peptide and the Hck SH# are weakly cooperative events.