MULTINUCLEAR NMR PROBES OF BIOLOGICAL SYSTEMS

长期目标是开发和应用最先进的核磁共振 确定溶液结构性质的技术 普遍分布的参与金属稳态的蛋白质， 金属硫蛋白，MT。 此信息对于阐明至关重要 MT 在金属调节中作用的分子机制。 安 拟议研究的一个组成部分是贡献 用于大分子研究的新型 NMR 方法的调整/完善。 虽然最近可以通过以下方式获得详细的结构信息 含 Cd、Zn 哺乳动物的 NMR 和 X 射线晶体学方法 MT，类似的结构信息不适用于含 Cu 的 哺乳动物 MT 的形式，也不是来自其他物种的 MT （酿酒酵母、粗糙脉孢菌）。 具体核磁共振实验 建议使用银离子作为化学和磁性（自旋 I=1/2) 同晶金属离子替代 Cu 测定 这种金属形式的 MT 的溶液结构。 核磁共振方法也将被使用 用于海洋中 MT 的完整解决方案结构测定 甲壳类动物、美洲龙虾和蓝蟹，它们将 二价金属离子Cd和Zn，和/或一价金属离子Cu。 从这些来源中选择的 MT 样本，其中包括重组体 中国仓鼠 MT 的形式，将同时和/或随后使用 在旨在描述动力学和 与该蛋白质的生理作用相关的机制特征 金属稳态。 由于金属的相似性，人们很容易推测： 转录因子和 MT 中的结合基序，后者 可能在细胞修复过程中具有金属调节功能， 生长、分化。

The long-term objectives are to develop and apply state-of-the-art NMR techniques to the determination of the solution structural properties of a ubiquitously distributed protein involved in metal homeostasis, metallothioneins, MTs. This information is essential for the elucidation of the molecular mechanism of action of MT in metal regulation. An integral component of the proposed research is to contribute the adaptation/refinement of novel new NMR methods for macromolecular studies. While detailed structural information has recently become available through NMR and X-ray crystallographic methods for the Cd, Zn containing mammalian MT, similar structural information is not available for the Cu+ containing forms of the mammalian MT, nor for the MTs from other species (Saccharomyces cerevisiae, Neurospora crassa). Specific NMR experiments are proposed using the Ag+ ion as a chemically and magnetically (spin I=1/2) isomorphic metal ion substitute for Cu+ for the determination of the solution structure of this metal form of MT. NMR methods will also be used for the full solution structure determination of MTs from the marine crustaceans, the American lobster and the blue crab, which bind the divalent metal ions, Cd and Zn, and/or the monovalent metal ion, Cu. Selected MT samples from these sources, which will include recombinant forms of Chinese hamster MT, will concurrently and/or subsequently be used in experiments whose objectives are to delineate the kinetics and mechanistic features associated with this protein's physiological role in metal homeostasis. It is tempting to speculate that because of the analogies of the metal- binding motifs in both the transcription factors and MT, that the latter could have a metalloregulatory function in cellular repair processes, growth, and differentiation.