STRUCTURAL KINETIC STUDIES BY NUCLEAR MAGNETIC RESONANCE

描述：人ADP核糖基化因子1（ARF 1）的构象 将通过溶液状态NMR进行研究。 场地自行车纯四重奏 共振将首次应用于蛋白质，以允许直接 Zn++、Ca++、Mg++和其它物种与它们相互作用的光谱 四周 ARF 1是一种参与形成的小G蛋白， 非网格蛋白包被的囊泡的调节。 ARF 1是一个大型 在这些囊泡表面发现了一种称为涂层剂的复合物。 的GTP ARF 1的形式是形成包衣体所必需的，GDP形式促进 囊泡融合 ARF蛋白具有额外的N-末端序列， 是螺旋形的，并在N-末端通过豆蔻酰化修饰。 是希望 了解存在于细胞中的ARF 1的形式，GDP或GTP连接， 以及它们如何通过肉豆蔻酰基与磷脂相互作用。 最初，将在GDP和GTP中研究非豆蔻酰化蛋白质 形式，以及缺乏独特的ARF N-末端的那些形式， 顺序 人们还希望研究这种蛋白质的肉豆蔻酰化形式 通过用最少量的洗涤剂溶解它。 这些蛋白质和许多其他蛋白质都含有上述离子 作为结构元素、活性催化中心或 使者 几乎没有人直接用化学方法研究这些离子。 当与蛋白质结合时的光谱。 纯四重共振可以确定 离子周围的非对称电子电荷分布，并可用于 推断其结合中涉及的共价相互作用的程度。 是 提出发展一种光谱学的形式， 对冷冻蛋白质溶液和单晶的研究敏感， 从而为研究周围环境的变化提供了一种全新的方法 这些离子的直接。 它将基于场循环核磁共振，其中 将冷冻样品快速移入和移出500 MHz磁体 在一次实验运行中多次。 该方法已被证明 以前在金属合金和小分子的研究中。 许多其他种类 也可以用这种方法研究许多问题，如键的取向 含氘核或17 O的基质，或 与蛋白质结合的含硼抑制剂。

DESCRIPTION: The conformations of human ADP ribosylation factor 1 (ARF1) are to be studied by solution state NMR. Field cycling pure quadruple resonance will be applied for the first time to proteins, to permit direct spectroscopy of Zn++, Ca++, Mg++ and other species in interaction with their surroundings. ARF 1 is a small G protein implicated in formation and regulation of non-clathrin coated vesicles. ARF 1 is a member of a large complex called coatamer found on the surface of these vesicles. The GTP form of ARF 1 is required for coatamer formation, and the GDP form promotes vesicle fusion. The ARF proteins have an extra N-terminal sequence, which is helical and modified by myristoylation at the N-terminus. It is hoped to understand the forms of ARF 1 that exist in the cell, GDP- or GTP-ligated, and how they interact with phospholipid through the myristoyl group. Initially, the non-myristoylated protein will be studied in the GDP and GTP forms, and also in those forms lacking the distinctive ARF N-terminal sequence. It is also hoped to study the myristoylated forms of the protein by solubilizing it with a minimum amount of detergent. These proteins, and many others, contain ions such as those mentioned above playing roles as structural elements, active catalytic centers, or messengers. There has been almost no study of these ions directly by spectroscopy when bound to proteins. Pure quadruple resonance can determine the non-symmetric electron charge distribution around an ion, and be used to infer the degree of covalent interaction involved in its binding. It is proposed to develop a form of this spectroscopy that will be sufficiently sensitive for studies of frozen protein solutions and single crystals, thereby providing a completely new way to study changes in the surroundings of these ions directly. It will be based on field-cycling NMR, in which a frozen sample will be pneumatically moved into and out of a 500 MHz magnet many times in a single experimental run. This method has been demonstrated previously in studies of metal alloys and small molecules. Many other kinds of problems might also be studied by the method, such as bond orientations of substrates containing deuterons or 17O, or the spectroscopy of boron-containing inhibitors bound to proteins.