STRUCTURAL KINETIC STUDIES BY NUCLEAR MAGNETIC RESONANCE

描述：人ADP核糖化因子1(ARF1)的构象 将通过溶液状态核磁共振进行研究。田野自行车纯四人组 将首次将共振应用于蛋白质，以允许直接 锌、钙、镁等离子与其相互作用的光谱 周围的环境。ARF1是一种小G蛋白，参与形成和 非蛋白酪蛋白包被囊泡的调节。ARF 1是一个大型 在这些小泡的表面发现了一种名为Coatamer的复合体。《GTP》 大衣编队需要ARF1形式，GDP形式促进 囊泡融合。ARF蛋白具有额外的N-末端序列，该序列 是螺旋的，并通过N-末端的肉豆蔻酰化修饰。人们希望能够 了解存在于细胞中的ARF1的形式，GDP或GTP连接， 以及它们如何通过肉豆蔻基与磷脂相互作用。 最初，非肉豆蔻化蛋白将在国内生产总值和总蛋白中进行研究。 形式，以及那些缺乏独特ARF N-末端的形式 序列。人们还希望研究这种蛋白质的肉豆蔻酰化形式。 用最少量的洗涤剂溶解它。 这些蛋白质和许多其他蛋白质含有上述离子。 作为结构元素、活性催化中心或 信使。对这些离子的直接研究几乎没有。 与蛋白质结合时的光谱学。纯粹的四重共振可以确定 离子周围的非对称电子电荷分布，可用于 推断其结合所涉及的共价相互作用的程度。它是 建议开发一种形式的这种光谱学，将充分 对冷冻蛋白质溶液和单晶的研究敏感， 从而为研究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，其中一个 冷冻的样品将以气动方式移入和移出500 MHz的磁铁 在一次实验中跑了很多次。这种方法已经得到了验证。 以前从事金属合金和小分子的研究。许多其他种类的 的问题也可以用这种方法来研究，比如键的取向。 含氚或17O的底物，或光谱学 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.