CONSTRUCTION OF LOW TEMPERATURE SOLID STATE NMR PROBE

蛋白质结构研究新方法的发展 固体核磁共振测定是本研究的重点。这个 正在开发的方法依赖于蛋白质的几个特征 系统。应在适当的时间范围内固定该系统 保持与取向相关的自旋相互作用和单轴 与外加磁场平行排列的蛋白质的取向 字段必须存在。结合到的蛋白质满足这些标准 在玻璃板上机械取向的脂质双层。我们是 目前正在评估其他自发定向到 磁场。这些系统由以下混合物组成 形成双层圆盘的磷脂和洗涤剂 垂直于外加磁场的长轴。这些 圆盘被称为双圆盘。发现圆盘在以下情况下翻转 某些顺磁性离子被添加到双极管中。这是定向的 处于有利位置的圆盘，其长轴与 应用领域。当蛋白质被插入到这些双细胞中时， 可以进行与蛋白质骨架结构有关的测量 从酰胺基位的15N和1H取向。几种膜 蛋白质已经成功地并入双细胞系统。 这些包括丝状噬菌体FD的外壳蛋白， 汞转运蛋白Mert，HIV-1辅助蛋白Vpu. 和来自乙酰胆碱受体的M2肽。表征 这些蛋白质中的一种正在使用双细胞技术进行 固体核磁共振结构测定。定向的其他方法 二倍体圆盘正在被追逐。EF手的使用，钙 结合蛋白质基序以结合顺磁性 金属正在接受测试。这个系统将引导顺磁金属 到指定的结合位置，用 适当的轴，并让蛋白质研究没有任何 与顺磁离子的相互作用。

The development of a new method for protein structure determination by solid-state NMR is the focus of this research. The methods being developed rely on several features of the protein system. The system should be immobilized on a time scale appropriate to preserve the orientation dependent spin interactions and a uni-axis of orientation of the protein aligned parallel to the applied magnetic field must be present. These criteria are met for proteins bound to lipid bilayers oriented mechanically on glass plates. We are currently evaluating other systems that orient spontaneously in magnetic fields. These systems are composed of mixtures of phospholipids and detergents that form bilayer discs that orient with the long axis perpendicular to the applied magnetic field. These discs are referred to as bicelles. The discs were found to flip when certain paramagnetic ions were added to the bicelles. This orients the discs in the favorable position with the long axis parallel to the applied field. When proteins are inserted into these bicelles, measurements pertaining to the protein backbone structure can be made from the 15N and 1H orientations at the amide sites. Several membrane proteins have been successfully incorporated into the bicelle system. These include the coat protein from the filamentous bacteriophage fd, the mercury transport protein merT, the HIV-1 accessory protein Vpu and the M2 peptide from the acetycholine receptor. Characterization of these proteins is proceeding using the bicelle technique for solid-state NMR structure determination. Other methods of orienting the bicelle discs are being pursued. The use of an EF hand, calcium binding protein motif for the purpose of binding the paramagnetic metal is being tested. This system will direct the paramagnetic metal to the designated site of binding, orient the bicelle with the appropriate axis and leave the protein to be studied free of any interaction with the paramagnetic ion.