CONSTRUCTION OF LOW TEMPERATURE SOLID STATE NMR PROBE

一种新的蛋白质结构分析方法的发展 固体核磁共振测定是本研究的重点。 的 正在开发的方法依赖于蛋白质的几个特征， 系统 系统应在适当的时间范围内固定 为了保持取向相关的自旋相互作用和单轴 蛋白质的方向平行于所施加的磁场 字段必须存在。 对于结合至以下的蛋白质，符合这些标准： 在玻璃板上机械取向的脂双层。 我们 目前正在评估其他系统， 磁场 这些系统由以下物质的混合物组成： 磷脂和去污剂形成双层圆盘， 长轴垂直于所施加的磁场。 这些 盘被称为双胞。 发现光盘翻转时， 将某些顺磁性离子加入到双胞中。 这是定向的 在有利的位置与长轴平行的光盘 应用领域 当蛋白质被插入这些双胞时， 可以进行与蛋白质骨架结构有关的测量 从酰胺位点的15 N和1H取向。 几个膜 蛋白质已经成功地结合到Bicelle系统中。 这些包括来自丝状噬菌体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 这些蛋白质正在使用bicelle技术进行， 固态NMR结构测定。 其他定向方法 正在追查比塞勒光盘。 使用EF手，钙 结合蛋白基序，用于结合顺磁性 金属正在测试中。 这个系统会引导顺磁性金属 到指定的结合位点，将bicelle与 适当的轴，并离开蛋白质的任何研究 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.