NOVEL PREPARATION METHOD FOR ALIGNED MEMBRANE PROTEINS

这项研究计划是针对一个最重要的和困难的问题与膜蛋白质结构测定的NMR定向组件，即样品制备。已经表明，取向样品的质量对NMR实验的结果具有显著的影响，其随后影响使用数据进行结构测定的能力。此外，虽然NMR通常被认为是一种非破坏性技术，但长时间的实验和重复的射频脉冲不可避免地导致样品温度升高，从而导致样品降解。样品制备技术的进展，如添加金属离子的双层制剂和螯合剂的双分子组装提出。此外，定向样品的新的制备方法，是适应朗缪尔-布洛杰特方法的可能性将进行探讨。 对现有制备程序的这些修改以及样品制备的新方法旨在改善定向蛋白质/磷脂组装体的总体温度稳定性和寿命。将通过固态NMR评估通过这些拟议技术制备的样品的总体质量。两个膜蛋白，外壳蛋白的fd噬菌体和Vpu的HIV-1，以这些方式制备的结构，将通过各种固态NMR技术进行研究。

This research proposal is aimed at one of the most important and difficult problems associated with membrane protein structure determination by NMR of oriented assemblies; namely sample preparation. it has been shown that the quality of the oriented sample has a dramatic effect on the results of the NMR experiment that subsequently affects the ability to use the data for structure determination. Additionally, although NMR is generally considered a non-destructive technique, long experiment times and repetitive radio frequency pulses inevitably lead to increased sample temperature and hence sample degradation. Advances in sample preparation techniques such as the addition of metal ions to bilayer preparations and chelating agents to bicelle assemblies are proposed. Additionally, the possibility of orienting samples by novel preparation methods that are adaptations of Langmuir-Blodgett methodologies will be explored. These amendments to already existing preparative procedures as well as new methods of sample preparation are aimed at improving the overall temperature stability and longevity of the oriented protein/phospholipid assemblies. The overall quality of the samples prepared by these proposed techniques will be assessed by solid-state NMR. The structures of two membrane proteins, the coat protein of fd bacteriophage and Vpu of HIV-l, prepared in these ways, will be investigated by a variety of solid-state NMR techniques.