NOVEL PREPARATION METHOD FOR ALIGNED MEMBRANE PROTEINS

本研究针对定向组装核磁共振法测定膜蛋白结构中最重要也是最困难的问题之一，即样品的制备。结果表明，定向样品的质量对核磁共振实验的结果有很大的影响，从而影响了利用数据确定结构的能力。此外，尽管核磁共振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会导致样品温度上升，从而导致样品退化。提出了样品制备技术的进展，如在双层制剂中添加金属离子和在双柱组件中添加螯合剂。此外，还将探索通过适应朗缪尔-布洛杰特方法的新制备方法来定向样品的可能性。这些对现有制备程序以及新的样品制备方法的修正旨在提高定向蛋白质/磷脂组件的整体温度稳定性和寿命。用这些拟议技术制备的样品的整体质量将通过固态核磁共振进行评估。用这些方法制备的两种膜蛋白，Fd噬菌体的外壳蛋白和HIV-L的VPU，将用各种固体核磁共振技术研究其结构。

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.