NOVEL PREPARATION METHOD FOR ALIGNED MEMBRANE PROTEINS

本研究计划针对膜蛋白定向组装体的核磁共振结构测定中最重要和最困难的问题之一；即样品制备。结果表明，取向样品的质量对核磁共振实验结果有显著影响，从而影响使用数据进行结构测定的能力。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导致样品温度升高，从而导致样品降解。提出了在双分子层制备中加入金属离子和在双分子组件中加入螯合剂等样品制备技术的进展。此外，定向样品的可能性由新的制备方法是适应Langmuir-Blodgett方法将被探索。这些对现有制备程序的修订以及样品制备的新方法旨在提高定向蛋白质/磷脂组装的整体温度稳定性和寿命。通过这些建议的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 - 1的Vpu）的结构将通过各种固态核磁共振技术进行研究。

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.