NOVEL PREPARATION METHOD FOR ALIGNED MEMBRANE PROTEINS

对齐膜蛋白的新制备方法

• 批准号: 6525566
• 负责人: GAIL E FANUCCI
• 金额: $ 4.62万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6525566
• 关键词: human immunodeficiency virus 1; lipid bilayer membrane; membrane proteins; method development; nuclear magnetic resonance spectroscopy; phospholipids; protein structure; surface property; tissue /cell preparation; virus envelope

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.

该研究建议针对与膜蛋白结构确定定向组件的最重要和最困难的问题之一。即样品制备。已经表明，面向样品的质量对NMR实验的结果具有巨大的影响，该结果随后影响使用数据进行结构确定的能力。另外，尽管NMR通常被认为是一种非破坏性技术，但长期实验时间和重复的射频脉冲不可避免地会导致样品温度升高，从而导致样品降解。提出了样品制备技术的进步，例如将金属离子添加到双层制剂中，并提出了螯合剂为贝塞尔组件。此外，还将探索通过新的制备方法来定位样品的可能性，这些方法将探索Langmuir-Blodgett方法的适应性。 这些修正案对已经存在的制备程序以及样品制备的新方法旨在改善定向蛋白/磷脂组件的整体温度稳定性和寿命。这些提出的技术制备的样品的总体质量将由固态NMR评估。以这些方式制备的两种膜蛋白（FD噬菌体的外套蛋白和HIV-L的VPU）的结构将由各种固态NMR技术研究。

项目成果

Drug-membrane interactions studied in phospholipid monolayers adsorbed on nonporous alkylated microspheres.

研究了吸附在无孔烷基化微球上的磷脂单层的药物-膜相互作用。

• DOI: 10.1177/1087057106297063
• 发表时间: 2007
• 期刊: Journal of biomolecular screening
• 作者: Lukacova,Viera;Peng,Ming;Fanucci,Gail;Tandlich,Roman;Hinderliter,Anne;Maity,Bikash;Manivannan,Ethirajan;Cook,GregoryR;Balaz,Stefan
• 通讯作者: Balaz,Stefan