EXTENSION OF SOLUTION NMR TO 50 KDA PROTEINS AND BEYOND

说明(改编自摘要)： 核磁共振(核磁共振)光谱学仍然是核心 高分辨率模式的确定技术 蛋白质、核酸及其复合体的结构和动力学。 然而，已知的蛋白质中有很大一部分 通过分析基因组序列是无法获得的 溶液核磁共振方法。这是因为它们太大了，要么是 因为它们需要与大型程序集关联 因此，翻滚速度太慢，无法获得最佳的核磁共振性能。 这项提案寻求资金来开发一种新的方法来呈现核磁共振 大分子蛋白质的松弛特性 现代三重共振及其相关核磁共振的有效应用 技巧。基本的方法是简单地排列蛋白质 分子作为一种小得多的蛋白质翻滚。这样做的中心目标是 建议：开发低粘度反胶束体系 能够在水中溶解100 kDa的蛋白质的液体。这个 申请人计算蛋白质在反胶束中的增溶作用 溶解在液化的丁烷、丙烷或乙烷中，会随着 相关时间足够短，以允许完整的电池 将应用现有的三重共振技术，即使没有好处 氢化的结果。需要中等压力，最高可达50巴 来液化这些烷烃。压力下的光学光谱学将是 用作一种快速分析方法。一个高分辨率的探测器将用来 表征各种测试蛋白质的核磁共振特性。应该 这一策略被证明是成功的，它可以提供一个通用的、灵活的和 使用高分辨率溶液核磁共振的极其有效的方法 表征大小达100 kDa的蛋白质的技术，还可以 提供一种检查膜相关膜或完整膜的方法 蛋白质。

DESCRIPTION (Adapted from abstract): Nuclear magnetic resonance (NMR) spectroscopy continues to be a central technique in the determination of high resolution models of the structure and dynamics of proteins, nucleic acids, and their complexes. Nevertheless, a significant fraction of the proteins that are known through the analysis of the genomic sequence are inaccessible to solution NMR methods. This is because they are too large, either by themselves and because they require association with large assemblies of lipids, and therefore tumble too slowly for optimal NMR performance. This proposal seeks funds to develop a new approach to rendering the NMR relaxation properties of large proteins amenable to the comprehensive and efficient application of modern triple resonance and related NMR techniques. The basic approach is to arrange simply for the protein molecule to tumble as a much smaller protein. The central goal of this proposal is: To develop a reverse micelle system in a low viscosity fluid capable of solvating, in water, proteins as large as 100 kDa. The applicant calculates that solubilization of proteins in reverse micelles dissolved in liquefied butane, propane, or ethane will tumble with sufficiently short correlation times to allow the full battery of existing triple resonance techniques to be applied, even without benefit of deuteration. Modest pressures ranging up to 50 bar will be required to liquefy these alkanes. Optical spectroscopy under pressure will be employed as a rapid assay. A high resolution probe will be used to characterize the NMR properties of the various test proteins. Should this strategy prove successful, it could provide a general, flexible and extremely powerful approach to using high resolution solution NMR techniques to characterize proteins up to 100 kDa in size and may also offer a route to examining membrane associated or integral membrane proteins.