EXTENSION OF SOLUTION NMR TO 50 KDA PROTEINS AND BEYOND

描述（改编自摘要）： 核磁共振（NMR）光谱仍然是一个核心的 高分辨率模型的确定技术 蛋白质、核酸及其复合物的结构和动力学。 然而，已知的蛋白质中有很大一部分 通过基因组序列的分析， 溶液NMR方法。这是因为它们太大了， 因为它们需要与大型程序集相关联， 因此，对于最佳NMR性能而言，翻滚太慢。 该提案寻求资金，以开发一种新的方法， 大蛋白质的弛豫特性适合于综合 现代三重共振及相关核磁共振技术的有效应用 技术.基本的方法是简单地安排蛋白质 分子翻滚成一个小得多的蛋白质。这个项目的中心目标是 建议：开发一种低粘度的反胶束体系， 能够在水中溶解大至100 kDa的蛋白质的流体。的 申请人计算了蛋白质在反胶束中的溶解 溶解在液化丁烷、丙烷或乙烷中的液体会翻滚， 足够短的相关时间，以允许完整的电池 现有的三重共振技术，即使没有好处， 氘代。将需要高达50 bar的适度压力 来分解这些烷烃。压力下的光学光谱学将是 用作快速测定。高分辨率探头将用于 表征各种测试蛋白质的NMR性质。应该 这一战略被证明是成功的，它可以提供一个通用的，灵活的， 使用高分辨率溶液NMR的一种非常强大的方法 技术来表征大小高达100 kDa的蛋白质， 提供了一种检查膜相关或完整膜的途径， proteins.

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.