Membrane Protein Stability

膜蛋白稳定性

• 批准号: 6526027
• 负责人: JAMES U BOWIE
• 金额: $ 22.52万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6526027
• 关键词: Escherichia coli; Halobacteriaceae; X ray crystallography; bacterial genetics; bacteriorhodopsins; calorimetry; chemical kinetics; chemical stability; crystallization; detergents; diacylglycerols; enzyme induction /repression; gene mutation; mass spectrometry; model design /development; permease; phosphotransferases; physical model; point mutation; protein denaturation; protein folding; protein structure function; racemization; solubility; structural biology; thermodynamics

Although a quarter of all genes encode membrane proteins and the vast majority of therapeutic drugs target membrane proteins, remarkably little is known about membrane protein structure and function. This knowledge gap has occurred largely because often poor behavior of membrane proteins in detergent solution that leads to many intractable technical problems. We recently discovered that single point mutations in a marginally stable membrane protein, diacylglycerol kinase, can dramatically improve its stability in detergent. Moreover, stabilizing mutations are not rare. The goal of the work in this proposal is to learn more about how mutations stabilize membrane proteins in detergent. This work could lead to both practical improvements in our ability to handle membrane proteins and also provide fundamental insight into membrane protein structure. Aim I. Identify and characterize additional stabilizing mutations in diacylglycerol kinase and bacteriorhodopsin. This aim will not only provide fodder for subsequent aims, but by identifying a large number of mutations, it may be possible to develop predictive rules regarding side-chain types and sequence positions that are likely to stabilize. Aim II. Solve structures of stable mutants. We will learn in atomic detail how many of the mutants stabilize, providing new insights into how membrane protein structures are held together. Aim III. Examine the mechanism of inactivation in detergent solution for diacylglycerol kinase, and bacteriorhodopsin. This work will lead to a better understanding of why certain mutations stabilize and may also aid future detergent improvements by highlighting weaknesses in existing detergents. Aim IV. Develop a general screen for stabilizing mutations. We have devised a strategy for stability mutant screening that does not depend on a rapid activity assay. This method could bring the benefits of high stability to essentially any membrane protein of interest.

尽管所有基因中的四分之一编码膜蛋白和绝大多数治疗药物的靶向膜蛋白，但对膜蛋白结构和功能知之甚少。 这种知识差距很大程度上是因为膜蛋白在洗涤剂溶液中的行为往往差，导致许多棘手的技术问题。 我们最近发现，在边缘稳定的膜蛋白（二酰基甘油激酶）中的单点突变可以显着提高其在洗涤剂中的稳定性。 而且，稳定突变并不罕见。 该提案中工作的目的是了解更多有关突变如何稳定洗涤剂中的膜蛋白的信息。 这项工作可能会导致我们处理膜蛋白的能力的实际改进，还可以提供对膜蛋白结构的基本见解。 AIM I.确定并表征二酰基甘油激酶和细菌紫红质中的其他稳定突变。这个目标不仅可以为后续目标提供饲料，而且通过确定大量突变，可以制定有关侧链类型和可能稳定的序列位置的预测性规则。目标II。解决稳定突变体的结构。 我们将学习原子能的细节，有多少突变体稳定了多少突变体，从而提供了有关膜蛋白结构如何结合在一起的新见解。目标三。检查洗涤剂溶液中的二酰基甘油激酶和细菌紫红素的灭活机制。这项工作将使人们更好地理解某些突变稳定的原因，还可以通过突出现有洗涤剂的弱点来帮助未来的洗涤剂改善。目标IV。为稳定突变开发通用屏幕。我们已经设计了一种稳定突变体筛选的策略，该策略不取决于快速活动测定。 这种方法可以为任何感兴趣的膜蛋白带来高稳定性的好处。