Membrane Protein Stability

膜蛋白稳定性

• 批准号: 6637245
• 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/6637245
• 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.

尽管四分之一的基因编码膜蛋白，并且绝大多数治疗药物都靶向膜蛋白，但人们对膜蛋白的结构和功能知之甚少。 这种知识差距的出现很大程度上是因为膜蛋白在洗涤剂溶液中的不良行为常常导致许多棘手的技术问题。 我们最近发现，一种稍微稳定的膜蛋白二酰甘油激酶的单点突变可以显着提高其在洗涤剂中的稳定性。 此外，稳定突变并不罕见。 该提案的工作目标是更多地了解突变如何稳定洗涤剂中的膜蛋白。 这项工作不仅可以实际提高我们处理膜蛋白的能力，还可以提供对膜蛋白结构的基本了解。目标 I. 鉴定并表征二酰基甘油激酶和细菌视紫红质中的其他稳定突变。这一目标不仅将为后续目标提供素材，而且通过识别大量突变，有可能制定有关可能稳定的侧链类型和序列位置的预测规则。目标二。解决稳定突变体的结构。 我们将详细了解有多少突变体稳定下来，为膜蛋白结构如何结合在一起提供新的见解。目标三。检查二酰甘油激酶和细菌视紫红质在洗涤剂溶液中的失活机制。这项工作将有助于更好地理解为什么某些突变会稳定下来，并且还可能通过强调现有洗涤剂的弱点来帮助未来洗涤剂的改进。目标四。开发稳定突变的通用筛选。我们设计了一种不依赖于快速活性测定的稳定性突变体筛选策略。 这种方法可以为基本上任何感兴趣的膜蛋白带来高稳定性的好处。