Engineering Soluble Aggregation-Prone and Membrane-Bound Proteins

工程化可溶性易聚集和膜结合的蛋白质

• 批准号: 7137979
• 负责人: Gregory A. Weiss
• 金额: $ 26.19万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7137979
• 关键词: X ray crystallography; binding proteins; bioengineering /biomedical engineering; biotechnology; caveolins; chemical aggregate; high throughput technology; lipid bilayer membrane; liposomes; nitric oxide synthase; nuclear magnetic resonance spectroscopy; phage display; protein engineering; protein kinase A; protein structure; site directed mutagenesis; solubility; structural biology; surface plasmon resonance

DESCRIPTION: (provided by applicant): Structural biology and, in recent years, structural proteomics have yielded tremendous insight into protein mechanism and function. However, a high percentage of proteins remain off-limits to high-throughput structure determination methods. For example, solving structures of membrane-bound proteins is a difficult and idiosyncratic art. Furthermore, proteins susceptible to aggregation are simply not amenable to current methods for structure determination. The first long-term goal of the research proposed here is the development of a high-throughput method for converting insoluble proteins, both membrane-bound and oligomerization-prone, to soluble proteins upon which the powerful tools of structural biology can be brought to bear. An equally important second long-term goal is to elucidate and understand the characteristics of protein structure leading to aggregate and membrane bound states. Determining the structures of previously unattainable targets will expedite the development of therapeutics for a host of diseases, such as disorders resulting from amyloid fibril formation, a specific type of protein aggregation. Specifically, the experiments proposed here focus on the caveolin-1, a key regulator of signal transduction. Caveolin-1 binds to a large number of different cellular proteins, and can inhibit key enzymes, including protein kinase A (PKA) and endothelial nitric oxide synthase (eNOS). Such activities allow selections for functional, yet more soluble, caveolin-1 variants. As both an aggregation-prone and membrane-associated protein, caveolin-1 provides an ideal system for the planned experiments. In essence, one series of experiments will uncover molecular determinants for both aggregation and membrane binding. In the first specific aim, the determinants of solubility, aggregation, and membrane-binding will be investigated during experiments aimed at engineering soluble variants of caveolin. The structure of the soluble variant will be determined by solution phase NMR in the second specific aim. This structure will be compared to a structure determined by solid-state NMR of an aggregated variant of caveolin. Structural insight will be then guide mutagenesis experiments aimed at testing the mechanistic basis for protein aggregation and membrane-binding.

描述：（由申请人提供）：结构生物学和近年来的结构蛋白质组学已经对蛋白质机制和功能产生了巨大的洞察力。然而，高比例的蛋白质仍然无法使用高通量结构测定方法。例如，解析膜结合蛋白的结构是一门困难且特殊的艺术。此外，易聚集的蛋白质根本不适合当前的结构测定方法。这里提出的研究的第一个长期目标是开发一种高通量方法，将不溶性蛋白质（膜结合的和易于寡聚化的蛋白质）转化为可溶性蛋白质，从而可以利用结构生物学的强大工具。同样重要的第二个长期目标是阐明和理解导致聚集和膜结合状态的蛋白质结构的特征。确定以前无法实现的目标的结构将加快许多疾病治疗方法的开发，例如淀粉样原纤维形成（一种特定类型的蛋白质聚集）引起的疾病。具体来说，这里提出的实验重点关注信号转导的关键调节因子 Caveolin-1。 Caveolin-1 与大量不同的细胞蛋白结合，并可以抑制关键酶，包括蛋白激酶 A (PKA) 和内皮一氧化氮合酶 (eNOS)。此类活动允许选择有功能且更可溶的 Caveolin-1 变体。作为一种易于聚集的膜相关蛋白，caveolin-1 为计划的实验提供了理想的系统。本质上，一系列实验将揭示聚集和膜结合的分子决定因素。在第一个具体目标中，将在旨在工程化小窝蛋白可溶性变体的实验中研究溶解度、聚集和膜结合的决定因素。在第二个具体目标中，可溶变体的结构将通过溶液相NMR确定。该结构将与通过固态 NMR 测定的小窝蛋白聚集变体的结构进行比较。然后，结构洞察将指导旨在测试蛋白质聚集和膜结合的机制基础的诱变实验。