Engineering Soluble Aggregation-Prone and Membrane-Bound Proteins

结构生物学和近年来的结构蛋白质组学已经对蛋白质产生了巨大的洞察力 机制和功能。然而，高比例的蛋白质仍然是高通量的禁区 结构测定方法例如，解决膜结合蛋白质的结构是一个困难的问题。 此外，易于聚集的蛋白质根本不适合当前的技术。 结构测定方法。这里提出的研究的第一个长期目标是 开发了一种高通量方法，用于转化膜结合和 寡聚化倾向，可溶性蛋白质，结构生物学的强大工具可以带来 去承受同样重要的第二个长期目标是阐明和理解 蛋白质结构导致聚集和膜结合状态。确定以前的结构 无法达到的目标将加速许多疾病的治疗方法的发展， 由淀粉样纤维形成引起，淀粉样纤维是一种特殊类型的蛋白质聚集。 具体来说，这里提出的实验集中在小窝蛋白-1，一个关键的信号调节器 转导Caveolin-1与大量不同的细胞蛋白质结合，并能抑制关键酶， 包括蛋白激酶A（PKA）和内皮型一氧化氮合酶（eNOS）。这些活动使 选择功能性的、更可溶的小窝蛋白-1变体。作为一个聚合倾向和 膜相关蛋白caveolin-1为计划的实验提供了理想的系统。在 本质上，一系列的实验将揭示聚集和膜的分子决定因素， 约束力 在第一个具体目标中，溶解度、聚集和膜结合的决定因素将是 在旨在工程化小窝蛋白的可溶性变体的实验中进行了研究。的结构 在第二个具体目标中，可溶性变体将通过溶液相NMR测定。该结构将 与通过固态NMR测定的小窝蛋白的聚集变体的结构相比。结构 洞察力将指导诱变实验，旨在测试蛋白质的机制基础， 聚集和膜结合。

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.