Engineering Soluble Aggregation-Prone and Membrane-Bound Proteins

描述：(申请人提供)：结构生物学和近年来，结构蛋白质组学对蛋白质机制和功能产生了巨大的洞察力。然而，很高比例的蛋白质仍然是高通量结构测定方法的禁区。例如，解决膜结合蛋白质的结构是一门困难而独特的艺术。此外，容易聚集的蛋白质根本不适用于目前的结构确定方法。这项研究的第一个长期目标是开发一种高通量的方法，将膜结合和易齐聚的不溶性蛋白质转化为可溶蛋白质，从而可以利用强大的结构生物学工具。另一个同样重要的长期目标是阐明和理解导致聚集态和膜结合态的蛋白质结构特征。确定以前无法达到的目标的结构将加快一系列疾病的治疗方法的发展，例如由淀粉样原纤维形成引起的疾病，淀粉样纤维形成是一种特定类型的蛋白质聚集。具体地说，这里提出的实验重点是小窝蛋白-1，一个关键的信号转导调节因子。小窝蛋白-1能结合大量不同的细胞蛋白，并能抑制包括蛋白激酶A(PKA)和内皮型一氧化氮合酶(ENOS)在内的关键酶。这种活性允许选择功能性的、但更易溶的小窝蛋白-1变异体。作为一种易于聚集和膜相关的蛋白质，小窝蛋白-1为计划中的实验提供了一个理想的系统。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，将在旨在工程可溶变体的小窝蛋白的实验中研究决定溶解性、聚集性和膜结合的因素。在第二个特定目标中，将通过溶液相核磁共振确定可溶变异体的结构。这种结构将与由小窝蛋白聚合体的固体核磁共振确定的结构进行比较。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突变实验。

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.