Engineering Soluble Aggregation-Prone and Membrane-Bound Proteins

描述：（由申请人提供）：近年来，结构生物学和结构蛋白质组学对蛋白质的机制和功能有了深刻的了解。然而，高比例的蛋白质仍然是高通量结构测定方法的禁区。例如，解决膜结合蛋白的结构是一门困难而独特的艺术。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（包括膜结合蛋白和易寡聚化蛋白）转化为可溶蛋白，从而使结构生物学的强大工具能够发挥作用。第二个同样重要的长期目标是阐明和理解导致聚集体和膜结合状态的蛋白质结构的特征。确定以前无法达到的靶点的结构将加速许多疾病的治疗方法的开发，例如由淀粉样纤维形成（一种特定类型的蛋白质聚集）引起的疾病。具体来说，这里提出的实验集中在信号转导的关键调节因子caveolin-1上。Caveolin-1与大量不同的细胞蛋白结合，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）。这样的活动允许选择功能性的，但更容易溶解的小洞蛋白-1变体。作为一种易于聚集和膜相关的蛋白，小窝蛋白-1为计划中的实验提供了理想的系统。本质上，一系列的实验将揭示聚集和膜结合的分子决定因素。在第一个特定目标中，将在旨在工程溶性小窝蛋白变体的实验中研究溶解度、聚集性和膜结合的决定因素。可溶变体的结构将在第二个特定目标中通过液相核磁共振确定。这种结构将与洞穴蛋白聚合变体的固态核磁共振确定的结构进行比较。然后，结构洞察力将指导诱变实验，旨在测试蛋白质聚集和膜结合的机制基础。

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.