Engineering Soluble Aggregation-Prone and Membrane-Bound Proteins

产品说明：（申请人提供）：结构生物学和近年来的结构蛋白质组学对蛋白质的作用机制和功能有了深刻的认识。然而，高百分比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个困难的和特殊的艺术。此外，蛋白质容易聚集是简单地不服从目前的方法用于结构测定。本文提出的研究的第一个长期目标是开发一种高通量方法，用于将膜结合和寡聚化倾向的不溶性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目标的结构将加快许多疾病的治疗方法的开发，例如由淀粉样蛋白原纤维形成（一种特定类型的蛋白质聚集）引起的疾病。具体来说，这里提出的实验集中在小窝蛋白-1，信号转导的关键调节器。Caveolin-1与大量不同的细胞蛋白质结合，并且可以抑制关键酶，包括蛋白激酶A（PKA）和内皮型一氧化氮合酶（eNOS）。这样的活性允许选择功能性的、更可溶的小窝蛋白-1变体。Caveolin-1作为一种聚集性蛋白和膜结合蛋白，为实验提供了一个理想的体系。本质上，一系列实验将揭示聚集和膜结合的分子决定因素。在第一个具体的目标，溶解度，聚集和膜结合的决定因素将在实验期间进行调查，旨在工程化的小窝蛋白的可溶性变体。在第二个具体目标中，可溶性变体的结构将通过溶液相NMR确定。将该结构与通过小窝蛋白的聚集变体的固态NMR确定的结构进行比较。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.