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: 7259408
• 负责人: Gregory A. Weiss
• 金额: $ 29.85万
• 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/7259408
• 关键词: Alzheimer' s Disease; Arts; Atherosclerosis; Binding; Binding Proteins; Capsid Proteins; Caveolins; Cell surface; Cells; Characteristics; Cyclic AMP-Dependent Protein Kinases; Data; Development; Diabetes Mellitus; Disease; Drug Design; Engineering; Enzymes; Figs - dietary; Goals; Inflammation; Lead; Length; Ligands; Liposomes; Lung; Malignant Neoplasms; Measurement; Mediating; Membrane; Membrane Proteins; Methods; Modeling; Molecular; Mutagenesis; Mutation; Numbers; Phage Display; Phase; Portraits; Prostate; Proteins; Proteomics; Publications; Publishing; Quality Control; Research; Research Personnel; Role; Scanning; Series; Shapes; Shotguns; Signal Transduction; Solubility; Solutions; Structure; Surface Plasmon Resonance; System; Testing; Therapeutic; Ursidae Family; Variant; Work; amyloid fibril formation; base; caveolin 1; flasks; human NOS3 protein; improved; insight; malignant breast neoplasm; programs; protein aggregate; protein aggregation; protein function; protein structure; research study; solid state; structural biology; therapeutic target; tool

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）：结构生物学以及近年来，结构蛋白质组学对蛋白质机制和功能产生了深刻的见解。然而，高比例的蛋白质仍然是高通量结构测定方法的禁止。例如，解决膜结合蛋白的结构是一种困难而特质的艺术。此外，易感聚集的蛋白质根本不适合当前的结构确定方法。此处提出的研究的第一个长期目标是开发一种高通量方法，用于转化膜结合和寡聚化的不溶性蛋白，以易溶蛋白，可以将结构生物学强大的工具带给耐种。同样重要的第二长期目标是阐明和了解导致骨料和膜结合状态的蛋白质结构的特征。确定以前无法实现的靶标的结构将加快针对多种疾病的疗法发展，例如由淀粉样蛋白纤维形成引起的疾病，淀粉样蛋白纤维形成是一种特定类型的蛋白质聚集。具体而言，此处提出的实验集中在Caveolin-1上，Caveolin-1是信号转导的关键调节剂。 Caveolin-1与大量不同的细胞蛋白结合，并可以抑制包括蛋白激酶A（PKA）和内皮一氧化氮合酶（ENOS）在内的关键酶。此类活动允许选择功能性但更可溶的小窝蛋白-1变体。作为易于聚集的蛋白质和膜相关的蛋白，Caveolin-1为计划实验提供了理想的系统。本质上，一系列实验将发现聚集和膜结合的分子决定因素。在第一个具体目的中，将在旨在工程可溶解可溶蛋白的实验过程中研究溶解度，聚集和膜结合的决定因素。可溶性变体的结构将由第二个特定目标中的溶液相NMR确定。该结构将与由小窝蛋白聚集变体的固态NMR确定的结构进行比较。然后，将进行结构洞察力，以指导诱变实验，以测试蛋白质聚集和膜结合的机械基础。