A Protein Footprinting Toolbox

蛋白质足迹工具箱

• 批准号: 6602924
• 负责人: PEHR A HARBURY
• 金额: $ 26.49万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6602924
• 关键词: analytical chemistry; analytical method; binding sites; biotechnology; chemical fingerprinting; conformation; high throughput technology; method development; protein binding; protein folding; protein protein interaction; protein sequence; proteomics; solutions; structural biology

DESCRIPTION (provided by applicant): Our goal is to develop a protein footprinting toolbox for measuring at single amino-acid resolution the structures, energetics and conformational changes of large proteins in complex mixtures. The tools will be inexpensive, will require no specialized equipment, and will be practically workable for any biochemistry laboratory. Complete data on a single protein will be obtained in parallel from a single experiment. A form of the tools suitable for high-throughput proteomics applications will be implemented. Understanding protein conformation, interactions and ligand binding is essential to all biological inquiry. Measurement of these properties with large proteins in complex mixtures, characteristic of the environments in which proteins normally operate, is very difficult to achieve due to the lack of suitable tools. Our preliminary studies report a novel biochemical technique, misincorporation proton-alkyl exchange (MPAX) that can be used to footprint protein structure at single amino-acid resolution. MPAX exploits translational misincorporation of cysteine residues to generate probes for physical analysis, and chemical modification strategies to read out the local structural environment of the probes. We apply MPAX to the triosephosphate isomerase (b/a) 8 barrel, accurately determining its substrate binding site, a protein-protein interaction surface, the solvent-accessible protein surface, the stability of the barrel, and its unfolding pathway. Because MPAX requires only microgram quantities of material and is not limited by protein size, it is ideally suited for solution structural studies of large and poorly behaved macromolecules. In this application, we propose to extend the basic footprinting capabilities of MPAX to include measurement of pair wise residue proximity in proteins, measurement of the rates of conformational changes in proteins, and measurement of structure for proteins that can only be produced in a eukaryotic expression system.

描述（由申请人提供）：我们的目标是开发一个蛋白质足迹工具箱，用于在复杂混合物中大型蛋白质的结构，能量和构象变化在单个氨基酸分辨率下进行测量。这些工具将是便宜的，不需要专业设备，并且对于任何生物化学实验室实际上都可以工作。单个蛋白质的完整数据将与单个实验并联获得。将实施适合高通量蛋白质组学应用程序的工具的一种形式。 了解蛋白质构象，相互作用和配体结合对于所有生物学探究至关重要。通过缺乏合适的工具，很难实现这些特性，这些特性具有复杂混合物中大蛋白的特征，在其中蛋白质正常运行的环境的特征很难实现。我们的初步研究报告了一种新型的生化技术，失物质子 - 烷基交换（MPAX），可用于单氨基酸分辨率的足迹蛋白结构。 MPAX利用了半胱氨酸残基的翻译失误，以生成用于物理分析的探针，以及化学修饰策略，以读取探针的局部结构环境。我们将MPAX应用于三氧磷酸异构酶（B/A）8桶，准确地确定其底物结合位点，蛋白质 - 蛋白质相互作用表面，溶剂可访问的蛋白质表面，桶的稳定性及其展开的途径。由于MPAX仅需要微克量的材料量，并且不受蛋白质大小的限制，因此它非常适合大型且表现不佳的大分子的结构研究。在此应用中，我们建议将MPAX的基本足迹能力扩展到包括对蛋白质的配对残留物接近度的测量，测量蛋白质的构象变化速率以及对蛋白质结构的测量系统。