A Protein Footprinting Toolbox

蛋白质足迹工具箱

• 批准号: 6890868
• 负责人: PEHR A HARBURY
• 金额: $ 26.6万
• 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/6890868
• 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种蛋白质足迹工具箱，用于以单个氨基酸分辨率测量复杂混合物中大蛋白质的结构、能量和构象变化。这些工具价格低廉，不需要专门的设备，并且适用于任何生物化学实验室。单个蛋白质的完整数据将从单个实验中并行获得。将实施一种适合高通量蛋白质组学应用的工具。 了解蛋白质构象、相互作用和配体结合对于所有生物学研究至关重要。由于缺乏合适的工具，对复杂混合物中的大蛋白质（蛋白质正常运行环境的特征）的这些特性进行测量非常困难。我们的初步研究报告了一种新型生化技术，即错误掺入质子烷基交换（MPAX），可用于以单个氨基酸分辨率追踪蛋白质结构。 MPAX 利用半胱氨酸残基的翻译错误掺入来生成用于物理分析的探针，并利用化学修饰策略来读取探针的局部结构环境。我们将 MPAX 应用于磷酸丙糖异构酶 (b/a) 8 桶，准确测定其底物结合位点、蛋白质-蛋白质相互作用表面、溶剂可及的蛋白质表面、桶的稳定性及其解折叠途径。由于 MPAX 仅需要微克量的材料，并且不受蛋白质大小的限制，因此非常适合大型且性能较差的大分子的溶液结构研究。在此应用中，我们建议扩展 MPAX 的基本足迹功能，包括测量蛋白质中的成对残基接近度、测量蛋白质中的构象变化率以及测量只能在真核表达系统中产生的蛋白质的结构。