A Protein Footprinting Toolbox

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、能量学和构象变化。这些工具将是廉价的，将不需要专门的设备，并将实际上适用于任何生物化学实验室。将从单个实验中平行获得单个蛋白质的完整数据。一种适合高通量蛋白质组学应用的工具将被实现。 了解蛋白质构象、相互作用和配体结合对所有生物学研究都是必不可少的。由于缺乏合适的工具，测量复杂混合物中的大蛋白质的这些特性（蛋白质正常运作的环境特征）非常难以实现。我们的初步研究报告了一种新的生物化学技术，错误掺入质子-烷基交换（MPAX），可用于足迹蛋白质结构在单个氨基酸的分辨率。MPAX利用半胱氨酸残基的翻译错误掺入来产生用于物理分析的探针，以及化学修饰策略来读出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，溶剂可接近的蛋白质表面，桶的稳定性及其展开途径。由于MPAX只需要微克数量的材料，并且不受蛋白质大小的限制，因此它非常适合于大分子和行为不佳的大分子的溶液结构研究。在本申请中，我们建议扩展MPAX的基本足迹能力，包括测量蛋白质中成对残基的接近度，测量蛋白质中构象变化的速率，以及测量只能在真核表达系统中产生的蛋白质的结构。

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.