A Protein Footprinting Toolbox

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，用于在单氨基酸分辨率下测量复杂混合物中大型蛋白质的结构，能量学和构象变化。这些工具价格低廉，不需要专门的设备，而且对任何生物化学实验室都切实可行。单个蛋白质的完整数据将从单个实验中并行获得。将实现一种适合高通量蛋白质组学应用的工具形式。

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.