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. ? ?
| Shi, Xuesong; Bonilla, Steve; Herschlag, Daniel et al. (2015) Quantifying Nucleic Acid Ensembles with X-ray Scattering Interferometry. Methods Enzymol 558:75-97 |
| Shi, Xuesong; Herschlag, Daniel; Harbury, Pehr A B (2013) Structural ensemble and microscopic elasticity of freely diffusing DNA by direct measurement of fluctuations. Proc Natl Acad Sci U S A 110:E1444-51 |
| Bernstein, Rachel; Schmidt, Kierstin L; Harbury, Pehr B et al. (2011) Structural and kinetic mapping of side-chain exposure onto the protein energy landscape. Proc Natl Acad Sci U S A 108:10532-7 |
| Boas, F Edward; Harbury, Pehr B (2008) Design of protein-ligand binding based on the molecular-mechanics energy model. J Mol Biol 380:415-24 |
| Mathew-Fenn, Rebecca S; Das, Rhiju; Harbury, Pehr A B (2008) Remeasuring the double helix. Science 322:446-9 |
| Boas, F Edward; Harbury, Pehr B (2007) Potential energy functions for protein design. Curr Opin Struct Biol 17:199-204 |
