Structural biology plays a central role in modern molecular bioscience, enabling both a greater understanding and new mechanisms of manipulation of biomolecular action. However, despite tremendous development in tools for the generation of high resolution molecular models, large families of proteins are still poorly represented in databases of protein structure due to limitations of current technology. One method that has been used successfully to qualitatively study the structure of several of these families is hydroxyl radical protein footprinting (HRPF). HRPF is an emerging technology that has been used to study changes in protein topography by measuring changes in the apparent rate of reaction between hydroxyl radicals generated in situ and amino acid side chains on the protein surface. While this technology has been used successfully to study challenging problems in protein structure (e.g. membrane protein topography, glycoprotein-protein interactions, protein oligomerization and aggregation, protein interactions with heterogeneous ligand mixtures), such studies have always been comparative, detecting relative changes in protein topography from one conformation to another. Quantitative descriptions of protein structure have not been achieved due to a lack of knowledge of the link between HRPF reactivities and biophysical properties of the protein. Here, we propose to leverage preliminary data to develop amino acid-resolution HRPF (HR-HRPF) into a quantitative measurement of protein topography, accurately measuring the average solvent accessible surface areas (
Molecular models of biomolecular structures have led to a revolution in the way we understand and manipulate the molecules of life. However, due to limitations of current technology, large gaps remain in our understanding of the three-dimensional structure of biomolecules, including many that play essential roles in human health. Here, we propose to develop a robust technology coupling protein chemistry, mass spectrometry, and computational modeling of protein structure to give biochemists a new method for generating reliable molecular models of protein structure.
|Riaz, Mohammad; Misra, Sandeep K; Sharp, Joshua S (2018) Towards high-throughput fast photochemical oxidation of proteins: Quantifying exposure in high fluence microtiter plate photolysis. Anal Biochem 561-562:32-36|