Measuring Protein Topology in Conformationally Heterogeneous Mixtures by Top-Down Hydroxyl Radical Protein Footprinting

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With support from the Chemical Measurement and Imaging Program, Dr. Joshua Sharp and his group at the University of Mississippi are developing analytical tools to advance the ability to determine the three-dimensional shape of biomolecules (including proteins) - information essential for understanding biology at the molecular level. Of particular interest are challenging applications involving proteins that fluctuate between multiple shapes. The Sharp group is developing powerful new methods to characterize shapes coexisting in solution. As part of this technology development, they are incorporating hands-on research training opportunities for postdoctoral research associates and undergraduate students recruited via minority outreach programs at the University of Mississippi.This project is developing methodology that can provide new insights into structurally heterogeneous proteins, including (but not limited to) conformationally dynamic proteins and those encountered in protein aggregation. The methods can also illuminate the structural consequences of protein sequence variations and post-translational modifications. The approach combines advances in high resolution hydroxyl radical protein footprinting (HR-HRPF) with top-down tandem mass spectrometry (MS/MS) analyses. In HRPF, the rate of reaction of amino acid side chains with hydroxyl radicals generated in situ is used to assess the side chain?s solvent accessibility. Previous work has shown that different non-covalent conformations co-existing in a mixture experience differing amounts of oxidation when subjected to HRPF. However, "bottom-up" HR-HRPF as currently practiced only detects an overall average of all conformers; structural details are lost upon digestion and bottom-up analysis. By using top-down analyses with HR-HRPF, conformers can be separated based on the extent of overall modification. Development of this technology is focusing on two systems with controllable conformational changes. Mixtures of wild-type CXCL4 (a tetramer) with the K50E mutant of CXCL4 (a dimer) are being used to test the ability to characterize the dimer and tetramer independently in solution, using isotopic labeling of the mutant to separate the conformers by mass and verify results from the unlabeled samples. A second test system probes the differential hydroxyl radical reactivity of the unfolded and folded states of ubiquitin to differentiate the conformers based on the number of oxidation events that occur in each protein. The knowledge derived from this project will benefit researchers working at the forefront of some of the most difficult problems in structural biology - the detailed characterization of dynamic populations of conformations including aggregation, inherently disordered proteins, and protein misfolding/unfolding. This technology would also play a powerful role in the analysis of the structural consequences of protein sequence variants and protein post-translational modifications.