Biology subjects

Sharp, J. S.

Publications and source records attributed to Sharp, J. S..

3 recordsLinked to original sources

bioRxiv2018

Real Time Normalization of Fast Photochemical Oxidation of Proteins Experiments by Inline Adenine Radical Dosimetry

Hydroxyl radical protein footprinting (HRPF) is a powerful method for measuring protein topography, allowing researchers to monitor events that alter the solvent accessible surface of a protein (e.g. ligand binding, aggregation, conformational changes, etc.) by measuring changes in the apparent rate of reaction of portions of the protein to hydroxyl radicals diffusing in solution. Fast Photochemical Oxidation of Proteins (FPOP) offers an ultra-fast benchtop method for performing HRPF, photolyzing hydrogen peroxide using a UV laser to generate high concentrations of hydroxyl radicals that are consumed on roughly a microsecond timescale. The broad reactivity of hydroxyl radicals means that almost anything added to the solution (e.g. ligands, buffers, excipients, etc.) will scavenge hydroxyl radicals, altering their half-life and changing the effective radical concentration experienced by the protein. Similarly, minute changes in peroxide concentration, laser fluence, and buffer composition can alter the effective radical concentration, making reproduction of data challenging. Here, we present a simple method for radical dosimetry that can be carried out as part of the FPOP workflow, allowing for measurement of effective radical concentration in real time. Additionally, by modulating the amount of radical generated, we demonstrate that FPOP HRPF experiments carried out in buffers with widely differing levels of hydroxyl radical scavenging capacity can be normalized on the fly, yielding statistically indistinguishable results for the same conformer. This method represents a major step in transforming FPOP into a robust and reproducible technology capable of probing protein structure in a wide variety of contexts.

biochemistry↗

bioRxiv2017

Mapping of the Fondaparinux Binding Site of JR-FL gp120 by High Resolution Hydroxyl Radical Protein Footprinting and Computational Docking

The adhesion of HIV gp120 antigen to human cells is modulated in part by interactions with heparan sulfate. The HXB2 strain of gp120 has been shown to interact with heparin primarily through the V3 loop, although other domains including the C-terminal domain were also implicated. However, the JR-FL strain (representative of CCR5-interacting strains that make up newest infections) was shown to have a drastically lowered affinity to heparin due to the loss of several basic residues in the V3 loop, and deletion of the V3 loop in JR-FL gp120 was shown to abrogate some, but not all, heparin binding. Here, we use high resolution hydroxyl radical protein footprinting to measure the changes in protein surface oxidation levels that result from the binding of a model heparin fragment (fondaparinux). Protection in both the V3 loop and the N-terminus of JR-FP gp120 is observed. The well-defined composition of fondaparinux allowed us to perform docking simulations, which showed two clusters of fondaparinux binding: the V3 loop, and a domain consisting of the N- and C-termini. Together, the experimental and theoretical results indicate the heparin/heparan sulfate binding sites on JR-FL gp120 and the efficient interaction of fondaparinux, a widely exploited therapeutic carbohydrate, on gp120.

biochemistry↗

bioRxiv2017

Quantitative Protein Topography Measurements By High Resolution Hydroxyl Radical Protein Footprinting Enable Accurate Molecular Model Selection

We report an integrated workflow that allows mass spectrometry-based high-resolution hydroxyl radical protein footprinting (HR-HRPF) measurements to accurately measure the absolute average solvent accessible surface area ( ) of amino acid side chains. This approach is based on application of multi-point HR-HRPF, electron-transfer dissociation (ETD) tandem MS (MS/MS) acquisition, measurement of effective radical doses by radical dosimetry, and proper normalization of the inherent reactivity of the amino acids. The accuracy of the resulting measurements was tested by using well-characterized protein models. Moreover, we demonstrated the ability to use measurements from HR-HRPF to differentiate molecular models of high accuracy (< 3[A] backbone RMSD) from models of lower accuracy (> 4[A] backbone RMSD). The ability of data from HR-HRPF to differentiate molecular model quality was found to be comparable to that of data obtained from X-ray crystal structures, indicating the accuracy and utility of HR-HRPF for evaluating the accuracy of computational models.

biochemistry↗