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

2017 ◽  
Author(s):  
Sandeep K. Misra ◽  
Amika Sood ◽  
Paulo A. Soares ◽  
Vitor H. Pomin ◽  
Robert J. Woods ◽  
...  
Keyword(s):  
High Resolution ◽  
Hydroxyl Radical ◽  
Heparan Sulfate ◽  
Surface Oxidation ◽  
V3 Loop ◽  
Heparin Binding ◽  
Protein Footprinting ◽  
Computational Docking ◽  
Docking Simulations ◽  
Hydroxyl Radical Protein Footprinting

AbstractThe 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.

scholarly journals High Structural Resolution Hydroxyl Radical Protein Footprinting Reveals an Extended Robo1-Heparin Binding Interface

2015 ◽  
Vol 290 (17) ◽  
pp. 10729-10740 ◽  
Author(s):  
Zixuan Li ◽  
Heather Moniz ◽  
Shuo Wang ◽  
Annapoorani Ramiah ◽  
Fuming Zhang ◽  
...  
Keyword(s):  
Hydroxyl Radical ◽  
Heparin Binding ◽  
Protein Footprinting ◽  
Binding Interface ◽  
Hydroxyl Radical Protein Footprinting

High-Resolution Hydroxyl Radical Protein Footprinting: Biophysics Tool for Drug Discovery

2018 ◽  
Vol 47 (1) ◽  
pp. 315-333 ◽  
Author(s):  
Janna Kiselar ◽  
Mark R. Chance
Keyword(s):  
Protein Structure ◽  
High Resolution ◽  
Hydroxyl Radical ◽  
Solvent Accessibility ◽  
Side Chains ◽  
Protein Footprinting ◽  
Hydroxyl Radical Protein Footprinting ◽  
Wide Range ◽  
Hydroxyl Radical Footprinting

Hydroxyl radical footprinting (HRF) of proteins with mass spectrometry (MS) is a widespread approach for assessing protein structure. Hydroxyl radicals react with a wide variety of protein side chains, and the ease with which radicals can be generated (by radiolysis or photolysis) has made the approach popular with many laboratories. As some side chains are less reactive and thus cannot be probed, additional specific and nonspecific labeling reagents have been introduced to extend the approach. At the same time, advances in liquid chromatography and MS approaches permit an examination of the labeling of individual residues, transforming the approach to high resolution. Lastly, advances in understanding of the chemistry of the approach have led to the determination of absolute protein topologies from HRF data. Overall, the technology can provide precise and accurate measures of side-chain solvent accessibility in a wide range of interesting and useful contexts for the study of protein structure and dynamics in both academia and industry.

scholarly journals Quantitative Protein Topography Measurements by High Resolution Hydroxyl Radical Protein Footprinting Enable Accurate Molecular Model Selection

2017 ◽  
Author(s):  
Boer Xie ◽  
Amika Sood ◽  
Robert J. Woods ◽  
Joshua S. Sharp
Keyword(s):  
High Resolution ◽  
Hydroxyl Radical ◽  
Computational Models ◽  
Electron Transfer Dissociation ◽  
Molecular Model ◽  
Solvent Accessible Surface Area ◽  
Protein Footprinting ◽  
Backbone Rmsd ◽  
Hydroxyl Radical Protein Footprinting ◽  
Lower Accuracy

AbstractWe 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 (<SASA>) 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 <SASA> measurements was tested by using well-characterized protein models. Moreover, we demonstrated the ability to use <SASA> measurements from HR-HRPF to differentiate molecular models of high accuracy (< 3Å backbone RMSD) from models of lower accuracy (> 4Å backbone RMSD). The ability of <SASA> data from HR-HRPF to differentiate molecular model quality was found to be comparable to that of <SASA> data obtained from X-ray crystal structures, indicating the accuracy and utility of HR-HRPF for evaluating the accuracy of computational models.

scholarly journals Accurate protein structure prediction with hydroxyl radical protein footprinting data

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sarah E. Biehn ◽  
Steffen Lindert
Keyword(s):  
Protein Structure ◽  
Hydroxyl Radical ◽  
Protein Structure Prediction ◽  
Structure Prediction ◽  
Tertiary Structure ◽  
Solvent Exposure ◽  
Protein Footprinting ◽  
Hydroxyl Radical Protein Footprinting ◽  
Predict Protein Structure ◽  
Ab Initio Models

AbstractHydroxyl radical protein footprinting (HRPF) in combination with mass spectrometry reveals the relative solvent exposure of labeled residues within a protein, thereby providing insight into protein tertiary structure. HRPF labels nineteen residues with varying degrees of reliability and reactivity. Here, we are presenting a dynamics-driven HRPF-guided algorithm for protein structure prediction. In a benchmark test of our algorithm, usage of the dynamics data in a score term resulted in notable improvement of the root-mean-square deviations of the lowest-scoring ab initio models and improved the funnel-like metric Pnear for all benchmark proteins. We identified models with accurate atomic detail for three of the four benchmark proteins. This work suggests that HRPF data along with side chain dynamics sampled by a Rosetta mover ensemble can be used to accurately predict protein structure.

scholarly journals Probing the solution structure of Factor H using hydroxyl radical protein footprinting and cross-linking

2016 ◽  
Vol 473 (12) ◽  
pp. 1805-1819 ◽  
Author(s):  
Anna Baud ◽  
Florence Gonnet ◽  
Isabelle Salard ◽  
Maxime Le Mignon ◽  
Alexandre Giuliani ◽  
...  
Keyword(s):  
Hydroxyl Radical ◽  
Solution Structure ◽  
Binding Capacity ◽  
Fluid Phase ◽  
Factor H ◽  
Cross Linking ◽  
Complement Protein ◽  
Protein Footprinting ◽  
Protein Factor ◽  
Hydroxyl Radical Protein Footprinting

The control protein Factor H (FH) is a crucial regulator of the innate immune complement system, where it is active on host cell membranes and in the fluid phase. Mutations impairing the binding capacity of FH lead to severe autoimmune diseases. Here, we studied the solution structure of full-length FH, in its free state and bound to the C3b complement protein. To do so, we used two powerful techniques, hydroxyl radical protein footprinting (HRPF) and chemical cross-linking coupled with mass spectrometry (MS), to probe the structural rearrangements and to identify protein interfaces. The footprint of C3b on the FH surface matches existing crystal structures of C3b complexed with the N- and C-terminal fragments of FH. In addition, we revealed the position of the central portion of FH in the protein complex. Moreover, cross-linking studies confirmed the involvement of the C-terminus in the dimerization of FH.

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