Aliphatic peptidyl hydroperoxides as a source of secondary oxidation in hydroxyl radical protein footprinting

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.

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

Annual Review of Biophysics ◽

10.1146/annurev-biophys-070317-033123 ◽

2018 ◽

Vol 47 (1) ◽

pp. 315-333 ◽

Cited By ~ 16

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.

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The utilization of the search engine, Bolt, to decrease search time and increase peptide identifications in hydroxyl radical protein footprinting‐based workflows

PROTEOMICS ◽

10.1002/pmic.202000295 ◽

2021 ◽

pp. 2000295 ◽

Cited By ~ 1

Author(s):

Emily E. Chea ◽

Amol Prakash ◽

Lisa M. Jones

Keyword(s):

Hydroxyl Radical ◽

Search Engine ◽

Search Time ◽

Protein Footprinting ◽

Hydroxyl Radical Protein Footprinting

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

Biochemical Journal ◽

10.1042/bcj20160225 ◽

2016 ◽

Vol 473 (12) ◽

pp. 1805-1819 ◽

Cited By ~ 7

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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