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.

Interaction of C3b2–IgG complexes with complement proteins properdin, factor B and factor H: implications for amplification

2000 ◽  
Vol 349 (1) ◽  
pp. 217-223
Author(s):  
Emiliana JELEZAROVA ◽  
Anna VOGT ◽  
Hans U. LUTZ
Keyword(s):  
Present Report ◽  
Alternative Pathway ◽  
Fluid Phase ◽  
Factor H ◽  
Complement Protein ◽  
Factor B ◽  
Physiological Conditions ◽  
Amplification Loop ◽  
Target Molecules ◽  
Properdin Factor B

Nascent C3b can form ester bonds with various target molecules on the cell surface and in the fluid phase. Previously, we showed that C3b2-IgG complexes represent the major covalent product of C3 activation in serum [Lutz, Stammler, Jelezarova, Nater and Späth (1996) Blood 88, 184-193]. In the present report, binding of alternative pathway proteins to purified C3b2-IgG complexes was studied in the fluid phase by using biotinylated IgG for C3b2-IgG generation and avidin-coated plates to capture complexes. Up to seven moles of properdin ‘monomer’ bound per mole of C3b2-IgG at physiological conditions in the absence of any other complement protein. At low properdin/C3b2-IgG ratios bivalent binding was preferred. Neither factor H nor factor B affected properdin binding. On the other hand, properdin strongly stimulated factor B binding. Interactions of all three proteins with C3b2-IgG exhibited pH optima. An ionic strength optimum was most pronounced for properdin, while factor B binding was largely independent of the salt concentration. C3b2-IgG complexes were powerful precursors of the alternative pathway C3 convertase. In the presence of properdin, C3 convertase generated from C3b2-IgG cleaved about sevenfold more C3 than the enzyme generated on C3b. C3b2-IgG complexes could therefore maintain the amplification loop of complement longer than free C3b.

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

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.

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.

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