Quantitative Mapping of Protein Structure by Hydroxyl Radical Footprinting-Mediated Structural Mass Spectrometry: A Protection Factor Analysis

Abstract

Measurements from hydroxyl radical footprinting (HRF) provide rich information about the solvent accessibility of amino acid side chains of a protein. Traditional HRF data analyses focus on comparing the difference in the modification/footprinting rate of a specific site to infer structural changes across two protein states, e.g., between a free and ligand-bound state. However, the rate information itself is not fully used for the purpose of comparing different protein sites within a protein on an absolute scale. To provide such a cross-site comparison, we present a new, to our knowledge, data analysis algorithm to convert the measured footprinting rate constant to a protection factor (PF) by taking into account the known intrinsic reactivity of amino acid side chain. To examine the extent to which PFs can be used for structural interpretation, this PF analysis is applied to three model systems where radiolytic footprinting data are reported in the literature. By visualizing structures colored with the PF values for individual peptides, a rational view of the structural features of various protein sites regarding their solvent accessibility is revealed, where high-PF regions are buried and low-PF regions are more exposed to the solvent. Furthermore, a detailed analysis correlating solvent accessibility and local structural contacts for gelsolin shows a statistically significant agreement between PF values and various structure measures, demonstrating that the PFs derived from this PF analysis readily explain fundamental HRF rate measurements. We also tested this PF analysis on alternative, chemical-based HRF data, showing improved correlations of structural properties of a model protein barstar compared to examining HRF rate data alone. Together, this PF analysis not only permits a novel, to our knowledge, approach of mapping protein structures by using footprinting data, but also elevates the use of HRF measurements from a qualitative, cross-state comparison to a quantitative, cross-site assessment of protein structures in the context of individual conformational states of interest.

Incorporation of a Reporter Peptide in FPOP Compensates for Adventitious Scavengers and Permits Time-Dependent Measurements

“…This will require calibrating a reporter peptide’s rate constant against a standard reaction. Chance has identified a similar goal for synchrotron footprinting [21]. This would facilitate cross-laboratory comparisons of FPOP results and enable its use to determine coarse-grained HOS of proteins.…”

Quantitative Protein Topography Analysis and High-Resolution Structure Prediction Using Hydroxyl Radical Labeling and Tandem-Ion Mass Spectrometry (MS)*

“…where R i is the relative chemical reactivity for residue-type i to solution generated hydroxyl radicals, using proline as the internal reference, and k fp is the measured rate constant for the residue (46). Table I shows the calculated PFs using both y-and b-ion rate data from CaM.…”

“…After normalization, we examined both a biophysical approach based on first principles and a regression learning approach based on statistical evaluation of the data (random forest regression) to predict structure from the data and then compared and contrasted the methods. Recently, we introduced the concept of the protection factor (PF), where reactivity measures at the peptide level from MS1 based data were first normalized based on known reactivity data from the literature, such that the rate constants across different peptides could be compared on an absolute scale (46). The corrected rates were used to predict the surface or interior locations of peptides using gelsolin as an example.…”

Theoretical modeling of multiprotein complexes by iSPOT: Integration of small-angle X-ray scattering, hydroxyl radical footprinting, and computational docking

“…Note that log P f is previously demonstrated to be strongly correlated with the so-called solvent accessible surface area SA i for each amino acid (Huang et al, 2015; Kaur et al, 2015). In other words, the φ 2 score is an average difference of solvent accessibility between each docked conformation and its target crystal structure to measure the goodness of fit with simulated footprinting data.…”

“…It provides information about the shape of a full complex (Bernado and Blackledge, 2010; Koch et al, 2003; Putnam et al, 2007; Yang, 2014), but it is known that over-fitting of experimental curves may yield incorrect structural models (Rambo and Tainer, 2013). Another example is the use of the so-called hydroxyl radical footprinting (Huang et al, 2015), which probes the solvent accessibility of surface residues (Hambly and Gross, 2005; Huang et al, 2015; Kaur et al, 2015; Maleknia and Downard, 2014; Sharp et al, 2004; Tullius and Dombroski, 1986; Xu and Chance, 2007). By itself, it is not able to predict the correct structure of a protein-protein complex (Kamal and Chance, 2008).…”