Abstract
Hydroxyl radical footprinting based MS for protein structure assessment has the goal of understanding ligand induced conformational changes and macromolecular interactions, for example, protein tertiary and quaternary structure, but the structural resolution provided by typical peptide-level quantification is limiting. In this work, we present experimental strategies using tandem-MS fragmentation to increase the spatial resolution of the technique to the single residue level to provide a high precision tool for molecular biophysics research. Overall, in this study we demonstrated an eightfold increase in structural resolution compared with peptide level assessments. In addition, to provide a quantitative analysis of residue based solvent accessibility and protein topography as a basis for high-resolution structure prediction; we illustrate strategies of data transformation using the relative reactivity of side chains as a normalization strategy and predict side-chain surface area from the footprinting data. We tested the methods by examination of Ca(+2)-calmodulin showing highly significant correlations between surface area and side-chain contact predictions for individual side chains and the crystal structure. Tandem ion based hydroxyl radical footprinting-MS provides quantitative high-resolution protein topology information in solution that can fill existing gaps in structure determination for large proteins and macromolecular complexes.
Highlights
From the ‡Center for Proteomics and Bioinformatics, Case Western Reserve University School of Medicine, 10009 Euclid Ave, Cleveland, Ohio, 44109
1 The abbreviations used are: HRF, Hydroxyl radical footprinting; CaM, Calmodulin; Dose Response (DR), Dose response; fSASA, fractional solvent accessible surface area; HDX-MS, Hydrogen-deuterium exchange MS; lution data enabled by the similar reactivity of the OH radical with each and every backbone position has helped solve important problems in the nucleic acids field, such as understanding RNA folding and ribosome assembly [1,2,3,4,5]
Hydrogen-deuterium exchange MS (HDX-MS) is suited to measure secondary and tertiary structure stability through backbone exchange, whereas HRF-MS has been effective at measuring the relative solvent accessibility of specific amino acid side chains mediated by intramolecular tertiary and intermolecular quaternary structure interactions
Summary
Sample Preparation and Synchrotron X-ray Radiolysis—Recombinant bovine calmodulin (CaM) was purified as described previously [28]. A survey MS experiment on a CaM sample exposed to the x-ray beam for 20 ms was carried out to detect a wide distribution of oxidatively modified species Using these detected m/z values; we generated a “dynamic inclusion” target list containing a total of 62 unique m/z species and their expected retention time values. For each fragment ion j in F, ion abundances from MS2 level experiments are recorded to generate tandem SIC plots for unmodified and modified fragment forms of the modified peptide. This can be viewed as the “electronic” extraction of multiple transitions in a “pseudoSRM” experiment [33, 34]. Leave-one-out validation was performed to perform prediction on a single residue, while training on the remaining 39 residues
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