Structural proteomics approaches using mass spectrometry are increasingly used in biology to examine the composition and structure of macromolecules. Hydroxyl radical-mediated protein footprinting using mass spectrometry has recently been developed to define structure, assembly, and conformational changes of macromolecules in solution based on measurements of reactivity of amino acid side chain groups with covalent modification reagents. Accurate measurements of side chain reactivity are achieved using quantitative liquid-chromatography-coupled mass spectrometry, whereas the side chain modification sites are identified using tandem mass spectrometry. In addition, the use of footprinting data in conjunction with computational modeling approaches is a powerful new method for testing and refining structural models of macromolecules and their complexes. In this review, we discuss the basic chemistry of hydroxyl radical reactions with peptides and proteins, highlight various approaches to map protein structure using radical oxidation methods, and describe state-of-the-art approaches to combine computational and footprinting data.
Protein footprinting utilizing hydroxyl radicals coupled with mass spectrometry has become a powerful technique for mapping the solvent accessible surface of proteins and examining protein-protein interactions in solution. Hydroxyl radicals generated by radiolysis or chemical methods efficiently react with many amino acid residue side chains, including the aromatic and sulfur-containing residues along with proline and leucine, generating stable oxidation products that are valuable probes for examining protein structure. In this study, we examine the radiolytic oxidation chemistry of histidine, lysine, and arginine for comparison with their metal-catalyzed oxidation products. Model peptides containing arginine, histidine, and lysine were irradiated using white light from a synchrotron X-ray source or a cesium-137 gamma-ray source. The rates of oxidation and the radiolysis products were primarily characterized by electrospray mass spectrometry including tandem mass spectrometry. Arginine is very sensitive to radiolytic oxidation, giving rise to a characteristic product with a 43 Da mass reduction as a result of the loss of guanidino group and conversion to gamma-glutamyl semialdehyde, consistent with previous metal-catalyzed oxidation studies. Histidine was oxidized to generate a mixture of products with characteristic mass changes primarily involving rupture of and addition to the imidazole ring. Lysine was converted to hydroxylysine or carbonylysine by radiolysis. The development of methods to probe these residues due to their high frequency of occurrence, their typical presence on the protein surface, and their frequent participation in protein-protein interactions considerably extends the utility of protein footprinting.
The formation of individual tertiary contacts of the Tetrahymena L-21 Sca I ribozyme has been monitored by hydroxyl radical footprinting and its global conformation by analytical ultracentrifugation as a function of monovalent ion concentration in the absence of divalent ions. Advanced methods of data analysis, which allow the hydroxyl radical reactivity of every nucleotide to be quantified, permit monitoring of each and every structural element of the RNA. Monovalent ion-mediated global compaction of the ribozyme is accompanied by the formation of native tertiary contacts; most native tertiary contacts are evident except several that are located near where divalent ions are observed in crystallographic structures. Non-native tertiary contacts are also observed at low but not high concentrations of monovalent ions. In light of recent studies that have shown that the presence of monovalent ions greatly accelerates the Mg2+-dependent folding of the Tetrahymena ribozyme, the present studies suggest that Na+ concentration changes not only the starting position of the RNA on its folding funnel but also pushes it deep into the well by forming native tertiary contacts and, thus, favoring fast and correct folding pathways.
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