No abstract

Hydroxyl radical-mediated protein footprinting is a convenient and sensitive technique for mapping solvent-accessible surfaces of proteins and examining the structure and dynamics of biological assemblies. In this study, the reactivities and tendencies to form easily detectable products for all 20 (common) amino acid side chains along with cystine are directly compared using various standards. Although we have previously reported on the oxidation of many of these residues, this study includes a detailed examination of the less reactive residues and better defines their usefulness in hydroxyl radical-mediated footprinting experiments. All 20 amino amides along with cystine and a few tripeptides were irradiated by gamma-rays, the products were analyzed by electrospray mass spectrometry, and rate constants of modification were measured. The reactivities of amino acid side chains were compared based on their loss of mass spectral signal normalized to the rate of loss for Phe or Pro that were radiolyzed simultaneously to serve as internal standards. In this way, accurate quantitation of relative rates could be assured. A reactivity order of amino acid side chains was obtained as Cys > Met > Trp > Tyr > Phe > cystine > His > Leu, Ile > Arg, Lys, Val > Ser, Thr, Pro > Gln, Glu > Asp, Asn > Ala > Gly. Ala and Gly are far too unreactive to be useful probes in typical experiments and Asp and Asn are unlikely to be useful as well. Although Ser and Thr are more reactive than Pro, which is known to be a useful probe, their oxidation products are not easily detectable. Thus, it appears that 14 of the 20 side chains (plus cystine) are most likely to be useful in typical experiments. Since these residues comprise approximately 65% of the sequence of a typical protein, the footprinting approach provides excellent coverage of the side-chain reactivity for proteins.

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.

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