Radiolytic modification and reactivity of amino acid residues serving as structural probes for protein footprinting

G Xu, MR Chance - Analytical chemistry, 2005 - ACS Publications
G Xu, MR Chance
Analytical chemistry, 2005ACS Publications
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
detectible 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 …
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 detectible 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 γ-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 detectible. 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 ∼65% of the sequence of a typical protein, the footprinting approach provides excellent coverage of the side-chain reactivity for proteins.
ACS Publications