Unfolding of apomyoglobin helices by synchrotron radiolysis and mass spectrometry

The synchrotron X-ray protein radiolysis technique is based on a quantitati ve determination of the extent and the site of millisecond radiolytic oxida tion of amino-acid side chains by mass spectrometry. The amino acids most s usceptible to radiolytic oxidation are cysteine, methionine, phenyl-alanine , tyrosine, tryptophan, proline, histidine, and leucine. These residues ser ve as reactive markers within a protein structure that can be used to monit or changes in solvent accessibility during folding or as part of macromolec ular interactions. To monitor the unfolding, the extent of radiolytic produ cts of side chains of reactive amino acids is quantitatively measured by ma ss spectrometry as a function of the denaturant concentration following pro teolysis. This approach provides site-specific unfolding isotherms for vari ous segments of a protein without the use of mutation or labeling technique s. Application of this technique to the equilibrium urea unfolding of apomy oglobin at pH 7.8 has demonstrated the cooperative unfolding of helices A t o C consistent with midpoints, DeltaG, and m values derived from fluorescen ce data. The G helix, in contrast, showed a local unfolding behavior. The s imilarity of the thermodynamic data derived by this synchrotron-based metho d for helix A (containing two oxidizable tryptophan residues) to that of th e fluorescence data indicates that the limited oxidation of proteins by exp osure to X-rays on millisecond timescales does not alter the structure of a pomyglobin. This supports the viability of the method for the study of prot ein folding and the mapping of protein interaction sites.