Unfolding of apomyoglobin helices by synchrotron radiolysis and mass spectrometry.
The synchrotron X-ray protein radiolysis technique is based on a quantitative determination of the extent and the site of millisecond radiolytic oxidation of amino-acid side chains by mass spectrometry. The amino acids most susceptible to radiolytic oxidation are cysteine, methionine, phenylalanine, tyrosine, tryptophan, proline, histidine, and leucine. These residues serve as reactive markers within a protein structure that can be used to monitor changes in solvent accessibility during folding or as part of macromolecular interactions. To monitor the unfolding, the extent of radiolytic products of side chains of reactive amino acids is quantitatively measured by mass spectrometry as a function of the denaturant concentration following proteolysis. This approach provides site-specific unfolding isotherms for various segments of a protein without the use of mutation or labeling techniques. Application of this technique to the equilibrium urea unfolding of apomyoglobin at pH 7.8 has demonstrated the cooperative unfolding of helices A to C consistent with midpoints, DeltaG, and m values derived from fluorescence data. The G helix, in contrast, showed a local unfolding behavior. The similarity of the thermodynamic data derived by this synchrotron-based method for helix A (containing two oxidizable tryptophan residues) to that of the fluorescence data indicates that the limited oxidation of proteins by exposure to X-rays on millisecond timescales does not alter the structure of apomyglobin. This supports the viability of the method for the study of protein folding and the mapping of protein interaction sites.
Maleknia
S D
SD
Sciformatics, Sydney, NSW 2001, Australia. maleknia@scinformatics.com
Downard
K M
KM
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D017510:Protein Folding; D013329:Structure-Activity Relationship; D017356:Synchrotrons; D014508:Urea / Q000737:chemistry
D001059:Apoproteins; D009211:Myoglobin; C010857:apomyoglobin; D014508:Urea