title: Protein surface mapping using diethylpyrocarbonate with mass spectrometric detection.
abstract: The reliability and information content of diethylpyrocarbonate (DEPC) as a covalent probe of protein surface structure has been improved when used appropriately with mass spectrometric detection. Using myoglobin, cytochrome c, and beta-2-microglobulin as model protein systems, we demonstrate for the first time that DEPC can modify Ser and Thr residues in addition to His and Tyr residues. This result expands the capability of DEPC as a structural probe because about 25% of the sequence of the average protein can now be covered using this covalent labeling reagent. In addition, we establish a new approach based on mass spectrometry to ensure the structural integrity of proteins during amino acid-specific covalent labeling reactions. This approach involves monitoring the extent of modification as a function of reagent concentration and allows any small-scale or local perturbations caused by the covalent label to be readily identified and avoided. Results indicate that these dose-response plots are much more reliable and generally applicable probes of possible protein structural changes than fluorescence or circular dichroism spectroscopies. These dose-response plots also provide a means of quantitatively comparing the reactivity of each modified residue. On the basis of comparisons to known X-ray crystal structures, we find that the solvent accessibility of the reactive atom in the side chain and the presence of a nearby charged residue most affect modification rates. Finally, this improved surface mapping method has been used to determine the effect of Cu(II) binding on the structure of beta-2-microglobulin. Results confirm that Cu(II) binds His31, but not any of the other three His residues, and changes the solvent accessibility of residues near His31 and near the N-terminus.
authors.affiliation: Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA.
authors.forename: Vanessa Leah
authors.initials: VL
authors.lastname: Mendoza
authors.forename: Richard W
authors.initials: RW
authors.lastname: Vachet
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry; D045304:Cytochromes c / Q000737:chemistry; D004047:Diethyl Pyrocarbonate / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D009211:Myoglobin / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013499:Surface Properties; D001613:beta 2-Microglobulin / Q000737:chemistry
chemical_list: D000596:Amino Acids; D009211:Myoglobin; D011506:Proteins; D001613:beta 2-Microglobulin; D003300:Copper; D045304:Cytochromes c; D004047:Diethyl Pyrocarbonate