title: Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping--a novel approach to assess intermolecular protein contacts.
abstract: The intermolecular contact regions between monomers of the homodimeric DNA binding protein ParR and the interaction between the glycoproteins CD28 and CD80 were investigated using a strategy that combined chemical cross-linking with differential MALDI-MS analyses. ParR dimers were modified in vitro with the thiol-cleavable cross-linker 3,3'-dithio-bis(succinimidylproprionate) (DTSSP), proteolytically digested with trypsin and analyzed by MALDI-MS peptide mapping. Comparison of the peptide maps obtained from digested cross-linked ParR dimers in the presence and absence of a thiol reagent strongly supported a "head-to-tail" arrangement of the monomers in the dimeric complex. Glycoprotein fusion constructs CD28-IgG and CD80-Fab were cross-linked in vitro by DTSSP, characterized by nonreducing SDS-PAGE, digested in situ with trypsin and analyzed by MALDI-MS peptide mapping (+/- thiol reagent). The data revealed the presence of an intermolecular cross-link between the receptor regions of the glycoprotein constructs, as well as a number of unexpected but nonetheless specific interactions between the fusion domains of CD28-IgG and the receptor domain of CD80-Fab. The strategy of chemical cross-linking combined with differential MALDI-MS peptide mapping (+ thiol reagent) enabled localization of the interface region(s) of the complexes studied and clearly demonstrates the utility of such an approach to obtain structural information on interacting noncovalent complexes.
authors.affiliation: Department of Molecular Biology, University of Southern Denmark, Odense. bennett@pr-group.sdu.dk
authors.forename: K L
authors.initials: KL
authors.lastname: Bennett
authors.forename: M
authors.initials: M
authors.lastname: Kussmann
authors.forename: P
authors.initials: P
authors.lastname: Björk
authors.forename: M
authors.initials: M
authors.lastname: Godzwon
authors.forename: M
authors.initials: M
authors.lastname: Mikkelsen
authors.forename: P
authors.initials: P
authors.lastname: Sørensen
authors.forename: P
authors.initials: P
authors.lastname: Roepstorff
mesh_terms: D000595:Amino Acid Sequence; D018122:B7-1 Antigen / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins; D001665:Binding Sites; D018106:CD28 Antigens / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D018122:B7-1 Antigen; D001426:Bacterial Proteins; D018106:CD28 Antigens; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; C086169:ParR protein, bacteria; D012097:Repressor Proteins; D008239:Lysine; D003545:Cysteine