Chemical cross-linking with thiol-cleavable reagents combined with differential mass spectrometric peptide mapping--a novel approach to assess intermolecular protein contacts.
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.
Bennett
K L
KL
Department of Molecular Biology, University of Southern Denmark, Odense. bennett@pr-group.sdu.dk
Kussmann
M
M
Björk
P
P
Godzwon
M
M
Mikkelsen
M
M
Sørensen
P
P
Roepstorff
P
P
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
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