Structural studies of the sBBI/trypsin non-covalent complex using covalent modification and mass spectrometry.
BACKGROUND
The study of protein recognition sites is crucial for understanding the mechanisms of protein interaction. Mass spectrometry can be a method of choice for the investigation of the contact surface within the protein non-covalent complexes.


METHODS
Probing the reactivity of essential amino acid residues of soybean Bowman-Birk inhibitor (sBBI) within the non-covalent sBBI/bovine trypsin complex was performed using covalent labeling by the BS3 cross-linker and charge tag with a quaternary ammonium group in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analysis.


RESULTS
Significant modulation of the reactivity of essential K16 and S17 residues in the sBBI molecule upon binding to trypsin was established. The studies of sBBI proteolytic peptides with the same structure but carrying different labels using metastable dissociation in LIFT mode demonstrated that fragmentation pathways were oriented by used modification (BS3 cross-linker or charge tag).


CONCLUSIONS
The effectiveness of the mass spectrometric approach including covalent modification for exploring protein-protein interaction sites has been demonstrated. The alteration of the reactivity of functionally important amino acid residues in the sBBI molecule is most likely related to changes in their microenvironment. It has been suggested that in the presence of charge tags fragmentation in LIFT mode proceeds through the formation of salt bridges between quaternary ammonium groups and acidic residues due to the occurrence of zwitterions (including basic and acidic residues). Despite the presence of one or several charge tags, fragmentation takes place yielding modulated bi /yj ion series depending on the positions of the tags.
CEA/Institut de Génomique/Centre National de Génotypage, Evry, France; CEA/Institut de Génomique/Centre National de Séquençage, Evry, France.
Ekaterina
E
Darii
Guanalini
G
Saravanamuthu
Ivo G
IG
Gut
Jean-Claude
JC
Tabet
D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011392:Proline / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000737:chemistry* / Q000378:metabolism
D010455:Peptides; D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D011392:Proline; D014357:Trypsin