Detecting protein-protein interactions in the intact cell of Bacillus subtilis (ATCC 6633).
The salt bridge, paired group-specific reagent cyanogen (ethanedinitrile; C(2)N(2)) converts naturally occurring pairs of functional groups into covalently linked products. Cyanogen readily permeates cell walls and membranes. When the paired groups are shared between associated proteins, isolation of the covalently linked proteins allows their identity to be assigned. Examination of organisms of known genome sequence permits identification of the linked proteins by mass spectrometric techniques applied to peptides derived from them. The cyanogen-linked proteins were isolated by polyacrylamide gel electrophoresis. Digestion of the isolated proteins with proteases of known specificity afforded sets of peptides that could be analyzed by mass spectrometry. These data were compared with those derived theoretically from the Swiss Protein Database by computer-based comparisons (Protein Prospector; http://prospector.ucsf.edu). Identification of associated proteins in the ribosome of Bacillus subtilis strain ATCC 6633 showed that there is an association homology with the association patterns of the ribosomal proteins of Haloarcula marismortui and Thermus thermophilus. In addition, other proteins involved in protein biosynthesis were shown to be associated with ribosomal proteins.
Winters
Michael S
MS
Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221-0172, USA.
Day
R A
RA
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009570:Nitriles / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D009570:Nitriles; D010455:Peptides; D012269:Ribosomal Proteins; C011206:cyanogen