Determination of peptide regions exposed at the surface of the bacterial ribosome with antibodies against synthetic peptides.
We synthesized six peptides corresponding to regions that are predicted to be surface-exposed of the following ribosomal proteins: protein L2, positions (D263-K272); protein L5, positions (I136-G150); protein L25, positions (Q75-D90); protein S3, positions (Q222-K232) derived from Escherichia coli; and protein L2, positions (K257-K275), and protein S3, positions (R130-T150) from Bacillus stearothermophilus. These peptides were employed to raise ribosomal protein-cross-reactive antibodies. The anti-peptide antisera reacted specifically with their parent proteins, as demonstrated by immunoblotting experiments. In a competition assay proteins L2 from E. coli and B. stearothermophilus as well as proteins L5 and L25 from E. coli were found to be accessible to the respective anti-peptide antibodies in the 50S subunits, but not in 70S ribosomes, proving their location at the 50S interface which is covered by the 30S subunit in the 70S complex. Two of the anti-peptide antisera directed against sequences deduced from protein S3 of E. coli and B. stearothermophilus reacted with 30S subunits as well as with 70S ribosomes, demonstrating their location at the backside, which is exposed to solvent. Thus, by the strategy applied specific short peptide stretches were located at the surface of the ribosome.
Herfurth
E
E
Max-Delbrück-Centrum für Molekulare Medizin, Abteilung Proteinchemie, Berlin, Germany.
Wittmann-Liebold
B
B
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000737:chemistry*; D000918:Antibody Specificity; D001426:Bacterial Proteins / Q000737:chemistry* / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000737:chemistry / Q000378:metabolism; D015151:Immunoblotting; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000276:immunology; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000276:immunology; D012270:Ribosomes / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000276:immunology
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D010455:Peptides; D012269:Ribosomal Proteins; D012710:Serum Albumin, Bovine