Peptide environment of the peptidyl transferase center from Escherichia coli 70 S ribosomes as determined by thermoaffinity labeling with dihydrospiramycin.
In an attempt to gain information about the peptidyl transferase center at the peptide level we cross-linked the spiramycin derivative dihydrospiramycin to its functional binding site in the 70 S ribosome of Escherichia coli. In this manner ribosomal proteins S12, S14, L17, L18, L27 and L35 were found specifically affinity-labeled. Proteolytic fragmentation of these proteins, separation by C18 reversed-phase high performance liquid chromatography of the peptide mixtures, and subsequent sequence analysis of labeled peptides revealed peptide regions at positions Ala1-Lys9 and Tyr116-Lys119 of S12, Leu47-Asp53 of protein S14, Ser6-Lys35 of protein L17, Ala57-Lys63 of protein L18, Ala5-Lys18 and Val66-Lys71 of protein L27, and Thr5-Lys11 of protein L35. This approach is a valuable tool to characterize the binding site of spiramycin as well as the peptidyl transferase center at the molecular level.
Bischof
O
O
Max-Delbrück-Centrum für Molekulare Medizin, Abteilung Proteinchemie, Berlin-Buch, Germany.
Urlaub
H
H
Kruft
V
V
Wittmann-Liebold
B
B
D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001667:Binding, Competitive; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010458:Peptidyl Transferases / Q000032:analysis / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure; D015572:Spiramycin / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology
D000345:Affinity Labels; D000900:Anti-Bacterial Agents; D010446:Peptide Fragments; D012269:Ribosomal Proteins; D015572:Spiramycin; D010458:Peptidyl Transferases