Cross-linked amino acids in the protein pairs L3-L19 and L23-L29 of Bacillus stearothermophilus ribosomes after treatment with diepoxybutane.
Treatment of native 50 S ribosomal subunits of Bacillus stearothermophilus with the homobifunctional cross-linking reagent diepoxybutane generated two cross-linked protein pairs, L3-L19 and L23-L29, which were isolated and identified. The analysis of the cross-linking sites at the amino acid level in both protein pairs is presented. Using a combination of sequence analysis and mass spectrometry it could be demonstrated that His-28 in protein L3 and the N-terminal amino acids Met-1, His-2, and His-3 in protein L19 are involved in forming the cross-link L3-L19. Within the pair L23-L29 Met-1 in protein L23 and Lys-4 in protein L29 were identified as cross-linking sites employing a similar approach. Comparison of our data with results derived from other cross-linking experiments showed that in general the structural organization of the ribosomes in eubacteria (the Gram-positive B. stearothermophilus and the Gram-negative Escherichia coli) has been conserved to quite an extent during evolution but that the fine structures differ slightly. By mass spectrometry the specificity of diepoxybutane and its cleaving mechanism using sodium periodate could be examined. In addition the complete amino acid sequence of protein L19 of B. stearothermophilus has been determined and revealed 58% identical amino acid residues to the homologous E. coli protein L19.
Herwig
S
S
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin, Germany.
Kruft
V
V
Eckart
K
K
Wittmann-Liebold
B
B
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004852:Epoxy Compounds / Q000494:pharmacology*; D001411:Geobacillus stearothermophilus / Q000737:chemistry* / Q000187:drug effects; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D012269:Ribosomal Proteins / Q000737:chemistry*; D012270:Ribosomes / Q000737:chemistry* / Q000187:drug effects; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D003432:Cross-Linking Reagents; D004852:Epoxy Compounds; D012269:Ribosomal Proteins; C007366:diepoxybutane