Identification of cross-linked amino acids in the protein pair HmaL23-HmaL29 from the 50S ribosomal subunit of the archaebacterium Haloarcula marismortui.
50S ribosomal subunits from the extreme halophilic archaebacterium Haloarcula marismortui were treated with the homobifunctional protein-protein cross-linking reagents diepoxybutane (4 A) and dithiobis(succinimidyl propionate) (12 A). The dominant product with both cross-linking reagents was identified on the protein level as HmaL23-HmaL29, which is homologous to the protein pair L23-L29 from Escherichia coli [Walleczek, J., Martin, T., Redl, B., Stöffler-Meilicke, M., & Stöffler, G. (1989) Biochemistry 28, 4099-4105] and from Bacillus stearothermophilus [Brockmöller, J., & Kamp, R. M. (1986) Biol. Chem. Hoppe-Seyler 367, 925-935]. To reveal the exact cross-linking site in HmaL23-HmaL29, the cross-linked complex was purified on a preparative scale by conventional and high-performance liquid chromatography. After endoproteolytic fragmentation of the protein pair, the amino acids engaged in cross-link formation were unambiguously identified by N-terminal sequence analysis and mass spectrometry of the cross-linked peptides. The cross-link is formed between lysine-57 in the C-terminal region of HmaL29 and the alpha-amino group of the N-terminal serine in protein HmaL23, irrespective of the cross-linking reagent. This result demonstrates that the N-terminal region of protein HmaL23 and the C-terminal domain of HmaL29 are highly flexible so that the distance between the two polypeptide chains can vary by at least 8 A. Comparison of our cross-linking results with those obtained with B. stearothermophilus revealed that the fine structure within this ribosomal domain is at least partially conserved.
Bergmann
U
U
Max-Planck-Institut für Molekulare Genetik, Abteilung Wittmann, Berlin, Dahlem, Germany.
Wittmann-Liebold
B
B
D001105:Archaea / Q000737:chemistry*; D019843:Archaeal Proteins; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D015180:Electrophoresis, Gel, Two-Dimensional; D004852:Epoxy Compounds; D008239:Lysine; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012270:Ribosomes / Q000737:chemistry*; D012694:Serine; D013388:Succinimides
D019843:Archaeal Proteins; D003432:Cross-Linking Reagents; D004852:Epoxy Compounds; C080399:HmaL23 protein, Haloarcula marismortui; C080400:HmaL29 protein, Haloarcula marismortui; D010446:Peptide Fragments; D012269:Ribosomal Proteins; D013388:Succinimides; D012694:Serine; C007366:diepoxybutane; C011240:dithiobis(succinimidylpropionate); D008239:Lysine