The ribosomal protein HmaS7 from the 30S subunit of the extreme halophilic archaeum Haloarcula marismortui was isolated by semi-preparative RP-HPLC.The complete amino-acid sequence of this protein was determined by automated microsequence analysis of appropriate peptide fragments from several proteinase digests. The entire protein consists of 205 amino acids with a corresponding molecular mass of 22580 Da.The modification at the amino-terminal amino acid was deblocked so that the N-terminal amino acids could be sequenced and the type of the modification was identified as an acetyl group by electrospray mass spectrometry of suitable peptides. Homology studies of HmaS7 showed similarities to ribosomal proteins derived from organisms of all three urkingdoms, such as to EcoS7, HmoS7, MvaS7, SacS7 and RatS7; due to the strong sequence homologies found within the archaebacterial ribosomal proteins we conclude that the protein sequence which was determined for S7 from Methanococcus vannielii by nucleotide sequencing of the gene should be about 20 or 30 amino acids longer than previously published
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.