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. 1996 May;5(5):969–972. doi: 10.1002/pro.5560050519

1H and 15N nuclear magnetic resonance assignment and secondary structure of the cytotoxic ribonuclease alpha-Sarcin.

R Campos-Olivas 1, M Bruix 1, J Santoro 1, A Martínez del Pozo 1, J Lacadena 1, J G Gavilanes 1, M Rico 1
PMCID: PMC2143403  PMID: 8732769

Abstract

The ribosome-inactivating protein alpha-Sarcin (alpha S) is a 150-residue fungal ribonuclease that, after entering sensitive cells, selectively cleaves a single phosphodiester bond in an universally conserved sequence of the major rRNA to inactivate the ribosome and thus exert its cytotoxic action. As a first step toward establishing the structure-dynamics-function relationships in this system, we have carried out the assignment of the 1H and 15N NMR spectrum of alpha S on the basis of homonuclear (1H-1H) and heteronuclear (1H-15N) two-dimensional correlation spectra of a uniformly 15N-labeled sample, and two selectively 15N-labeled (Tyr and Phe) samples, as well as a single three-dimensional experiment. The secondary structure of alpha S, as derived from the characteristic patterns of dipolar connectivities between backbone protons, conformational chemical shifts, and the protection of backbone amide protons against exchange, consists of a long N-terminal beta-hairpin, a short alpha-helical segment, and a C-terminal beta-sheet of five short strands arranged in a + 1, + 1, + 1, + 1 topology, connected by long loops in which the 13 Pro residues are located.

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Selected References

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  1. Endo Y., Glück A., Chan Y. L., Tsurugi K., Wool I. G. RNA-protein interaction. An analysis with RNA oligonucleotides of the recognition by alpha-sarcin of a ribosomal domain critical for function. J Biol Chem. 1990 Feb 5;265(4):2216–2222. [PubMed] [Google Scholar]
  2. Endo Y., Huber P. W., Wool I. G. The ribonuclease activity of the cytotoxin alpha-sarcin. The characteristics of the enzymatic activity of alpha-sarcin with ribosomes and ribonucleic acids as substrates. J Biol Chem. 1983 Feb 25;258(4):2662–2667. [PubMed] [Google Scholar]
  3. Jiménez M. A., Nieto J. L., Herranz J., Rico M., Santoro J. 1H NMR and CD evidence of the folding of the isolated ribonuclease 50-61 fragment. FEBS Lett. 1987 Sep 14;221(2):320–324. doi: 10.1016/0014-5793(87)80948-1. [DOI] [PubMed] [Google Scholar]
  4. Kumar A., Ernst R. R., Wüthrich K. A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules. Biochem Biophys Res Commun. 1980 Jul 16;95(1):1–6. doi: 10.1016/0006-291x(80)90695-6. [DOI] [PubMed] [Google Scholar]
  5. Lacadena J., Mancheño J. M., Martinez-Ruiz A., Martínez del Pozo A., Gasset M., Oñaderra M., Gavilanes J. G. Substitution of histidine-137 by glutamine abolishes the catalytic activity of the ribosome-inactivating protein alpha-sarcin. Biochem J. 1995 Jul 15;309(Pt 2):581–586. doi: 10.1042/bj3090581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Lacadena J., Martínez del Pozo A., Barbero J. L., Mancheño J. M., Gasset M., Oñaderra M., López-Otín C., Ortega S., García J., Gavilanes J. G. Overproduction and purification of biologically active native fungal alpha-sarcin in Escherichia coli. Gene. 1994 May 3;142(1):147–151. doi: 10.1016/0378-1119(94)90370-0. [DOI] [PubMed] [Google Scholar]
  7. Mancheño J. M., Gasset M., Lacadena J., Martínez del Pozo A., Oñaderra M., Gavilanes J. G. Predictive study of the conformation of the cytotoxic protein alpha-sarcin: a structural model to explain alpha-sarcin-membrane interaction. J Theor Biol. 1995 Feb 7;172(3):259–267. doi: 10.1006/jtbi.1995.0022. [DOI] [PubMed] [Google Scholar]
  8. Martinez S. E., Smith J. L. Crystallization and preliminary characterization of mitogillin, a ribosomal ribonuclease from Aspergillus restrictus. J Mol Biol. 1991 Apr 5;218(3):489–492. doi: 10.1016/0022-2836(91)90692-y. [DOI] [PubMed] [Google Scholar]
  9. Martínez del Pozo A., Gasset M., Oñaderra M., Gavilanes J. G. Conformational study of the antitumor protein alpha-sarcin. Biochim Biophys Acta. 1988 Apr 14;953(3):280–288. doi: 10.1016/0167-4838(88)90036-2. [DOI] [PubMed] [Google Scholar]
  10. Sacco G., Drickamer K., Wool I. G. The primary structure of the cytotoxin alpha-sarcin. J Biol Chem. 1983 May 10;258(9):5811–5818. [PubMed] [Google Scholar]
  11. Wishart D. S., Sykes B. D., Richards F. M. Relationship between nuclear magnetic resonance chemical shift and protein secondary structure. J Mol Biol. 1991 Nov 20;222(2):311–333. doi: 10.1016/0022-2836(91)90214-q. [DOI] [PubMed] [Google Scholar]
  12. Yamasaki K., Muto Y., Ito Y., Wälchli M., Miyazawa T., Nishimura S., Yokoyama S. A 1H-15N NMR study of human c-Ha-ras protein: biosynthetic incorporation of 15N-labeled amino acids. J Biomol NMR. 1992 Jan;2(1):71–82. doi: 10.1007/BF02192801. [DOI] [PubMed] [Google Scholar]

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