IIB   20738
INSTITUTO DE INVESTIGACIONES BIOLOGICAS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Structural features of a polypeptide related to ubiquitin from Natrialba magadii
Autor/es:
ORDÓÑEZ, M.V.; GUILLEN CASAS, J.; VILLALAÍN, J.; NERCESSIAN, D.; CONDE, R.D.
Lugar:
Carlos Paz, Córdoba
Reunión:
Congreso; VI Congreso Argentino de Microbiología General SAMIGE; 2009
Institución organizadora:
Sociedad Argentina de Microbiología General
Resumen:
<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:ES; mso-fareast-language:ES;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Ubiquitin is a highly conserved protein in eukaryotes. Also, many ubiquitin-like proteins (Ubls) and ubiquitin like domain containing proteins (UbDs) have been described in both prokaryotes and eukaryotes. Usually these proteins share high structural homology but little sequence identity with ubiquitin. Also, because are part of homologous conjugation pathways as that of ubiquitin, prokaryotic Ubls like ThiS and MoaD are functionally related to ubiquitin. All Ubls and UbDs proteins have a β-grasp fold comprised mainly by a β-strand with 5 antiparallel β-sheets with one α-helix region. We are involved in studying the presence of Ubls and UbDs proteins in halophilic archaea and their evolutionary relationship with ubiquitin. Previously, we isolated by PCR a 400bps (p400) DNA fragment from Natrialba magadii genome whose amino acidic sequence presents a 3D structure similar to ubiquitin by bioinformatic tests. Here we describe the biochemical-structural characterization of this polypeptide after expressing the construction pET24b::p400 in E. coli cells rosetta strain. As previously shown, the recombinant P400 polypeptide (P400r) appears in inclusion bodies from where can be partially purified by Ni-NTA affinity chromatography. In this work we show that it can be better purified after SDS-PAGE separation of total E. coli rosetta proteins followed by Zinc-Imidazol reverse staining. P400r was eluted from the gel slice with a Tris-glycine buffer with 90% yield. After renaturalization in presence of a buffer containing 1.5M KCl, the purified protein was recognized by the anti-ubiquitin antibody. Then, the secondary structure of P400r was analyzed by Fourier Tranformed Infrared Spectroscopy. The resulting amide I spectrum showed that its conformation is mainly composed by β-sheets with a low level of α-helix. This preliminary result is in good agreement with both the predicted 3D model obtained and that expected for proteins with ubiquitin like fold. Finally, the recent publication of the complete genome sequence of Nab. magadii allowed us to gain information about the complete protein to which P400 belongs. The pBlast with P400 sequence against this genome revealed that it suits to a major protein of 262 amino acids. P400 is near the N-t region that also has a predicted signal peptide site at the N-teminus that would direct the protein to its secretion. This signal peptide is similar to that described for lipoproteins from bacteria, which usually are anchored to the cell membrane in the periplasm. Given these results we hypothesize that P400 is an ubiquitin like domain of a major protein that is secreted and anchored to the membrane in Nab. magadii cells. The physiological role of this protein is still to be determined. Supported by UNMdP and CONICET.