Secondary structure determination by FTIR of an archaeal ubiquitin-like polypeptide from Natrialba magadii

ORDOÑEZ, M. V.; GUILLEN, J.; NERCESSIAN, D.; VILLALAIN, J.; CONDE, R. D.

EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS

SPRINGER

Lugar: Berlin; Año: 2011 vol. 40 p. 1101 - 1107

0175-7571

The ubiquitin protein belongs to the beta-grasp fold family, characterized by four or five  beta-sheets with a single alpha-helical middle region. Ubiquitin-like proteins(Ubls) are structural homologues with low sequence iden-tity to ubiquitin and are widespread among both eukaryotes and prokaryotes. We previously demonstrated by bioinformatics that P400, a polypeptide from the haloalkaliphilic archaeon Natrialba magadii, has structural homology with both ubiquitin and Ubls. This work examines the secondary structure of P400 by Fourier transform infrared spectros-copy (FTIR). After expression in Escherichia coli, recombinant P400 (rP400) was separated by PAGE and eluted pure from zinc-imidazole reversely stained gels. The requirement of high salt concentration of this polypeptide to be folded was corroborated by intrinsic fluorescence spectrum. Our results show that fluorescence spectra of rP400 in 1.5 M KCl buffer shifts and decreases after ther-mal denaturation as well as after chemical treatment. rP400 was lyophilized and rehydrated in buffer containing 1.5 M KCl before both immunochemical and FTIR tests were per-formed. It was found that rP 400 reacts with anti-ubiquitin antibody after rehydration in the presence of high salt concentrations. On the other hand, like ubiquitin and Ubls, the amide I band for rP400 shows 10% more of its sequence to be involved in beta-sheet structures than in alpha-helix. These findings suggest that P400 is a structural homologue of the ubiquitin family proteins.