title: Secondary structure determination by FTIR of an archaeal ubiquitin-like polypeptide from Natrialba magadii.
abstract: The ubiquitin protein belongs to the β-grasp fold family, characterized by four or five β-sheets with a single α-helical middle region. Ubiquitin-like proteins (Ubls) are structural homologues with low sequence identity 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 spectroscopy (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 thermal 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 performed. It was found that rP400 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 β-sheet structures than in α-helix. These findings suggest that P400 is a structural homologue of the ubiquitin family proteins.
authors.affiliation: Instituto de Investigaciones Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, CONICET, CC: 1245, 7600, Mar del Plata, Argentina. mvordone@mdp.edu.ar
authors.forename: M V
authors.initials: MV
authors.lastname: Ordóñez
authors.forename: J
authors.initials: J
authors.lastname: Guillén
authors.forename: D
authors.initials: D
authors.lastname: Nercessian
authors.forename: J
authors.initials: J
authors.lastname: Villalaín
authors.forename: R D
authors.initials: RD
authors.lastname: Conde
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry*; D006216:Halobacteriaceae; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry*; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D025801:Ubiquitin / Q000737:chemistry*
chemical_list: D019843:Archaeal Proteins; D010455:Peptides; D025801:Ubiquitin