Structural studies of Azotobacter sp FA-8 protein PhaP

MARIELA MEZZINA; MATIAS DIPAOLA; DIANA E. WETZLER; MANUEL GODOY; LAURA RIAIGER IUSTMAN; JULIA PETTINARI

Mendoza

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2012

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Phasins are proteins that are normally associated with granules of Poly(3.hidroxybutrate)(PHB), a biodegradable polymer accumulates by many bacteria. PhaP from Azotobacter sp. FA-8 enhances growth and polymer production in PHB producing E.coli, and it also showed protective effect in a non PHB producing E.coli strain, resulting in increased growth and higher resustance to stress conditions. In order to further characterize this phasing and shed light on its possible mechanism of action, a structural analysis was performed. The gene phaP was cloned in an expression vector that introduce a six histidine tag and was purified by affinity chromatography using nickel column. SDS-PAGE in non reductive and reductive conditions revealed that the proteins forms dimmers. A MALDI TOF TOF analysis permits to verify that the bands observed correspond to PhaP in its dimeric and monomeric form an it also showed tha PhaP homodimer is a homodimer, formed by monomers linked by disulphide bonds that involve the only cisteine present in the protein sequence. In order to study the secondary structure of the protein a circular dischoism spectrum was perrformes. The analysis of the spectra revealed that PhaP is composed mainly by helix. The results will help us to elaborate hypothesis about possible mechanisms of action of PhaP and its interaction with other proteins that can be later tested experimentally.