Structural studies of Azotobacter sp FA-8 protein PHAP

MEZZINA MARIELA; DI PAOLA MATIAS; WESLER D; GODOY MANUEL; LAURA J. RAIGER IUSTMAN; PETTINARI JULIA

Mendoza

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB).; 2012

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB).

Phasins are proteins that are normally associated with granules of poly(3-hydroxybutyrate) (PHB), a biodegradable polymer accumulated 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 resistance to stress conditions. In order to further characterize this phasin and shed light on its possible mechanism of action, a structural analysis was performed. The gene phaP was cloned in an expression vector that introduces a six histidine tag and was purified by affinity chromatography using a nickel column. SDS-PAGE in non reductive and reductive conditions revealed that the protein forms dimers. A MALDI TOF TOF analysis permitted to verify that the bands observed correspond to PhaP in its dimeric and monomeric form and it also showed that PhaP 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 dichroism spectrum was performed. The analysis of the spectra revealed that PhaP is composed mainly by  hélix. These results will help us elaborate hypothesis about possible mechanisms of action of PhaP and its interaction with other proteins that can be later tested experimentally