Structural and in vitro chaperone assays help unravel the multiple roles of PhaP from Azotobacter sp FA-8

MEZZINA, MARIELA; WETZLER, DIANA; DI PAOLA, MATIAS; PETTINARI M. JULIA

Mar del Plata

Congreso; IX Congreso de la Sociedad Argentina de Microbiología General (SAMIGE 2013); 2013

Sociedad Argentina de Microbiología General (SAMIGE)

Phasins are proteins that are normally associated with granules of poly(3-hydroxybutyrate) (PHB), a biodegradable polymer accumulated by many bacteria as a reserve molecule. These proteins play an important role in the formation of the granule and are involved in regulatory functions. Phasins have been shown to enhance growth and polymer production in natural and recombinant PHB producers. The production of PHB causes a stress in recombinant Escherichia coli, revealed by an increase in the concentration of several heat stress proteins. PhaP from Azotobacter sp. FA-8 enhances growth and polymer in PHB producing E.coli, and it was also reported to have an unexpected protective effect in a non PHB producing E. coli strain, resulting in increased growth and higher resistance to stress conditions, such as heat and oxidation. These observations suggest that PhaP could act as a chaperone, opening the door to novel biotechnological applications for this protein, for example, the production of recombinant proteins and other heterologous products in E. coli. In order to characterize this protein and shed light on its possible mechanism of action, a structural analysis was performed. A DNA fragment containing the phaP coding sequence was cloned in an expression vector that allows its overexpression by addition of IPTG and that introduces a six-histidine tag, obtaining a 22.05 KDa fusion protein. This protein was purified by affinity chromatography using a nickel column. SDS-PAGE of the purified protein showed two major bands that were analized by MALDI TOF TOF, to verify that the aminoacid sequence of both corresponded to PhaP. In silico secondary structure prediction indicated that most of the protein was expected to adopt an  helix conformation. The secondary structure of the protein was experimentally assessed by circular dichroism (CD). The analysis of the spectra revealed that PhaP contains a great proportion of  helix. Treatment with 2,2,2-trifluoroethanol (TFE) induced a change in  helix composition, indicating the presence of disordered regions, suggesting that PhaP could have a flexible nature. As PhaP in its natural host binds to the surface of lipid (PHB) granules, changes in secondary structure were analyzed in the presence of sodium oleate, as a hydrophobic mimic of PHB. Chemical denaturation and thermal stability experiments were performed to further characterize the protein and to test its resistance to these conditions, given its role in protection against heat shock in in vivo experiments. Chaperone activity was studied in vitro using citrate synthase (CS) as a model substrate in thermal denaturation experiments, revealing a clear protective effect of PhaP. These results will help us elaborate hypothesis about possible mechanisms of action of PhaP and its interaction with other proteins that can be further tested in in vivo experiments.