CONICET | Buscador de Institutos y Recursos Humanos

Phasins are proteins associated with the surface of granules of poly(3-hydroxyalkanoates) (PHAs), biodegradable polymers accumulated by many bacteria as reserve molecules, affecting granule number and size. Many phasins have additional roles, such as regulatory functions, interacting both with nucleic acids and other proteins. Phasins have been shown to enhance growth and polymer production in natural and recombinant PHA producers, but the mechanisms have only been partially investigated. Apart from enhancing growth and polymer in PHA producing E.coli, PhaP from Azotobacter sp. FA-8 also has an unexpected protective effect in non PHA producing E. coli, resulting in increased growth and higher resistance to stress conditions, such as heat and oxidation. These observations suggested that PhaP could have a chaperone-like effect, and prompted a deeper analysis. In order to characterize this protein and shed light on its possible mechanism of action, a structural analysis was performed using purified his-tagged PhaP. Its secondary structure was experimentally assessed by circular dichroism (CD), revealing that PhaP contains a great proportion of a helix, in accordance with in silico predictions. Treatment with 2, 2, 2- trifluoroethanol (TFE) induced a change in a-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 (PHA) granules, changes in its secondary structure were analyzed in the presence of sodium oleate, as a hydrophobic mimic of PHA, showing that the protein increases its a-helix proportion.Chaperone activity was studied in vitro using citrate synthase (CS) as a model substrate in thermal and chemical denaturation experiments. Addition of PhaP significantly increased the resistance to thermal denaturation of CS and the refolding rate of CS unfolded by treatment with guanidine hydrochloride, showing a clear protective effect of PhaP in vitro. These results will help us elaborate hypothesis about possible mechanisms of action of PhaP, and open the door to novel biotechnological applications for this protein, for example, in the production of recombinant proteins and other heterologous products in E. coli.