The Many Faces of a Phasin: Structural Analysis and in vitro chaperone activity of PhaP from Azotobacter sp. FA8

MEZZINA, M. P; DI PAOLA, M.; WETZLER, D. E; PETTINARI, M. J

Boston

Congreso; 114th General Meeting of the American Society for Microbiology; 2014

American Society for Microbiology (ASM)

Phasins are proteins associated with the surface of granules of poly(3-hydroxyalkanoates) (PHAs), biodegradable polymers accumulated by many bacteria as reserve molecules. Apart from their well known effects on the number and size of intracellular PHA granules, many phasins have additional roles, such as regulatory functions, interacting both with nucleic acids and other proteins. Phasins enhance growth and polymer production in natural and recombinant PHA producers, but the mechanisms have only been partially investigated. Expression of PhaP from Azotobacter sp. FA-8 in E. coli not only increased growth and polymer production in PHA producing strains, but this phasin had also an unexpected protective effect in non - PHA producing E. coli, increasing both growth and 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. A structural analysis of this protein was performed using purified his-tagged PhaP to characterize it and shed light on its possible mechanism of action. Its secondary structure was experimentally assessed by circular dichroism (CD), revealing that PhaP contains a great proportion of α helix, in accordance with in silico predictions. Treatment with 2, 2, 2- trifluoroethanol (TFE) induced a change in α-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, resulting in an increase in the proportion of α-helix. 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.