Direct observation of polyhydroxyalkanoate granule-associated-proteins on native granules and on poly(3-hydroxybutyrate) single crystals by atomic force microscopy

摘要

Polyhydroxyalkanoate (PHA) granules isolated from Comamonas acidovorans revealed the presence of globular particles on the granule surface when imaged using atomic force microscopy (AFM). The globular particles appeared to form a monolayer on the granule surface. Repeated washing with pure water resulted in granules with smooth surface without any globular particles. Purified granule-associated-proteins and PHA single crystals were used to construct a model representing native granule surface for AFM imaging. For this purpose, PhaR and PhaP proteins, purified from Paracoccus denitrificans were adsorbed onto poly(3-hydro-xybutyrate) [P(3HB)] single crystals. PhaR is a 22 kDa protein involved in the regulation of PhaP (phasin) protein (16 kDa). Both purified proteins were morphologically different but adsorbed uniformly on the single crystal surface forming a monolayer. Immunogold-labeling was used to further confirm the adsorption and identity of PhaR protein on P(3HB) single crystals. The study shows for the first time, that proteins associated with PHA granules can be imaged directly and characterised using AFM. In addition, AFM images obtained in this study provide direct evidence for the binding of PhaR and PhaP proteins to the hydro-phobic PHA surface.