Investigations on the binding of the major phasin PhaP1 from Ralstonia eutropha H16 to poly(3-hydroxybutyrate) granules

The surface of polyhydroxybutyrate (PHB) storage granules in bacteria is mainly covered by proteins referred to as phasins. The layer of phasins stabilizes the granules and prevents coalescence of separated granules in the cytoplasm and unspecific binding of other proteins to the hydrophobic surfaces of the granules. Phasin PhaP1 Reu is the major surface protein of PHB granules in Ralstonia eutropha H16 which occurs in addition to three homologues (PhaP2, PhaP3 and PhaP4) with binding capacity to PHB granules but at minor amounts. All four phasins lack a highly conserved domain, but share homologous hydrophobic regions. To identify the region of PhaP1 Reu , which is responsible for the binding of the protein to the granules, N-terminal and C-terminal fusions of EGFP with PhaP1 Reu or various regions of PhaP1 Reu were generated by recombinant techniques. The fusions were localized in the cells of various recombinant strains by fluorescence microscopy, and their presence in different subcellular protein fractions was determined by immunodetection of blotted proteins. The fusions were also analyzed for their in vitro binding capacity to isolated PHB granules. The results of these studies indicated that, unlike in the phasin of Rhodococcus ruber, no discrete binding motif exists but that instead several regions of PhaP1 Reu are contributing to the binding of this protein to the surface of the granules. The conclusions are supported by small angle X-ray scattering (SAXS) analysis of purified PhaP1 Reu , which revealed PhaP1 Reu as a planar, triangular protein occurring as trimer. This study provides new insights into the structure of the PHB granule surface and will also have impacts on potential biotechnological applications of phasin-fusion proteins and PHB granules in nanobiotechnology.