ABSTRACT
We report the isolation, functional reconstitution and photophysical characterisation of Rhodobacter sphaeroides photoactive yellow protein (PYP), of which the gene was recently cloned. Reconstitution of the his-tagged purified apo-protein with 4-hydroxy-cinnamic acid yields the characteristic blue absorbance at 446 nm, but surprisingly also an absorbance peak at 360 nm. This additional peak is not caused by binding of a second chromophore, as confirmed with mass spectroscopy. Moreover, reconstitution with the 'locked' analogue 7-hydroxy-coumarin-3-carboxylic acid yields only a single absorbance peak at 441 nm. The 446 nm and 360 nm species are part of a temperature- and pH-dependent equilibrium. Photoactivation of the protein leads to formation of a blue-shifted intermediate as in other PYPs, with a 100-fold increased groundstate recovery rate (k(pB-->pG)=500 s(-1)) compared to E-PYP.
Subject(s)
Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Photoreceptors, Microbial , Rhodobacter sphaeroides/chemistry , Apoproteins/chemistry , Apoproteins/genetics , Apoproteins/isolation & purification , Apoproteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Coumaric Acids/metabolism , Hydrogen-Ion Concentration , Kinetics , Mass Spectrometry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Rhodobacter sphaeroides/genetics , Spectrum Analysis , TemperatureABSTRACT
The photoactive yellow protein (pyp) gene has been isolated from Rhodobacter sphaeroides by probing with a homologous PCR-product. A sequence analysis shows that this pyp gene encodes a 124 AA protein with 48% identity to the three known PYPs. Downstream from pyp, a number of adjacent open reading frames were identified, including a gene encoding a CoA-ligase homologue (pCL). This latter protein is proposed to be involved in PYP chromophore activation, required for attachment to the apoprotein. We have demonstrated the presence of the chromophoric group, previously identified in PYP from Ectothiorhodospira halophila as trans 4-hydroxy cinnamic acid, in phototrophically cultured R. sphaeroides cells by capillary zone electrophoresis. The basic structure of the chromophore binding pocket in PYP has been conserved, as shown by a 3D model of R. sphaeroides PYP, constructed by homology-based molecular modelling. In addition, this model shows that R. sphaeroides PYP contains a characteristic, positively charged patch.
