title: Isolation, reconstitution and functional characterisation of the Rhodobacter sphaeroides photoactive yellow protein.
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.
authors: Laboratory for Microbiology, E.C. Slater Institute, BioCentrum, University of Amsterdam, Nieuwe Achtergracht 127, 1018 WS, Amsterdam, The Netherlands.
authors: A
authors: A
authors: Haker
authors: J
authors: J
authors: Hendriks
authors: T
authors: T
authors: Gensch
authors: K
authors: K
authors: Hellingwerf
authors: W
authors: W
authors: Crielaard
mesh_terms: D001059:Apoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D003373:Coumaric Acids / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013058:Mass Spectrometry; D020283:Photoreceptors, Microbial; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000737:chemistry* / Q000235:genetics; D013057:Spectrum Analysis; D013696:Temperature
chemical_list: D001059:Apoproteins; D001426:Bacterial Proteins; D003373:Coumaric Acids; D020283:Photoreceptors, Microbial; D011993:Recombinant Fusion Proteins; C050162:photoactive yellow protein, Bacteria