title: Characterization of photocycle intermediates in crystalline photoactive yellow protein.
abstract: The photocycle in photoactive yellow protein (PYP) crystals was studied by single-crystal absorption spectroscopy with experimental setups for low-temperature and time-resolved measurements. Thin and flat PYP crystals, suitable for light absorption studies, were obtained using special crystallization conditions. Illumination of PYP crystals at 100 K led to the formation of a photostationary state, which includes at least one hypsochromic and one bathochromic photoproduct that resemble PYP(H) and PYP(B), respectively. The effect of temperature, light color and light pulse duration on the occupancy of these low-temperature photoproducts was determined and appeared similar to that observed in solution. At room temperature a blueshifted photocycle intermediate was identified that corresponds to the blueshifted state of PYP (pB). Kinetic studies show that the decay of this blueshifted intermediate is biphasic at -12 degrees C and 15-fold faster than that observed in solution at room temperature. These altered pB decay kinetics confirm a model that holds that the photocycle in crystals takes place in a shortcut version. In this version the key structural events of the photocycle, such as photoisomerization and reversible protonation of the chromophore, take place, but large conformational changes in the surrounding protein are limited by constraints imposed by the crystal lattice.
authors.affiliation: Laboratory for Microbiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands. rkort@science.uva.nl
authors.forename: Remco
authors.initials: R
authors.lastname: Kort
authors.forename: Raimond B
authors.initials: RB
authors.lastname: Ravelli
authors.forename: Friedrich
authors.initials: F
authors.lastname: Schotte
authors.forename: Dominique
authors.initials: D
authors.lastname: Bourgeois
authors.forename: Wim
authors.initials: W
authors.lastname: Crielaard
authors.forename: Klaas J
authors.initials: KJ
authors.lastname: Hellingwerf
authors.forename: Michael
authors.initials: M
authors.lastname: Wulff
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D003460:Crystallization; D020607:Halorhodospira halophila / Q000737:chemistry; D007700:Kinetics; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D013053:Spectrophotometry; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; C050162:photoactive yellow protein, Bacteria