Premium
Characterization of Photocycle Intermediates in Crystalline Photoactive Yellow Protein † ¶
Author(s) -
Kort Remco,
Ravelli Raimond B.,
Schotte Friedrich,
Bourgeois Dominique,
Crielaard Wim,
Hellingwerf Klaas J.,
Wulff Michael
Publication year - 2003
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1562/0031-8655(2003)0780131copiic2.0.co2
Subject(s) - hypsochromic shift , chromophore , bathochromic shift , photoisomerization , chemistry , protonation , photochemistry , intermediate state , crystallography , kinetics , spectroscopy , absorption (acoustics) , crystallization , crystal (programming language) , absorption spectroscopy , ultrafast laser spectroscopy , isomerization , materials science , optics , fluorescence , organic chemistry , catalysis , ion , philosophy , physics , theology , quantum mechanics , computer science , composite material , programming language
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°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.