Distinguishing chromophore structures of photocycle intermediates of the photoreceptor PYP by transient fluorescence and energy transfer.
The cinnamoyl chromophore is the light-activated switch of the photoreceptor photoactive yellow protein (PYP) and isomerizes during the functional cycle. The fluorescence of W119, the only tryptophan of PYP, is quenched by energy transfer to the chromophore. This depends on the chromophore's transition dipole moment orientation and spectrum, both of which change during the photocycle. The transient fluorescence of W119 thus serves as a sensitive kinetic monitor of the chromophore's structure and orientation and was used for the first time to investigate the photocycle kinetics. From these data and measurements of the ps-fluorescence decay with background illumination (470 nm) we determined the fluorescence lifetimes of W119 in the I(1) and I (1') intermediates. Two coexisting distinct chromophore structures were proposed for the I(1) photointermediate from time-resolved X-ray diffraction ( Ihee, H., et al. Proc. Natl. Acad. Sci. U.S.A., 2005, 102, 7145 ): one with two hydrogen bonds to E46 and Y42, and a second with only one H-bond to Y42 and a different orientation. Only for the first of these is the calculated fluorescence lifetime of 0.22 ns in good agreement with the observed one of 0.26 ns. The second structure has a predicted lifetime of 0.71 ns. Thus, we conclude that in solution only the first I(1) structure occurs. The high resolution structure of the I(1') intermediate, the decay product of I(1) at alkaline pH, is still unknown. We predict from the observed lifetime of 1.3 ns that the chromophore structure of I(1') is quite similar to that of the I(2) intermediate, and I(1') should thus be considered as the alkaline (deprotonated) form of I(2).
Hoersch
Daniel
D
Biophysics Group, Department of Physics, Freie Universitat Berlin, Arnimallee 14, 14195 Berlin, Germany.
Otto
Harald
H
Cusanovich
Michael A
MA
Heyn
Maarten P
MP
D001426:Bacterial Proteins / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004735:Energy Transfer; D005453:Fluorescence; D020607:Halorhodospira halophila / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D020283:Photoreceptors, Microbial / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; C050162:photoactive yellow protein, Bacteria; D014364:Tryptophan