Chromophore structure in the photocycle of the cyanobacterial phytochrome Cph1.
The chromophore conformations of the red and far red light induced product states "Pfr" and "Pr" of the N-terminal photoreceptor domain Cph1-N515 from Synechocystis 6803 have been investigated by NMR spectroscopy, using specific 13C isotope substitutions in the chromophore. 13C-NMR spectroscopy in the Pfr and Pr states indicated reversible chemical shift differences predominantly of the C(4) carbon in ring A of the phycocyanobilin chromophore, in contrast to differences of C15 and C5, which were much less pronounced. Ab initio calculations of the isotropic shielding and optical transition energies identify a region for C4-C5-C6-N2 dihedral angle changes where deshielding of C4 is correlated with red-shifted absorption. These could occur during thermal reactions on microsecond and millisecond timescales after excitation of Pr which are associated with red-shifted absorption. A reaction pathway involving a hula-twist at C5 could satisfy the observed NMR and visible absorption changes. Alternatively, C15 Z-E photoisomerization, although expected to lead to a small change of the chemical shift of C15, in addition to changes of the C4-C5-C6-N2 dihedral angle could be consistent with visible absorption changes and the chemical shift difference at C4. NMR spectroscopy of a 13C-labeled chromopeptide provided indication for broadening due to conformational exchange reactions in the intact photoreceptor domain, which is more pronounced for the C- and D-rings of the chromophore. This broadening was also evident in the F2 hydrogen dimension from heteronuclear 1H-13C HSQC spectroscopy, which did not detect resonances for the 13C5-H, 13C10-H, and 13C15-H hydrogen atoms whereas strong signals were detected for the (13)C-labeled chromopeptide. The most pronounced 13C-chemical shift difference between chromopeptide and intact receptor domain was that of the 13C4-resonance, which could be consistent with an increased conformational energy of the C4-C5-C6-N2 dihedral angle in the intact protein in the Pr state. Nuclear Overhauser effect spectroscopy experiments of the 13C-labeled chromopeptide, where chromophore-protein interactions are expected to be reduced, were consistent with a ZZZssa conformation, which has also been found for the biliverdin chromophore in the x-ray structure of a fragment of Deinococcus radiodurans bacteriophytochrome in the Pr form.
van Thor
Jasper J
JJ
Laboratory of Molecular Biophysics, University of Oxford, Oxford OX1 3QU, United Kingdom. jasper@biop.ox.ac.uk
Mackeen
Mukram
M
Kuprov
Ilya
I
Dwek
Raymond A
RA
Wormald
Mark R
MR
D001426:Bacterial Proteins / Q000737:chemistry* / Q000528:radiation effects*; D000458:Cyanobacteria / Q000737:chemistry* / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D008027:Light; D018462:Photobiology / Q000379:methods; D010777:Photochemistry / Q000379:methods; D020283:Photoreceptors, Microbial; D010834:Phytochrome / Q000737:chemistry* / Q000528:radiation effects*; D011487:Protein Conformation / Q000528:radiation effects; D011494:Protein Kinases / Q000737:chemistry* / Q000528:radiation effects*; D011829:Radiation Dosage
D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; D010834:Phytochrome; D011494:Protein Kinases; C413623:Cph1 phytochrome protein, bacteria