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Chromophore structure in the photocycle of the cyanobacterial phytochrome Cph1

Chromophore structure in the photocycle of the cyanobacterial phytochrome Cph1
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 C4 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 13C-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.
0006-3495
1811-1822
van Thor, Jasper J.
29ffae56-08f0-4856-843a-e4043f8c9334
Mackeen, Mukram
679fcca0-8ae5-49ce-830a-2403dd3f3c2a
Kuprov, Ilya
bb07f28a-5038-4524-8146-e3fc8344c065
Dwek, Raymond A.
d8d9d5f8-f2c0-414e-b6b0-df77a33f0da4
Wormald, Mark R.
0d227cb3-0852-4ba1-90e6-cba89de5e994
van Thor, Jasper J.
29ffae56-08f0-4856-843a-e4043f8c9334
Mackeen, Mukram
679fcca0-8ae5-49ce-830a-2403dd3f3c2a
Kuprov, Ilya
bb07f28a-5038-4524-8146-e3fc8344c065
Dwek, Raymond A.
d8d9d5f8-f2c0-414e-b6b0-df77a33f0da4
Wormald, Mark R.
0d227cb3-0852-4ba1-90e6-cba89de5e994

van Thor, Jasper J., Mackeen, Mukram, Kuprov, Ilya, Dwek, Raymond A. and Wormald, Mark R. (2006) Chromophore structure in the photocycle of the cyanobacterial phytochrome Cph1. Biophysical Journal, 91 (5), 1811-1822. (doi:10.1529/biophysj.106.084335).

Record type: Article

Abstract

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 C4 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 13C-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.

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Published date: 1 September 2006
Organisations: Computational Systems Chemistry

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Local EPrints ID: 347390
URI: http://eprints.soton.ac.uk/id/eprint/347390
ISSN: 0006-3495
PURE UUID: c9b3c2b9-97fb-4402-a488-707470ad2cec
ORCID for Ilya Kuprov: ORCID iD orcid.org/0000-0003-0430-2682

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Date deposited: 27 Feb 2013 12:12
Last modified: 15 Mar 2024 03:43

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Contributors

Author: Jasper J. van Thor
Author: Mukram Mackeen
Author: Ilya Kuprov ORCID iD
Author: Raymond A. Dwek
Author: Mark R. Wormald

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