Chromophore structure in the photocycle of the cyanobacterial phytochrome Cph1

Jasper J. Van Thor, Muhammad Mukram Mohamed Mackeen, Ilya Kuprov, Raymond A. Dwek, Mark R. Wormald

Research output: Contribution to journalArticle

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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, C 15 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-C 6-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.

Original languageEnglish
Pages (from-to)1811-1822
Number of pages12
JournalBiophysical Journal
Volume91
Issue number5
DOIs
Publication statusPublished - 2006
Externally publishedYes

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Phytochrome
Magnetic Resonance Spectroscopy
Hydrogen
Spectrum Analysis
Deinococcus
Biliverdine
Synechocystis
Isotopes
Proteins
Carbon
Hot Temperature
X-Rays
Light

ASJC Scopus subject areas

  • Biophysics

Cite this

Chromophore structure in the photocycle of the cyanobacterial phytochrome Cph1. / Van Thor, Jasper J.; Mohamed Mackeen, Muhammad Mukram; Kuprov, Ilya; Dwek, Raymond A.; Wormald, Mark R.

In: Biophysical Journal, Vol. 91, No. 5, 2006, p. 1811-1822.

Research output: Contribution to journalArticle

Van Thor, Jasper J. ; Mohamed Mackeen, Muhammad Mukram ; Kuprov, Ilya ; Dwek, Raymond A. ; Wormald, Mark R. / Chromophore structure in the photocycle of the cyanobacterial phytochrome Cph1. In: Biophysical Journal. 2006 ; Vol. 91, No. 5. pp. 1811-1822.
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AU - Mohamed Mackeen, Muhammad Mukram

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AU - Dwek, Raymond A.

AU - Wormald, Mark R.

PY - 2006

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N2 - 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, C 15 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-C 6-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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