Dataset Information

Dynamic inhomogeneity in the photodynamics of cyanobacterial phytochrome Cph1.

ABSTRACT: Phytochromes are widespread red/far-red photosensory proteins well known as critical regulators of photomorphogenesis in plants. It is often assumed that natural selection would have optimized the light sensing efficiency of phytochromes to minimize nonproductive photochemical deexcitation pathways. Surprisingly, the quantum efficiency for the forward Pr-to-Pfr photoconversion of phytochromes seldom exceeds 15%, a value very much lower than that of animal rhodopsins. Exploiting ultrafast excitation wavelength- and temperature-dependent transient absorption spectroscopy, we resolve multiple pathways within the ultrafast photodynamics of the N-terminal PAS-GAF-PHY photosensory core module of cyanobacterial phytochrome Cph1 (termed Cph1?) that are primarily responsible for the overall low quantum efficiency. This inhomogeneity primarily reflects a long-lived fluorescent subpopulation that exists in equilibrium with a spectrally distinct, photoactive subpopulation. The fluorescent subpopulation is favored at elevated temperatures, resulting in anomalous excited-state dynamics (slower kinetics at higher temperatures). The spectral and kinetic behavior of the fluorescent subpopulation strongly resembles that of the photochemically compromised and highly fluorescent Y176H variant of Cph1?. We present an integrated, heterogeneous model for Cph1? that is based on the observed transient and static spectroscopic signals. Understanding the molecular basis for this dynamic inhomogeneity holds potential for rational design of efficient phytochrome-based fluorescent and photoswitchable probes.

SUBMITTER: Kim PW

PROVIDER: S-EPMC4018079 | biostudies-other | 2014 May

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Dynamic inhomogeneity in the photodynamics of cyanobacterial phytochrome Cph1.

Kim Peter W PW Rockwell Nathan C NC Martin Shelley S SS Lagarias J Clark JC Larsen Delmar S DS

Biochemistry 20140421 17

Phytochromes are widespread red/far-red photosensory proteins well known as critical regulators of photomorphogenesis in plants. It is often assumed that natural selection would have optimized the light sensing efficiency of phytochromes to minimize nonproductive photochemical deexcitation pathways. Surprisingly, the quantum efficiency for the forward Pr-to-Pfr photoconversion of phytochromes seldom exceeds 15%, a value very much lower than that of animal rhodopsins. Exploiting ultrafast excitat ...[more]

PMID: 24742290

Similar Datasets

Project description: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.

Project description:Phytochromes are dimeric biliprotein photoreceptors exhibiting characteristic red/far-red photocycles. Full-length cyanobacterial phytochrome Cph1 from Synechocystis 6803 is soluble initially but tends to aggregate in a concentration-dependent manner, hampering attempts to solve the structure using NMR and crystallization methods. Otherwise, the Cph1 sensory module (Cph1Δ2), photochemically indistinguishable from the native protein and used extensively in structural and other studies, can be purified to homogeneity in >10 mg amounts at mM concentrations quite easily. Bulk precipitation of full-length Cph1 by ammonium sulfate (AmS) was expected to allow us to produce samples for solid-state magic-angle spinning (MAS) NMR from dilute solutions before significant aggregation began. It was not clear, however, what effects the process of partial dehydration might have on the molecular structure. Here we test this by running solid-state MAS NMR experiments on AmS-precipitated Cph1Δ2 in its red-absorbing Pr state carrying uniformly (13)C/(15)N-labeled phycocyanobilin (PCB) chromophore. 2D (13)C-(13)C correlation experiments allowed a complete assignment of (13)C responses of the chromophore. Upon precipitation, (13)C chemical shifts for most of PCB carbons move upfield, in which we found major changes for C4 and C6 atoms associated with the A-ring positioning. Further, the broad spectral lines seen in the AmS (13)C spectrum reflect primarily the extensive inhomogeneous broadening presumably due to an increase in the distribution of conformational states in the protein, in which less free water is available to partake in the hydration shells. Our data suggest that the effect of dehydration process indeed leads to changes of electronic structure of the bilin chromophore and a decrease in its mobility within the binding pocket, but not restricted to the protein surface. The extent of the changes induced differs from the freezing process of the solution samples routinely used in previous MAS NMR and crystallographic studies. AmS precipitation might nevertheless provide useful protein structure/functional information for full-length Cph1 in cases where neither X-ray crystallography nor conventional NMR methods are available.

Dataset Information

Dynamic inhomogeneity in the photodynamics of cyanobacterial phytochrome Cph1.

Publications

Dynamic inhomogeneity in the photodynamics of cyanobacterial phytochrome Cph1.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets