Homogeneity of phytochrome Cph1 vibronic absorption revealed by resonance Raman intensity analysis.
Ontology highlight
ABSTRACT:
SUBMITTER: Spillane KM
PROVIDER: S-EPMC2880177 | biostudies-literature | 2009 Oct
REPOSITORIES: biostudies-literature
Publications
Homogeneity of phytochrome Cph1 vibronic absorption revealed by resonance Raman intensity analysis.
Spillane Katelyn M KM Dasgupta Jyotishman J Lagarias J Clark JC Mathies Richard A RA
Journal of the American Chemical Society 20091001 39
Phytochromes are an important class of red/far-red responsive photoreceptors that act as light-activated biological switches, ultimately driving growth and development in plants, bacteria, and fungi. The composition of the red-absorbing ground-state has been widely debated due to the presence of a shoulder feature on the blue edge of electronic absorption spectra, which many have attributed to the presence of multiple ground-state conformers. Here we use resonance Raman intensity analysis to cal ...[more]
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.
| S-EPMC1544288 | biostudies-literature
Project description: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.
| S-EPMC4018079 | biostudies-other
Project description:Phytochrome proteins utilize ultrafast photoisomerization of a linear tetrapyrrole chromophore to detect the ratio of red to far-red light. Femtosecond photodynamics in the PAS-GAF-PHY photosensory core of the Cph1 phytochrome from Synechocystis sp. PCC6803 (Cph1?) were resolved with a dual-excitation-wavelength-interleaved pump-probe (DEWI) approach with two excitation wavelengths (600 and 660 nm) at three pH values (6.5, 8.0, and 9.0). Observed spectral and kinetic heterogeneity in the excited-state dynamics were described with a self-consistent model comprised of three spectrally distinct populations with different protonation states (Pr-I, Pr-II, and Pr-III), each composed of multiple kinetically distinct subpopulations. Apparent partitioning among these populations is dictated by pH, temperature, and excitation wavelength. Our studies provide insight into photocycle initiation dynamics at physiological temperatures, implicate the low-pH/low-temperature Pr-I state as the photoactive state in vitro, and implicate an internal hydrogen-bonding network in regulating the photochemical quantum yield.
| S-EPMC6247788 | biostudies-literature
Project description:Femtosecond photodynamics of the Pfr form of the red/far-red phytochrome N-terminal PAS-GAF-PHY photosensory core module of the cyanobacterial phytochrome Cph1 (termed Cph1Δ) from Synechocystis were resolved with visible broadband transient absorption spectroscopy. Multiphasic generation dynamics via global target analysis revealed parallel evolution of two pathways with distinct excited- and ground-state kinetics. These measurements resolved two subpopulations: a majority subpopulation with fast excited-state decay and slower ground-state dynamics, corresponding to previous descriptions of Pfr dynamics, and a minority subpopulation with slower excited-state decay and faster ground-state primary dynamics. Both excited-state subpopulations generated the isomerized, red-shifted Lumi-Ff photoproduct (715 nm); subsequent ground-state evolution to a blue-shifted Meta-Fr population (635 nm) proceeded on 3 ps and 1.5 ns time scales for the two subpopulations. Meta-Fr was spectrally similar to a recently described photoinactive fluorescent subpopulation of Pr ((Fluor)Pr). Thus, the reverse Pfr to Pr photoconversion of Cph1Δ involves minor structural deformation of Meta-Fr to generate the fluorescent, photochemically refractory form of Pr, with slower subsequent equilibration with the photoactive Pr subpopulation ((Photo)Pr).
| S-EPMC4184438 | biostudies-literature
Project description:Photochemical interconversion between the red-absorbing (P(r)) and the far-red-absorbing (P(fr)) forms of the photosensory protein phytochrome initiates signal transduction in bacteria and higher plants. The P(r)-to-P(fr) transition commences with a rapid Z-to-E photoisomerization at the C(15)=C(16) methine bridge of the bilin prosthetic group. Here, we use femtosecond stimulated Raman spectroscopy to probe the structural changes of the phycocyanobilin chromophore within phytochrome Cph1 on the ultrafast time scale. The enhanced intensity of the C(15)-H hydrogen out-of-plane (HOOP) mode, together with the appearance of red-shifted C=C stretch and N-H in-plane rocking modes within 500 fs, reveal that initial distortion of the C(15)=C(16) bond occurs in the electronically excited I* intermediate. From I*, 85% of the excited population relaxes back to P(r) in 3 ps, whereas the rest goes on to the Lumi-R photoproduct consistent with the 15% photochemical quantum yield. The C(15)-H HOOP and skeletal modes evolve to a Lumi-R-like pattern after 3 ps, thereby indicating that the C(15)=C(16) Z-to-E isomerization occurs on the excited-state surface.
| S-EPMC2644115 | biostudies-literature
Project description:The ultrafast mechanisms underlying the initial photoisomerization (Pr → Lumi-R) in the forward reaction of the cyanobacterial photoreceptor Cph1 were explored with multipulse pump-dump-probe transient spectroscopy. A recently postulated multi-population model was used to fit the transient pump-dump-probe and dump-induced depletion signals. We observed dump-induced depletion of the Lumi-R photoproduct, demonstrating that photoisomerization occurs via evolution on both the excited- and ground-state electronic surfaces. Excited-state equilibrium was not observed, as shown via the absence of a dump-induced excited-state "Le Châtelier redistribution" of excited-state populations. The importance of incorporating the inhomogeneous dynamics of Cph1 in interpreting measured transient data is discussed.
| S-EPMC3798021 | biostudies-literature
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.
| S-EPMC4516977 | biostudies-literature
Project description:Ultrafast reactions activated by light absorption are governed by multidimensional excited-state (ES) potential energy surfaces (PESs), which describe how the molecular potential varies with the nuclear coordinates. ES PESs ad-hoc displaced with respect to the ground state can drive subtle structural rearrangements, accompanying molecular biological activity and regulating physical/chemical properties. Such displacements are encoded in the Franck-Condon overlap integrals, which in turn determine the resonant Raman response. Conventional spectroscopic approaches only access their absolute value, and hence cannot determine the sense of ES displacements. Here, we introduce a two-color broadband impulsive Raman experimental scheme, to directly measure complex Raman excitation profiles along desired normal modes. The key to achieve this task is in the signal linear dependence on the Frank-Condon overlaps, brought about by non-degenerate resonant probe and off-resonant pump pulses, which ultimately enables time-domain sensitivity to the phase of the stimulated vibrational coherences. Our results provide the tool to determine the magnitude and the sensed direction of ES displacements, unambiguously relating them to the ground state eigenvectors reference frame.
| S-EPMC9755279 | biostudies-literature
Project description:Phytochromes are dimeric photoreceptors that regulate a range of responses in plants and microorganisms through interconversion of red light-absorbing (Pr) and far-red light-absorbing (Pfr) states. Photoconversion between these states is initiated by light-driven isomerization of a bilin cofactor, which triggers protein structural change. The extent of this change, and how light-driven structural changes in the N-terminal photosensory region are transmitted to the C-terminal regulatory domain to initiate the signalling cascade, is unknown. We have used pulsed electron-electron double resonance (PELDOR) spectroscopy to identify multiple structural transitions in a phytochrome from Synechocystis sp. PCC6803 (Cph1) by measuring distances between nitroxide labels introduced into the protein. We show that monomers in the Cph1 dimer are aligned in a parallel 'head-to-head' arrangement and that photoconversion between the Pr and Pfr forms involves conformational change in both the N- and C-terminal domains of the protein. Cryo-trapping and kinetic measurements were used to probe the extent and temporal properties of protein motions for individual steps during photoconversion of Cph1. Formation of the primary photoproduct Lumi-R is not affected by changes in solvent viscosity and dielectric constant. Lumi-R formation occurs at cryogenic temperatures, consistent with their being no major structural reorganization of Cph1 during primary photoproduct formation. All remaining steps in the formation of the Pfr state are affected by solvent viscosity and dielectric constant and occur only at elevated temperatures, implying involvement of a series of long-range solvent-coupled conformational changes in Cph1. We show that signalling is achieved through ultrafast photoisomerization where localized structural change in the GAF domain is transmitted and amplified to cause larger-scale and slower conformational change in the PHY and histidine kinase domains. This hierarchy of timescales and extent of structural change orientates the histidine kinase domain to elicit the desired light-activated biological response.
| S-EPMC3530517 | biostudies-literature
Project description:A quantum mechanics (QM)/molecular mechanics (MM) hybrid method was applied to the Pr state of the cyanobacterial phytochrome Cph1 to calculate the Raman spectra of the bound PCB cofactor. Two QM/MM models were derived from the atomic coordinates of the crystal structure. The models differed in the protonation site of His(260) in the chromophore-binding pocket such that either the delta-nitrogen (M-HSD) or the epsilon-nitrogen (M-HSE) carried a hydrogen. The optimized structures of the two models display small differences specifically in the orientation of His(260) with respect to the PCB cofactor and the hydrogen bond network at the cofactor-binding site. For both models, the calculated Raman spectra of the cofactor reveal a good overall agreement with the experimental resonance Raman (RR) spectra obtained from Cph1 in the crystalline state and in solution, including Cph1 adducts with isotopically labeled PCB. However, a distinctly better reproduction of important details in the experimental spectra is provided by the M-HSD model, which therefore may represent an improved structure of the cofactor site. Thus, QM/MM calculations of chromoproteins may allow for refining crystal structure models in the chromophore-binding pocket guided by the comparison with experimental RR spectra. Analysis of the calculated and experimental spectra also allowed us to identify and assign the modes that sensitively respond to chromophore-protein interactions. The most pronounced effect was noted for the stretching mode of the methine bridge A-B adjacent to the covalent attachment site of PCB. Due a distinct narrowing of the A-B methine bridge bond angle, this mode undergoes a large frequency upshift as compared with the spectrum obtained by QM calculations for the chromophore in vacuo. This protein-induced distortion of the PCB geometry is the main origin of a previous erroneous interpretation of the RR spectra based on QM calculations of the isolated cofactor.
| S-EPMC2712200 | biostudies-literature