Absorption and emission spectroscopic investigation of thermal dynamics and photo-dynamics of the rhodopsin domain of the rhodopsin-guanylyl cyclase from the aquatic fungus Blastocladiella emersonii

A new class of rhodopsins covalently linked to an enzymatic domain was discovered recently. A member of this class of enzymerhodopsins, the rhodopsin-guanylyl cyclase (RhGC) was identified in the aquatic fungus Blastocladiella emersonii (BE). Characterization of RhGC showed that the second-messenger molecule cGMP (cyclic guanylyl monophosphate) is produced upon green light illumination. Here, the rhodopsin domain Rh (BE) of the rhodopsin-guanylyl cyclase RhGC was studied by absorption and emission spectroscopic methods. It was found that fresh thawed Rh (BE) was composed of a mixture of retinal – protein conformations. These retinal conformations are likely all-trans protonated retinal Schiff base (Ret_1), 13-cis protonated retinal Schiff base with repositioned counter ion (Ret_2), all-trans protonated retinal Schiff base with repositioned counter ion (Ret_3), and deprotonated all-trans retinal Schiff base (Ret_4). The Rh (BE) thermal denaturing was studied: An apparent protein melting temperature of m ≈ 49 °C was determined; the apparent protein melting time at room temperature (≈ 21.9 °C) was tm ≈ 1.45 h. Thermal retinal conformation restructuring with irreversible conversion likely to deprotonated 13-cis retinal Schiff base (Ret_4’) was observed. The photo-excitation of all-trans protonated retinal Schiff base (Ret_1) caused a primary photo-cycle dynamics involving excited-state picosecond all-trans – 13-cis isomerization (13-cis protonated retinal Schiff base Ret_5 formation) followed by ground-state sub-second intermediate retinal Schiff base formations (Ret_2, Ret_3, Ret_4) and sub-second to second recovery to the initial all-trans protonated retinal Schiff base (Ret_1). Long-time all-trans protonated retinal Schiff base photo-excitation caused irreversible (likely 13-cis) retinal Schiff base (Ret_4’) formation

Reaction dynamics of the chimeric channelrhodopsin C1C2

Channelrhodopsin (ChR) is a key protein of the optogenetic toolkit. C1C2, a functional chimeric protein of Chlamydomonas reinhardtii ChR1 and ChR2, is the only ChR whose crystal structure has been solved, and thus uniquely suitable for structure-based analysis. We report C1C2 photoreaction dynamics with ultrafast transient absorption and multi-pulse spectroscopy combined with target analysis and structure-based hybrid quantum mechanics/molecular mechanics calculations. Two relaxation pathways exist on the excited (S-1) state through two conical intersections Cl-1 and Cl-2, that are reached via clockwise and counter-clockwise rotations: (i) the C13=C14 isomerization path with 450 fs via Cl-1 and (ii) a relaxation path to the initial ground state with 2.0 ps and 11 ps via Cl-2, depending on the hydrogen-bonding network, hence indicating active-site structural heterogeneity. The presence of the additional conical intersection Cl-2 rationalizes the relatively low quantum yield of photoisomerization (30 +/- 3%), reported here. Furthermore, we show the photoreaction dynamics from picoseconds to seconds, characterizing the complete photocycle of C1C2

A framework for the cross-sectoral integration of multi-model impact projections: land use decisions under climate impacts uncertainties

Climate change and its impacts already pose considerable challenges for societies that will further increase with global warming (IPCC, 2014a, b). Uncertainties of the climatic response to greenhouse gas emissions include the potential passing of large-scale tipping points (e.g. Lenton et al., 2008; Levermann et al., 2012; Schellnhuber, 2010) and changes in extreme meteorological events (Field et al., 2012) with complex impacts on societies (Hallegatte et al., 2013). Thus climate change mitigation is considered a necessary societal response for avoiding uncontrollable impacts (Conference of the Parties, 2010). On the other hand, large-scale climate change mitigation itself implies fundamental changes in, for example, the global energy system. The associated challenges come on top of others that derive from equally important ethical imperatives like the fulfilment of increasing food demand that may draw on the same resources. For example, ensuring food security for a growing population may require an expansion of cropland, thereby reducing natural carbon sinks or the area available for bio-energy production. So far, available studies addressing this problem have relied on individual impact models, ignoring uncertainty in crop model and biome model projections. Here, we propose a probabilistic decision framework that allows for an evaluation of agricultural management and mitigation options in a multi-impactmodel setting. Based on simulations generated within the Inter-Sectoral Impact Model Intercomparison Project (ISI-MIP), we outline how cross-sectorally consistent multi-model impact simulations could be used to generate the information required for robust decision making. Using an illustrative future land use pattern, we discuss the trade-off between potential gains in crop production and associated losses in natural carbon sinks in the new multiple crop- and biome-model setting. In addition, crop and water model simulations are combined to explore irrigation increases as one possible measure of agricultural intensification that could limit the expansion of cropland required in response to climate change and growing food demand. This example shows that current impact model uncertainties pose an important challenge to long-term mitigation planning and must not be ignored in long-term strategic decision making

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Absorption and Emission Spectroscopic Investigation of Thermal Dynamics and Photo-Dynamics of the Rhodopsin Domain of the Rhodopsin-Guanylyl Cyclase from the Nematophagous Fungus Catenaria anguillulae

The rhodopsin-guanylyl cyclase from the nematophagous fungus Catenaria anguillulae belongs to a recently discovered class of enzymerhodopsins and may find application as a tool in optogenetics. Here the rhodopsin domain CaRh of the rhodopsin-guanylyl cyclase from Catenaria anguillulae was studied by absorption and emission spectroscopic methods. The absorption cross-section spectrum and excitation wavelength dependent fluorescence quantum distributions of CaRh samples were determined (first absorption band in the green spectral region). The thermal stability of CaRh was studied by long-time attenuation measurements at room temperature (20.5 °C) and refrigerator temperature of 3.5 °C. The apparent melting temperature of CaRh was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 62 ± 2 °C). The photocycle dynamics of CaRh was investigated by sample excitation to the first inhomogeneous absorption band of the CaRhda dark-adapted state around 590 nm (long-wavelength tail), 530 nm (central region) and 470 nm (short-wavelength tail) and following the absorption spectra development during exposure and after exposure (time resolution 0.0125 s). The original protonated retinal Schiff base PRSBall-trans in CaRhda photo-converted reversibly to protonated retinal Schiff base PRSBall-trans,la1 with restructured surroundings (CaRhla1 light-adapted state, slightly blue-shifted and broadened first absorption band, recovery to CaRhda with time constant of 0.8 s) and deprotonated retinal Schiff base RSB13-cis (CaRhla2 light-adapted state, first absorption band in violet to near ultraviolet spectral region, recovery to CaRhda with time constant of 0.35 s). Long-time light exposure of light-adapted CaRhla1 around 590, 530 and 470 nm caused low-efficient irreversible degradation to photoproducts CaRhprod. Schemes of the primary photocycle dynamics of CaRhda and the secondary photocycle dynamics of CaRhla1 are developed

Rhodopsin-cyclases for photocontrol of cGMP/cAMP and 2.3 Å structure of the adenylyl cyclase domain

The cyclic nucleotides cAMP and cGMP are important second messengers that orchestrate fundamental cellular responses. Here, we present the characterization of the rhodopsinguanylyl cyclase from Catenaria anguillulae (CaRhGC), which produces cGMP in response to green light with a light to dark activity ratio > 1000. After light excitation the putative signaling state forms with tau = 31 ms and decays with tau = 570 ms. Mutations (up to 6) within the nucleotide binding site generate rhodopsin-adenylyl cyclases (CaRhACs) of which the double mutated YFP-CaRhAC (E497K/C566D) is the most suitable for rapid cAMP production in neurons. Furthermore, the crystal structure of the ligand-bound AC domain (2.25 angstrom) reveals detailed information about the nucleotide binding mode within this recently discovered class of enzyme rhodopsin. Both YFP-CaRhGC and YFP-CaRhAC are favorable optogenetic tools for non-invasive, cell-selective, and spatio-temporally precise modulation of cAMP/cGMP with light

Light–Dark Adaptation of Channelrhodopsin Involves Photoconversion between the all-<i>trans</i> and 13-<i>cis</i> Retinal Isomers

Channelrhodopsins (ChR) are light-gated ion channels of green algae that are widely used to probe the function of neuronal cells with light. Most ChRs show a substantial reduction in photocurrents during illumination, a process named “light adaptation”. The main objective of this spectroscopic study was to elucidate the molecular processes associated with light–dark adaptation. Here we show by liquid and solid-state nuclear magnetic resonance spectroscopy that the retinal chromophore of fully dark-adapted ChR is exclusively in an all<i>-trans</i> configuration. Resonance Raman (RR) spectroscopy, however, revealed that already low light intensities establish a photostationary equilibrium between all<i>-trans</i>,15<i>-anti</i> and 13<i>-cis</i>,15-<i>syn</i> configurations at a ratio of 3:1. The underlying photoreactions involve simultaneous isomerization of the C(13)C(14) and C(15)N bonds. Both isomers of this DA_app state may run through photoinduced reaction cycles initiated by photoisomerization of only the C(13)C(14) bond. RR spectroscopic experiments further demonstrated that photoinduced conversion of the apparent dark-adapted (DA_app) state to the photocycle intermediates P500 and P390 is distinctly more efficient for the all-<i>trans</i> isomer than for the 13-<i>cis</i> isomer, possibly because of different chromophore–water interactions. Our data demonstrating two complementary photocycles of the DA_app isomers are fully consistent with the existence of two conducting states that vary in quantitative relation during light–dark adaptation, as suggested previously by electrical measurements

The relevance of uncertainty in future crop production for mitigation strategy planning

In order to achieve climate change mitigation, long-term decisions are required that must be reconciled with other societal goals that draw on the same resources. For example, ensuring food security for a growing population may require an expansion of crop land, thereby reducing natural carbon sinks or the area available for bio-energy production. Here, we show that current impact-model uncertainties pose an important challenge to long-term mitigation planning and propose a new risk-assessment and decision framework that accounts for competing interests. Based on cross-sectorally consistent simulations generated within the Inter-Sectoral Impact Model Intercomparison Project (ISI-MIP) we discuss potential gains and limitations of additional irrigation and trade-offs of the expansion of agricultural land as two possible response measures to climate change and growing food demand. We describe an illustrative example in which the combination of both measures may close the supply demand gap while leading to a loss of approximately half of all natural carbon sinks. We highlight current limitations of available simulations and additional steps required for a comprehensive risk assessment