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We developed three bathochromic, green‐light activatable, photolabile protecting groups based on a nitrodibenzofuran (NDBF) core with D‐π‐A push–pull structures. Variation of donor substituents (D) at the favored ring position enabled us to observe their impact on the photolysis quantum yields. Comparing our new azetidinyl‐NDBF (Az‐NDBF) photolabile protecting group with our earlier published DMA‐NDBF, we obtained insight into its excitation‐specific photochemistry. While the “two‐photon‐only” cage DMA‐NDBF was inert against one‐photon excitation (1PE) in the visible spectral range, we were able to efficiently release glutamic acid from azetidinyl‐NDBF with irradiation at 420 and 530 nm. Thus, a minimal change (a cyclization adding only one carbon atom) resulted in a drastically changed photochemical behavior, which enables photolysis in the green part of the spectrum., What a difference one atom makes: Azetidine (az) and dimethylamino (DMA) donor moieties differ only by one carbon atom in their molecular formula, but in the present study we observed huge differences in their impact on the photochemistry of D‐π‐A caging groups. Restriction of the degrees of movement freedom—due to the four‐membered ring—leads to a photocage that can be used within the “green gap” for the release of biologically relevant leaving groups (e.g., glutamic acid).

Photoactivatable compounds for example photoswitches or photolabile protecting groups (PPGs, photocages) for spatiotemporal light control, play a crucial role in different areas of research. For each application, parameters such as the absorption spectrum, solubility in the respective media and/or photochemical quantum yields for several competing processes need to be optimized. The design of new photochemical tools therefore remains an important task. In this study, we exploited the concept of excited‐state‐aromaticity, first described by N. Colin Baird in 1971, to investigate a new class of photocages, based on cyclic, ground‐state‐antiaromatic systems. Several thio‐ and nitrogen‐functionalized compounds were synthesized, photochemically characterized and further optimized, supported by quantum chemical calculations. After choosing the optimal scaffold, which shows an excellent uncaging quantum yield of 28 %, we achieved a bathochromic shift of over 100 nm, resulting in a robust, well accessible, visible light absorbing, compact new photocage with a clean photoreaction and a high quantum product (ϵ⋅Φ) of 893 M−1 cm−1 at 405 nm., The concept of excited state aromaticity was used to design a new class of photolabile protecting groups, based on cyclic, ground‐state‐antiaromatic systems. Supported by theoretical calculations, several thio‐ and nitrogen functionalized compounds were synthesized, characterized and further optimized, resulting in a well accessible, visible light absorbing, new photocage with a high uncaging cross section of 893 M−1 cm−1 at 405 nm.

The new class of microbial rhodopsins, called xenorhodopsins (XeRs),[1] extends the versatility of this family by inward H+ pumps.[2–4] These pumps are an alternative optogenetic tool to the light‐gated ion channels (e.g. ChR1,2), because the activation of electrically excitable cells by XeRs is independent from the surrounding physiological conditions. In this work we functionally and spectroscopically characterized XeR from Nanosalina (NsXeR).[1] The photodynamic behavior of NsXeR was investigated on the ps to s time scale elucidating the formation of the J and K and a previously unknown long‐lived intermediate. The pH dependent kinetics reveal that alkalization of the surrounding medium accelerates the photocycle and the pump turnover. In patch‐clamp experiments the blue‐light illumination of NsXeR in the M state shows a potential‐dependent vectoriality of the photocurrent transients, suggesting a variable accessibility of reprotonation of the retinal Schiff base. Insights on the kinetically independent switching mechanism could furthermore be obtained by mutational studies on the putative intracellular H+ acceptor D220., The unique behaviour of inward proton pumping xenorhodopsins (XeR) makes these microbial rhodopsins an alternative optogenetic tool to the passively transporting light‐gated ion channels (e.g. channelrhodopsin‐1 and channelrhodopsin‐2), because the activation of electrically excitable cells by XeRs is independent from the surrounding physiological conditions. Here, we functionally and spectroscopically characterized XeR from Nanosalina (NsXeR).

Regulation of complex biological networks has proven to be a key bottleneck in synthetic biology. Interactions between the structurally flexible RNA and various other molecules in the form of riboswitches have shown a high-regulation specificity and efficiency and synthetic riboswitches have filled the toolbox of devices in many synthetic biology applications. Here we report the development of a novel, small molecule binding RNA aptamer, whose binding is dependent on light-induced change of conformation of its small molecule ligand. As ligand we chose an azobenzene because of its reliable photoswitchability and modified it with chloramphenicol for a better interaction with RNA. The synthesis of the ligand ‘azoCm’ was followed by extensive biophysical analysis regarding its stability and photoswitchability. RNA aptamers were identified after several cycles of in vitro selection and then studied regarding their binding specificity and affinity toward the ligand. We show the successful development of an RNA aptamer that selectively binds to only the trans photoisomer of azoCm with a KD of 545 nM. As the aptamer cannot bind to the irradiated ligand (λ = 365 nm), a light-selective RNA binding system is provided. Further studies may now result in the engineering of a reliable, light-responsible riboswitch.

The ultrafast photoisomerization of model azobenzene macrocycles was studied by transient absorption spectroscopy. Our results reveal a strong dependence of the dynamics and the overall molecular properties on the geometric constraints and the intramolecular strain.

Proteorhodopsin (PR) is the most abundant retinal protein on earth and functions as a light-driven proton pump. Despite of extensive efforts, structural data for PR photointermediate states have not been obtained. Based on DNP-enhanced solid-state NMR, we were able to analyze the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the ground state in comparison to light induced, cryotrapped K- and M-states. A high M-state population could be achieved by preventing reprotonation of the Schiff base through a mutation of the primary proton donor (E108Q). Our data reveal unexpected large and alternating 13C chemical shift changes in the K-state propagating away from the Schiff base along the polyene chain. Furthermore, two different M-states have been observed reflecting the Schiff base reorientation after the de-protonation step. Our study provides novel insight into the photocycle of PR and also demonstrates the power of DNP-enhanced solid-state NMR to bridge the gap between functional and structural data and models.

The photoswitchable boron-dipyrromethene–dithienylethene molecular dyad is introduced as a prototype for the efficient fluorescence intensity modulation on the molecular level. The functionality of the system is based on the photochromism of the dithienylethene, which facilitates an efficient on- and off-switching of a Förster-type intramolecular energy transfer between the photoexcited BODIPY donor and the dithienylethene acceptor moiety. The switching behavior and dynamics of the molecular dyad are monitored by steady state and time-resolved spectroscopic methods. A quenching efficiency of up to 96% in the off-state is observed and explained by a drastically accelerated decay of the boron-dipyrromethene excited state due to the efficient energy transfer despite the orthogonal arrangement of donor and acceptor. An energy transfer time orders of magnitude shorter than the lifetime of the boron-dipyrromethene in the open state is determined.

The origin of coherent oscillations in CdSe quantum dots and in the CdSe/methylviologen electron transfer system is studied. In CdSe/methylviologen coherent phonons are triggered by the electron transfer from the quantum dot to methylviologen.

We examine the photoinduced excited state dynamics of pyrene modified adenosine, a versatile probe for folding and hybridization of ribonucleic acids. Measurements in different solvents revealed complex ultrafast dynamics, but high robustness since the overall fluorescence quantum yield (Φ f ) is hardly affected. The result is a strong fluorescent RNA-probe whose spectral properties change in a defined way upon environmental changes.

The dynamics of a water soluble spiropyran is investigated by means of femtosecond transient absorption spectroscopy in the visible and infrared spectral range revealing an ultrafast reversible switching behavior under aqueous conditions with a high fatigue resistance.

Femtosecond time-resolved absorption measurements were performed to investigate the influence of the pH, imidazole concentration, and point mutations on the isomerization process of Channelrhodopsin-2. Apart from the typical spectral characteristics of retinal isomerization, an additional absorption feature rises for the wild-type (wt) on a timescale from tens of ps to 1 ns within the spectral range of the photoproduct and is attributed to an equilibration between different K-intermediates. Remarkably, this absorption feature vanishes upon addition of imidazole or lowering the pH. In the latter case, the isomerization is dramatically slowed down, due to protonation of negatively charged amino acids within the retinal binding pocket, e.g., E123 and D253. Moreover, we investigated the influence of several point mutations within the retinal binding pocket E123T, E123D, C128T, and D156C. For E123T, the isomerization is retarded compared to wt and E123D, indicating that a negatively charged residue at this position functions as an effective catalyst in the isomerization process. In the case of the C128T mutant, all primary processes are slightly accelerated compared to the wt, whereas the isomerization dynamics for the D156C mutant is similar to wt after addition of imidazole.

and we show herein the de novo structure ofthegreenvariantofproteorhodopsinsolvedbysolutionNMRspectroscopy.The structure of PR (Figure 1) was solved in the short-chain lipid diC7PC (diheptanoyl-phosphocholine) combininglong-range NOEs with restraints derived from paramagneticrelaxation enhancement (PRE) and residual dipolar cou-plings (RDCs). The seven transmembrane helices are con-nected by short loops. Instead of the anti-parallel b-sheet thatis observed between helices B and C in other microbialrhodopsins, torsion angles derived from the protein backbonedihedral angle prediction program TALOS+ suggest that PRresidues G87–P90 form a short b-turn. The loop betweenhelices D and E is longer than predicted by the secondarystructure prediction program TMHMM.

Photoinduced electron transfer from organic dye molecules to semiconductor nanoparticles is the first and most important reaction step for the mechanism in the so called “wet solar cells” [1]. The time scale between the photoexcitation of the dye and the electron injection into the conduction band of the semiconductor colloid varies from a few tens of femtoseconds to nanoseconds, depending on the specific electron transfer parameters of the system, e.g., electronic coupling or free energy values of donor and acceptor molecules [2–10]. We show that visible pump/ white light probe is a very efficient tool to investigate the electron injection reaction allowing to observe simultaneously the relaxation of the excited dye, the injection process of the electron, the cooling of the injected electron and the charge recombination reaction.

Synthetic riboswitches are versatile tools for the study and manipulation of biological systems. Yet, the underlying mechanisms governing its structural properties and regulation under physiological conditions are poorly studied. We performed spectroscopic and calorimetric experiments to explore the folding kinetics and thermodynamics of the tetracycline-binding aptamer, which can be employed as synthetic riboswitch, in the range of physiological magnesium concentrations. The dissociation constant of the ligand-aptamer complex was found to strongly depend on the magnesium concentration. At physiological magnesium concentrations, tetracycline induces a significant conformational shift from a compact, but heterogeneous intermediate state toward the completely formed set of tertiary interactions defining the regulation-competent structure. Thus, the switching functionality of the tetracycline-binding aptamer appears to include both a conformational rearrangement toward the regulation-competent structure and its thermodynamic stabilization.

Proteorhodopsins are the most abundant retinal based photoreceptors and their phototrophic function might be relevant in marine ecosystems. Here, we describe their remarkable molecular properties with a special focus on the green absorbing variant. Its distinct features include a high pKa value of the primary proton acceptor stabilized through an interaction with a conserved histidine, a long-range interaction between the cytoplasmic EF loop and the chromophore entailing a particular mode of color tuning and a variable proton pumping vectoriality with complex voltage-dependence. The proteorhodopsin family represents a profound example for structure–function relationships. Especially the development of a biophysical understanding of green proteorhodopsin is an excellent example for the unique opportunities offered by a combined approach of advanced spectroscopic and electrophysiological methods. This article is part of a Special Issue entitled: Retinal Proteins—You can teach an old dog new tricks.

The ultrafast caroteonid to chlorophyll a energy transfer dynamics of the isolated fucoxanthin–chlorophyll proteins FCPa and FCPb from the diatom Cyclotella meneghiniana was investigated in a comprehensive study using transient absorption in the visible and near infrared spectral region as well as static fluorescence spectroscopy. The altered oligomerization state of both antenna systems results in a more efficient energy transfer for FCPa, which is also reflected in the different chlorophyll a fluorescence quantum yields. We therefore assume an increased quenching in the higher oligomers of FCPb. The influence of the carotenoid composition was investigated using FCPa and FCPb samples grown under different light conditions and excitation wavelengths at the blue (500nm) and red (550nm) wings of the carotenoid absorption. The different light conditions yield in altered amounts of the xanthophyll cycle pigments diadinoxanthin and diatoxanthin. Since no significant dynamic changes are observed for high light and low light samples, the contribution of the xanthophyll cycle pigments to the energy transfer is most likely negligible. On the contrary, the observed dynamics change drastically for the different excitation wavelengths. The analyses of the decay associated spectra of FCPb suggest an altered energy transfer pathway. For FCPa even an additional time constant was found after excitation at 500nm. It is assigned to the intrinsic lifetime of either the xanthophyll cycle carotenoids or more probable the blue absorbing fucoxanthins. Based on our studies we propose a detailed model explaining the different excitation energy transfer pathways in FCPa.

A two-domain elevator mechanism for sodium/proton antiport Chiara Lee, Hae Joo Kang, Christoph von Ballmoos, Simon Newstead, Povilas Uzdavinys, David L. Dotson, So Iwata, Oliver Beckstein, Alexander D. Cameron, David Drew Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, UK Centre for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden Center for Biological Physics, Department of Physics, Arizona State University, Tempe, AZ 85287-1504, USA Membrane Protein Laboratory, Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Chilton, Oxfordshire OX11 0DE, UK Research Complex at Harwell Rutherford, Appleton Laboratory, Harwell, Oxford, Didcot, Oxfordshire OX11 0FA, UK School of Life Sciences, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK E-mail: ddrew@dbb.su.se