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A small single molecule with multiple photoswitchable subunits, selectively and independently controllable by light of different wavelengths, is highly attractive for applications in multi-responsive materials and biological sciences. Herein, triple photoswitches are presented consisting of three independent azobenzene (AB) subunits that share a common central phenyl ring: the meta-trisazobenzenes (MTA). It is the unique meta-connectivity pattern leading to decoupling of all azo-subunits although they do overlap spatially. Based on this pattern, we design a triple MTA photoswitch, as proof-of-principle, with three different, electronically independent AB branches on the computer, which can be individually photo-excited to trigger ultra-fast E → Z isomerization at the selected AB branch.
Bei jeder chemischen Reaktion werden Bindungen gebrochen und andere neu geknüpft. Dabei ändert sich die Anordnung und eventuell Anzahl der Atome im Molekül. Voraussetzung hierfür sind Bewegungen der beteiligten Atome und Moleküle. Um chemische Umwandlungen in "Echtzeit" zu studieren, müssen Untersuchungen im Zeitbereich der Schwingungs- und Rotationsdynamik durchgeführt werden. Dazu nutzen Wissenschaftler des Instituts für Physikalische und Theoretische Chemie die Möglichkeiten der modernen Ultrakurzzeit-Lasertechnik.
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.
Pflanzen, aber auch einige Bakterien und Archäen verfügen über hocheffiziente Mechanismen, Licht in Energie umzuwandeln. Photovoltaik-Zellen reichen an die Perfektion dieser natürlichen Systeme noch lange nicht heran. Deshalb versuchen Forscher, mit ultraschnellen spektroskopischen Methoden der Natur in die Karten zu schauen und von ihr zu lernen.
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 (Φf) is hardly affected. The result is a strong fluorescent RNA-probe whose spectral properties change in a defined way upon environmental changes.
Dodecins, a group of flavin-binding proteins with a dodecameric quaternary structure, are able to incorporate two flavins within each of their six identical binding pockets building an aromatic tetrade with two tryptophan residues. Dodecin from the archaeal Halobacterium salinarum is a riboflavin storage device. We demonstrate that unwanted side reactions induced by reactive riboflavin species and degradation of riboflavin are avoided by ultrafast depopulation of the reactive excited state of riboflavin. Intriguingly, in this process, the staggered riboflavin dimers do not interact in ground and photoexcited states. Rather, within the tetrade assembly, each riboflavin is kept under the control of the respective adjacent tryptophan, which suggests that the stacked arrangement is a matter of optimizing the flavin load. We further identify an electron transfer in combination with a proton transfer as a central element of the effective excited state depopulation mechanism. Structural and functional comparisons of the archaeal dodecin with bacterial homologs reveal diverging evolution. Bacterial dodecins bind the flavin FMN instead of riboflavin and exhibit a clearly different binding pocket design with inverse incorporations of flavin dimers. The different adoption of flavin changes photochemical properties, making bacterial dodecin a comparably less efficient quencher of flavins. This supports a functional role different for bacterial and archaeal dodecins.
Thiophenylazobenzene: an alternative photoisomerization controlled by lone‐pair⋅⋅⋅π interaction
(2019)
Azoheteroarene photoswitches have attracted attention due to their unique properties. We present the stationary photochromism and ultrafast photoisomerization mechanism of thiophenylazobenzene (TphAB). It demonstrates impressive fatigue resistance and photoisomerization efficiency, and shows favorably separated (E)‐ and (Z)‐isomer absorption bands, allowing for highly selective photoconversion. The (Z)‐isomer of TphAB adopts an unusual orthogonal geometry where the thiophenyl group is perfectly perpendicular to the phenyl group. This geometry is stabilized by a rare lone‐pair⋅⋅⋅π interaction between the S atom and the phenyl group. The photoisomerization of TphAB occurs on the sub‐ps to ps timescale and is governed by this interaction. Therefore, the adoption and disruption of the orthogonal geometry requires significant movement along the inversion reaction coordinates (CNN and NNC angles). Our results establish TphAB as an excellent photoswitch with versatile properties that expand the application possibilities of AB derivatives.
Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identified a new subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterized a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and showed that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a new subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics.
African trypanosomes cause a parasitic disease known as sleeping sickness. Mitochondrial transcript maturation in these organisms requires a RNA editing reaction that is characterized by the insertion and deletion of U-nucleotides into otherwise non-functional mRNAs. Editing represents an ideal target for a parasite-specific therapeutic intervention since the reaction cycle is absent in the infected host. In addition, editing relies on a macromolecular protein complex, the editosome, that only exists in the parasite. Therefore, all attempts to search for editing interfering compounds have been focused on molecules that bind to proteins of the editing machinery. However, in analogy to other RNA-driven biochemical pathways it should be possible to stall the reaction by targeting its substrate RNAs. Here we demonstrate inhibition of editing by specific aminoglycosides. The molecules bind into the major groove of the gRNA/pre-mRNA editing substrates thereby causing a stabilization of the RNA molecules through charge compensation and an increase in stacking. The data shed light on mechanistic details of the editing process and identify critical parameters for the development of new trypanocidal compounds.
Microbial rhodopsins are omnipresent on Earth, however the vast majority of them remain uncharacterized. Here we describe a new rhodopsin clade from cold-adapted organisms and cold environments, such as glaciers, denoted as CryoRhodopsins (CryoRs). Our data suggest that CryoRs have photosensory activity. A distinguishing feature of the clade is the presence of a buried arginine residue close to the cytoplasmic face of its members. Combining single-particle cryo-electron microscopy and X-ray crystallography with the rhodopsin activation by light, we demonstrate that the arginine stabilizes a strongly blue-shifted intermediate of an extremely slow CryoRhodopsin photocycle. Together with extensive spectroscopic characterization, our investigations on CryoR1 and CryoR2 proteins reveal mechanisms of photoswitching in the newly identified clade and demonstrate principles of the adaptation of these rhodopsins to low temperatures.
Microbial rhodopsins are omnipresent on Earth, however the vast majority of them remain uncharacterized. Here we describe a new rhodopsin group from cold-adapted organisms and cold environments, such as glaciers, denoted as CryoRhodopsins (CryoRs). Our data suggest that CryoRs have dual functionality switching between inward transmembrane proton translocation and photosensory activity, both of which can be modulated with UV light. CryoR1 exhibits two subpopulations in the ground state, which upon light activation lead to transient photocurrents of opposing polarities. A distinguishing feature of the group is the presence of a buried arginine residue close to the cytoplasmic face of its members. Combining single-particle cryo-electron microscopy and X-ray crystallography with the rhodopsin activation by lit, we demonstrate that the arginine stabilizes a UV-absorbing intermediate of an extremely slow CryoRhodopsin photocycle. Together with extensive spectroscopic characterization, our investigations on CryoR1 and CryoR2 proteins reveal mechanisms of photoswitching in the newly identified group and demonstrate principles of the adaptation of these rhodopsins to low temperatures.Microbial rhodopsins are omnipresent on Earth, however the vast majority of them remain uncharacterized. Here we describe a new rhodopsin group from cold-adapted organisms and cold environments, such as glaciers, denoted as CryoRhodopsins (CryoRs). Our data suggest that CryoRs have dual functionality switching between inward transmembrane proton translocation and photosensory activity, both of which can be modulated with UV light. CryoR1 exhibits two subpopulations in the ground state, which upon light activation lead to transient photocurrents of opposing polarities. A distinguishing feature of the group is the presence of a buried arginine residue close to the cytoplasmic face of its members. Combining single-particle cryo-electron microscopy and X-ray crystallography with the rhodopsin activation by light, we demonstrate that the arginine stabilizes a UV-absorbing intermediate of an extremely slow CryoRhodopsin photocycle. Together with extensive spectroscopic characterization, our investigations on CryoR1 and CryoR2 proteins reveal mechanisms of photoswitching in the newly identified group and demonstrate principles of the adaptation of these rhodopsins to low temperatures.
Structural and functional consequences of the H180A mutation of the light-driven sodium pump KR2
(2022)
Krokinobacter eikastus rhodopsin 2 (KR2) is a light-driven pentameric sodium pump. Its ability to translocate cations other than protons and to create an electrochemical potential makes it an attractive optogenetic tool. Tailoring its ion pumping characteristics by mutations is therefore of great interest. In addition, understanding the functional and structural consequences of certain mutations helps to derive a functional mechanism of ion selectivity and transfer of KR2. Based on solid-state NMR spectroscopy, we report an extensive chemical shift resonance assignment of KR2 within lipid bilayers. This data set was then used to probe site-resolved allosteric effects of sodium binding, which revealed multiple responsive sites including the Schiff base nitrogen and the NDQ motif. Based on this data set, the consequences of the H180A mutation are probed. The mutant is silenced in the presence of sodium while in its absence, proton pumping is observed. Our data reveal specific long-range effects along the sodium transfer pathway. These experiments are complemented by time-resolved optical spectroscopy. Our data suggest a model in which sodium uptake by the mutant can still take place, while sodium release and backflow control are disturbed.
Flavins are employed to transform physical input into biological output signals. In this function, flavins catalyze a variety of light-induced reactions and redox processes. However, nature also provides flavoproteins with the ability to uncouple the mediation of signals. Such proteins are the riboflavin-binding proteins (RfBPs) with their function to store riboflavin for fast delivery of FMN and FAD. Here we present in vitro and in vivo data showing that the recently discovered archaeal dodecin is an RfBP, and we reveal that riboflavin storage is not restricted to eukaryotes. However, the function of the prokaryotic RfBP dodecin seems to be adapted to the requirement of a monocellular organism. While in eukaryotes RfBPs are involved in trafficking riboflavin, and dodecin is responsible for the flavin homeostasis of the cell. Although only 68 amino acids in length, dodecin is of high functional versatility in neutralizing riboflavin to protect the cellular environment from uncontrolled flavin reactivity. Besides the predominant ultrafast quenching of excited states, dodecin prevents light-induced riboflavin reactivity by the selective degradation of riboflavin to lumichrome. Coordinated with the high affinity for lumichrome, the directed degradation reaction is neutral to the cellular environment and provides an alternative pathway for suppressing uncontrolled riboflavin reactivity. Intriguingly, the different structural and functional properties of a homologous bacterial dodecin suggest that dodecin has different roles in different kingdoms of life.
The conformational dynamics induced by ligand binding to the tetracycline-binding aptamer is monitored via stopped-flow fluorescence spectroscopy and time-correlated single photon counting experiments. The fluorescence of the ligand is sensitive to changes within the tertiary structure of the aptamer during and after the binding process. In addition to the wild-type aptamer, the mutants A9G, A13U and A50U are examined, where bases important for regulation are changed to inhibit the aptamer’s function. Our results suggest a very fast two-step-mechanism for the binding of the ligand to the aptamer that can be interpreted as a binding step followed by a reorganization of the aptamer to accommodate the ligand. Binding to the two direct contact points A13 and A50 was found to occur in the first binding step. The exchange of the structurally important base A9 for guanine induces an enormous deceleration of the overall binding process, which is mainly rooted in an enhancement of the back reaction of the first binding step by several orders of magnitude. This indicates a significant loss of tertiary structure of the aptamer in the absence of the base A9, and underlines the importance of pre-organization on the overall binding process of the tetracycline-binding aptamer.
A novel thiazol‐based ratiometric dye for the detection of local pH values is synthesized, and its properties are characterized by a combination of optical spectroscopy, solid‐state NMR and DNP (dynamic nuclear polarization)‐enhanced solid‐state NMR. This novel dye covers a completely different sensitivity range with its acidic pKa value of 3.5 compared to other established dyes for ratiometric pH detection, such as SNARF. The dye is grafted to the surfaces of mesoporous silica materials, which enables, for the first time, direct in situ measurements of the local pH values in silica mesopores by a simple UV‐vis spectroscopy method. The obtained results, which are in good agreement with previous indirect techniques, indicate a background electrolyte‐dependent pKa shift of at least one pH unit under nanoconfined conditions compared to the pKa of the dye in bulk solution.