Förster resonance energy transfer within the neomycin aptamer.

Förster resonance energy transfer (FRET) measurements between two dyes is a powerful method to interrogate both structure and dynamics of biopolymers. The intensity of a fluorescence signal in a FRET measurement is dependent on both the distance and the relative orientation of the dyes. The latter can at the same time both complicate the analysis and give more detailed information. Here we present a detailed spectroscopic study of the energy transfer between the rigid FRET labels Çmf (donor) and tCnitro (quencher/acceptor) within the neomycin aptamer N1. The energy transfer originates from multiple emitting states of the donor and occurs on a low picosecond to nanosecond time-scale. To fully characterize the energy transfer, ultrafast transient absorption measurements were performed in conjunction with static fluorescence and time-correlated single photon counting (TCSPC) measurements, showing a clear distance dependence of both signal intensity and lifetime. Using a known NMR structure of the ligand-bound neomycin aptamer, the distance between the two labels was used to estimate κ2 and, therefore, make qualitative statements about the change in orientation after ligand binding with unprecedented temporal and spatial resolution. The advantages and potential applications of absorption-based methods using rigid labels for the characterization of FRET processes are discussed.

Vibrational Study of the Inward Proton Pump Xenorhodopsin NsXeR: Switch Order Determines Vectoriality.

Common proton pumps, e.g. HsBR and PR, transport protons out of the cell. Xenorhodopsins (XeR) were the first discovered microbial rhodopsins which come as natural inward proton pumps. In this work we combine steady-state (cryo-)FTIR and Raman spectroscopy with time-resolved IR and UV/Vis measurements to roadmap the inward proton transport of NsXeR and pinpoint the most important mechanistic features. Through the assignment of characteristic bands of the protein backbone, the retinal chromophore, the retinal Schiff base and D220, we could follow the switching processes for proton accessibility in accordance with the isomerization / switch / transfer model. The corresponding transient IR signatures suggest that the initial assignment of D220 as the proton acceptor needs to be questioned due to the temporal mismatch of the Schiff base and D220 protonation steps. The switching events in the K-L and MCP-MEC transitions are finely tuned by changes of the protein backbone and rearrangements of the Schiff base. This finely tuned mechanism is disrupted at cryogenic temperatures, being reflected in the replacement of the previously reported long-lived intermediate GS* by an actual redshifted (O-like) intermediate.

Small Warriors of Nature: Novel Red Emissive Chlorophyllin Carbon Dots Harnessing Fenton-Fueled Ferroptosis for In Vitro and In Vivo Cancer Treatment.

The appeal of carbon dots (CDs) has grown recently, due to their established biocompatibility, adjustable photoluminescence properties, and excellent water solubility. For the first time in the literature, copper chlorophyllin-based carbon dots (Chl-D CDs) are successfully synthesized. Chl-D CDs exhibit unique spectroscopic traits and are found to induce a Fenton-like reaction, augmenting photodynamic therapy (PDT) efficacies via ferroptotic and apoptotic pathways. To bolster the therapeutic impact of Chl-D CDs, a widely used cancer drug, temozolomide, is linked to their surface, yielding a synergistic effect with PDT and chemotherapy. Chl-D CDs' biocompatibility in immune cells and in vivo models showed great clinical potential.Proteomic analysis was conducted to understand Chl-D CDs' underlying cancer treatment mechanism. The study underscores the role of reactive oxygen species formation and pointed toward various oxidative stress modulators like aldolase A (ALDOA), aldolase C (ALDOC), aldehyde dehydrogenase 1B1 (ALDH1B1), transaldolase 1 (TALDO1), and transketolase (TKT), offering a deeper understanding of the Chl-D CDs' anticancer activity. Notably, the Chl-D CDs' capacity to trigger a Fenton-like reaction leads to enhanced PDT efficiencies through ferroptotic and apoptotic pathways. Hence, it is firmly believed that the inherent attributes of Chl-CDs can lead to a secure and efficient combined cancer therapy.

Enhancing the Potential of Fused Heterocycle-Based Triarylhydrazone Photoswitches.

Triarylhydrazones represent an attractive class of photochromic compounds offering many interesting features including high molar absorptivity, good addressability, and extraordinary thermal stability. In addition, unlike most other hydrazone-based photoswitches, they effectively absorb light above 365 nm. However, previously prepared triaryhydrazones suffer from low quantum yields of the Z→E photoisomerization. Here, we have designed a new subclass of naphthoyl-benzothiazole hydrazones that balance the most beneficial features of previously reported naphthoyl-quinoline and benzoyl-pyridine triarylhydrazones. These preserve the attractive absorption characteristics, exhibit higher thermal stability of the metastable form than the former and enhance the rate of the Z→E photoisomerization compared to the later, as a result of the weakening of the intramolecular hydrogen bonding between the hydrazone hydrogen and the benzothiazole moiety. Introducing the benzothiazole motif extends the tunability of the photochromic behaviour of hydrazone-based switches.

Enhancing the Potential of Fused Heterocycle-Based Triarylhydrazone Photoswitches.

Invited for the cover of this issue are Marek Cigán, Anna M. Grabarz and co-workers. The image depicts how a non-expert might imagine a "molecular photoswitch". Read the full text of the article at 10.1002/chem.202303509.

Wavelength Selective Photocontrol of Hybrid Azobenzene-Spiropyran Photoswitches with Overlapping Chromophores.

Compounds with multiple photoswitching units are appealing for complex photochemical control of molecular materials and nanostructures. Herein, we synthesized novel meta- and para- connected (related to the nitrogen of the indoline) azobenzene-spiropyran dyads, in which the central benzene unit is shared by both switches. We investigated their photochemistry using static and time-resolved transient absorption spectroscopy as well as quantum chemical calculations. In the meta-compound, the individual components are photochemically decoupled due to the meta-pattern. In the para-compound the spiro-connectivity leads to a bifunctional photoswitchable system with a red-shifted absorption. The azobenzene and the spiropyran can thus be addressed and switched independently by light of appropriate wavelength. Through the different connectivity patterns two different orthogonally photoswitchable systems have been obtained which are promising candidates for complex applications of light control.

Iminothioindoxyl Donors with Exceptionally High Cross Section for Protein Vibrational Energy Transfer.

Various protein functions are related to vibrational energy transfer (VET) as an important mechanism. The underlying transfer pathways can be experimentally followed by ultrafast Vis-pump/IR-probe spectroscopy with a donor-sensor pair of non-canonical amino acids (ncAAs) incorporated in a protein. However, so far only one donor ncAA, azulenylalanine (AzAla), exists, which suffers from a comparably low Vis extinction coefficient. Here, we introduce two novel donor ncAAs based on an iminothioindoxyl (ITI) chromophore. The dimethylamino-ITI (DMA-ITI) and julolidine-ITI (J-ITI) moieties overcome the limitation of AzAla with a 50 times higher Vis extinction coefficient. While ITI moieties are known for ultrafast photoswitching, DMA-ITI and J-ITI exclusively form a hot ground state on the sub-ps timescale instead, which is essential for their usage as vibrational energy donor. In VET measurements of donor-sensor dipeptides we investigate the performance of the new donors. We observe 20 times larger signals compared to the established AzAla donor, which opens unprecedented possibilities for the study of VET in proteins.

Assessing the Role of R120 in the Gating of CrChR2 by Time-Resolved Spectroscopy from Femtoseconds to Seconds.

The light-gated ion channel channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) is the most frequently used optogenetic tool in neurosciences. However, the precise molecular mechanism of the channel opening and the correlation among retinal isomerization, the photocycle, and the channel activity of the protein are missing. Here, we present electrophysiological and spectroscopic investigations on the R120H variant of CrChR2. R120 is a key residue in an extended network linking the retinal chromophore to several gates of the ion channel. We show that despite the deficient channel activity, the photocycle of the variant is intact. In a comparative study for R120H and the wild type, we resolve the vibrational changes in the spectral range of the retinal and amide I bands across the time range from femtoseconds to seconds. Analysis of the amide I mode reveals a significant impairment of the ultrafast protein response after retinal excitation. We conclude that channel opening in CrChR2 is prepared immediately after retinal excitation. Additionally, chromophore isomerization is essential for both photocycle and channel activities, although both processes can occur independently.

Magnesium Ion-Driven Folding and Conformational Switching Kinetics of Tetracycline Binding Aptamer: Implications for in vivo Riboswitch Engineering.

Engineering in vitro selected RNA aptamers into in vivo functional riboswitches represents a long-standing challenge in molecular biology. The highly specific aptamer domain of the riboswitch undergoes a conformational adjustment in response to ligand sensing, which in turn exerts the regulatory function. Besides essential factors like structural complexity and ligand binding kinetics, the active role of magnesium ions in stabilizing RNA tertiary structures and assisting in ligand binding can be a vital criterion. We present spectroscopic studies on the magnesium ion-driven folding of the Tetracycline binding aptamer. Using fluorescent labels, the aptamer pre-folding and subsequent ligand binding is monitored by magnesium titration experiments and time-resolved stopped-flow measurements. A minimum concentration of 0.5 mM magnesium is required to fold into a magnesium ion-stabilized binding-competent state with a preformed binding pocket. Tetracycline binding causes a pronounced conformational change that results in the establishment of the triple helix core motif, and that further propagates towards the closing stem. By a dynamic acquisition of magnesium ions, a kink motif is formed at the intersection of the triple helix and closing stem regions. This ultimately entails a stabilization of the closing stem which is discussed as a key element in the regulatory function of the Tetracycline aptamer.

Conserved tyrosine in phytochromes controls the photodynamics through steric demand and hydrogen bonding capabilities.

Using ultrafast spectroscopy and site-specific mutagenesis, we demonstrate the central role of a conserved tyrosine within the chromophore binding pocket in the forward (Pr â†’ Pfr) photoconversion of phytochromes. Taking GAF1 of the knotless phytochrome All2699g1 from Nostoc as representative member of phytochromes, it was found that the mutations have no influence on the early (<30 ps) dynamics associated with conformational changes of the chromophore in the excited state. Conversely, they drastically impact the extended protein-controlled excited state decay (>100 ps). Thus, the steric demand, position and H-bonding capabilities of the identified tyrosine control the chromophore photoisomerization while leaving the excited state chromophore dynamics unaffected. In effect, this residue operates as an isomerization-steric-gate that tunes the excited state lifetime and the photoreaction efficiency by modulating the available space of the chromophore and by stabilizing the primary intermediate Lumi-R. Understanding the role of such a conserved structural element sheds light on a key aspect of phytochrome functionality and provides a basis for rational design of optimized photoreceptors for biotechnological applications.

Mechanistic Elucidation of the Hula-Twist Photoreaction in Hemithioindigo.

The Hula-Twist (HT) photoreaction represents a fundamental photochemical pathway for bond isomerizations and is defined by the coupled motion of a double bond and an adjacent single bond. This photoreaction has been suggested as the defining motion for a plethora of light-responsive chromophores such as retinal within opsins, coumaric acid within photoactive yellow protein, or vitamin D precursors, and stilbenes in solution. However, due to the fleeting character of HT photoproducts a direct experimental observation of this coupled molecular motion was severely hampered until recently. To solve this dilemma, the Dube group has designed a molecular framework able to deliver unambiguous experimental evidence of the HT photoreaction. Using sterically crowded atropisomeric hemithioindigo (HTI) the HT photoproducts are rendered thermally stable and can be observed directly after their formation. However, following the ultrafast excited state process of the HT photoreaction itself has not been achieved so far and thus crucial information for an elementary understanding is still missing. In this work, we present the first ultrafast spectroscopy study of the HT photoreaction in HTI and probe the competition between different excited state processes. Together with extensive excited state calculations a detailed mechanistic picture is developed explaining the significant solvent effects on the HT photoreaction and revealing the intricate interplay between productive isomerizations and unproductive twisted intramolecular charge transfer (TICT) processes. With this study essential insights are thus gained into the mechanism of complex multibond rotations in the excited state, which will be of primary importance for further developments in this field.

Photochemistry of the Light-Driven Sodium Pump Krokinobacter eikastus Rhodopsin 2 and Its Implications on Microbial Rhodopsin Research: Retrospective and Perspective.

The discovery of the light-driven sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) in 2013 has changed the paradigm that cation transport in microbial rhodopsins is restricted to the translocation of protons. Even though this finding is already remarkable by itself, it also reignited more general discussions about the functional mechanism of ion transport. The unique composition of the retinal binding pocket in KR2 with a tight interaction between the retinal Schiff base and its respective counterion D116 also has interesting implications on the photochemical pathway of the chromophore. Here, we discuss the most recent advances in our understanding of the KR2 functionality from the primary event of photon absorption by all-trans retinal up to the actual protein response in the later phases of the photocycle, mainly from the point of view of optical spectroscopy. In this context, we furthermore highlight some of the ongoing debates on the photochemistry of microbial rhodopsins and give some perspectives for promising future directions in this field of research.

Watching the release of a photopharmacological drug from tubulin using time-resolved serial crystallography.

The binding and release of ligands from their protein targets is central to fundamental biological processes as well as to drug discovery. Photopharmacology introduces chemical triggers that allow the changing of ligand affinities and thus biological activity by light. Insight into the molecular mechanisms of photopharmacology is largely missing because the relevant transitions during the light-triggered reaction cannot be resolved by conventional structural biology. Using time-resolved serial crystallography at a synchrotron and X-ray free-electron laser, we capture the release of the anti-cancer compound azo-combretastatin A4 and the resulting conformational changes in tubulin. Nine structural snapshots from 1 ns to 100 ms complemented by simulations show how cis-to-trans isomerization of the azobenzene bond leads to a switch in ligand affinity, opening of an exit channel, and collapse of the binding pocket upon ligand release. The resulting global backbone rearrangements are related to the action mechanism of microtubule-destabilizing drugs.

Structural and functional consequences of the H180A mutation of the light-driven sodium pump KR2.

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.

RNA Probes for Visualization of Sarcin/ricin Loop Depurination without Background Fluorescence.

Protein synthesis via ribosomes is a fundamental process in all known living organisms. However, it can be completely stalled by removing a single nucleobase (depurination) at the sarcin/ricin loop of the ribosomal RNA. In this work, we describe the preparation and optimization process of a fluorescent probe that can be used to visualize depurination. Starting from a fluorescent thiophene nucleobase analog, various RNA probes that fluoresce exclusively in the presence of a depurinated sarcin/ricin-loop RNA were designed and characterized. The main challenge in this process was to obtain a high fluorescence signal in the hybridized state with an abasic RNA strand, while keeping the background fluorescence low. With our new RNA probes, the fluorescence intensity and lifetime can be used for efficient monitoring of depurinated RNA.

Influence of the PHY domain on the ms-photoconversion dynamics of a knotless phytochrome.

The ability of some knotless phytochromes to photoconvert without the PHY domain allows evaluation of the distinct effect of the PHY domain on their photodynamics. Here, we compare the ms dynamics of the single GAF domain (g1) and the GAF-PHY (g1g2) construct of the knotless phytochrome All2699 from cyanobacterium Nostoc punctiforme. While the spectral signatures and occurrence of the intermediates are mostly unchanged by the domain composition, the presence of the PHY domain slows down the early forward and reverse dynamics involving chromophore and protein binding pocket relaxation. We assign this effect to a more restricted binding pocket imprinted by the PHY domain. The photoproduct formation is also slowed down by the presence of the PHY domain but to a lesser extent than the early dynamics. This indicates a rate limiting step within the GAF and not the PHY domain. We further identify a pH dependence of the biphasic photoproduct formation hinting towards a pKa dependent tuning mechanism. Our findings add to the understanding of the role of the individual domains in the photocycle dynamics and provide a basis for engineering of phytochromes towards biotechnological applications.

Base-Free Synthesis and Photophysical Properties of New Schiff Bases Containing Indole Moiety.

Schiff bases represent an essential class in organic chemistry with antitumor, antiviral, antifungal, and antibacterial activities. The synthesis of Schiff bases requires the presence of an organic base as a catalyst such as piperidine. Base-free synthesis of organic compounds using a heterogeneous catalyst has recently attracted more interest due to the facile procedure, high yield, and reusability of the used catalyst. Herein, we present a comparative study to synthesize new Schiff bases containing indole moieties using piperidine as an organic base catalyst and Au@TiO2 as a heterogeneous catalyst. In both methods, the products were isolated in high yields and fully characterized using different spectral analysis techniques. The catalyst was reusable four times, and the activity was slightly decreased. The presence of Au increases the number of acidic sites of TiO2, resulting in C=O polarization. Yields of the prepared Schiff bases in the presence of Au@TiO2 and piperidine were comparable. However, Au@TiO2 is an easily separable and recyclable catalyst, which would facilitate the synthesis of organic compounds without applying any hazardous materials. Furthermore, the luminescence behavior of the synthesized Schiff bases exhibited spectral shape dependence on the substituent group. Interestingly, the compounds also displayed deep-blue fluorescence with Commission Internationale de l'Éclairage (CIE) coordinates of y < 0.1. Thus, these materials may contribute to decreasing the energy consumption of the emitting devices.

Inactivation of Competitive Decay Channels Leads to Enhanced Coumarin Photochemistry.

In the development of photolabile protecting groups, it is of high interest to selectively modify photochemical properties with structural changes as simple as possible. In this work, knowledge of fluorophore optimization was adopted and used to design new coumarin- based photocages. Photolysis efficiency was selectively modulated by inactivating competitive decay channels, such as twisted intramolecular charge transfer (TICT) or hydrogen-bonding, and the photolytic release of the neurotransmitter serotonin was demonstrated. Structural modifications inspired by the fluorophore ATTO 390 led to a significant increase in the uncaging cross section that can be further improved by the simple addition of a double bond. Ultrafast transient absorption spectroscopy gave insights into the underlying solvent-dependent photophysical dynamics. The chromophores presented here are excellently suited as new photocages in the visible wavelength range due to their simple synthesis and their superior photochemical properties.

Ultrafast separation of multiexcitons within core/shell quantum dot hybrid systems.

We investigated the electron transfer processes in methylene blue-CdTe and methylene blue-CdTe/CdSe complexes by steady state and femtosecond transient absorption spectroscopy by selective excitation of the quantum dot (QD) moiety. The ultrafast electron transfer is accelerated by the shell growth due to the separation of the charge carriers and the resulting increase of electron density in the shell. Transmission electron microscope images show that the successive addition of shell material deforms the spherical QDs until they adopt a tetrapodal shape. The increased donor-acceptor distance in the tetrapodal CdTe/CdSe QDs leads to a slower electron transfer. Photon flux dependent transient absorption measurements indicate the separation of two electrons for the QDs with a thin shell and thus demonstrate that charge carrier multiplication can be directly utilized for increased charge transfer in this type of QD hybrid system.

A long-lived fluorenyl cation: efficiency booster for uncaging and photobase properties.

The photochemistry of fluorenols has been of special interest for many years. This is because both the fluorenol and the fluorenyl cation are antiaromatic in the ground state due to their 4n π-electrons according to the Hückel rule. The photolysis reaction of various fluorene derivatives takes place via a cation intermediate and is preferred due to its excited state aromaticity. Here we present an extremely long-lived fluorenyl cation and its effects on the uncaging of various leaving groups. We analyze the relationship between uncaging quantum yields of fluorene-based cages and the longevity of their fluorenyl cations with different spectroscopic methods in the steady state and on an ultrafast time scale and find that the uncaging quantum yield rises with the stability of the cation. In contrast to previous reports, the cation can be observed on a time scale of minutes, even in moderately protic solvents as methanol and ethanol. The stability of this cation depends on the dimethylamino-substituents on the fluorene scaffold and the properties of the solvent. In addition, with bis-dimethylamino fluorenol, a photobase is introduced that expands the small group of known photoinduced hydroxide emitters.