Photo-isomerization of the isolated photoactive yellow protein chromophore: what comes before the primary step?

Photoactive proteins typically rely on structural changes in a small chromophore to initiate a biological response. While these changes often involve isomerization as the "primary step", preceding this is an ultrafast relaxation of the molecular framework caused by the sudden change in electronic structure upon photoexcitation. Here, we capture this motion for an isolated model chromophore of the photoactive yellow protein using time-resolved photoelectron imaging. It occurs in <150 fs and is apparent from a spectral shift of ∼70 meV and a change in photoelectron anisotropy. Electronic structure calculations enable the quantitative assignment of the geometric and electronic structure changes to a planar intermediate from which the primary step can then proceed.

Mechanism of Cyanine5 to Cyanine3 Photoconversion and Its Application for High-Density Single-Particle Tracking in a Living Cell.

Cyanine (Cy) dyes are among the most useful organic fluorophores that have found a wide range of applications in single-molecule and super-resolution imaging as well as in other biophysical studies. However, recent observations that blueshifted derivatives of Cy dyes are formed via photoconversion have raised concerns as to the potential artifacts in multicolor imaging. Here, we report the mechanism for the photoconversion of Cy5 to Cy3 that occurs upon photoexcitation during fluorescent imaging. Our studies show that the formal C2H2 excision from Cy5 occurs mainly through an intermolecular pathway involving a combination of bond cleavage and reconstitution while unambiguously confirming the identity of the fluorescent photoproduct of Cy5 to be Cy3 using various spectroscopic tools. The carbonyl products generated from singlet oxygen-mediated photooxidation of Cy5 undergo a sequence of carbon-carbon bond-breaking and -forming events to bring about the novel dye-to-dye transformation. We also show that the deletion of a two-methine unit from the polymethine chain, which results in the formation of blueshifted products, commonly occurs in other cyanine dyes, such as Alexa Fluor 647 (AF647) and Cyanine5.5. The formation of a blueshifted congener dye can obscure the multicolor fluorescence imaging, leading to misinterpretation of the data. We demonstrate that the potentially deleterious photoconversion, however, can be exploited to develop a new photoactivation method for high-density single-particle tracking in a living cell without using UV illumination and cell-toxic additives.

Photocontrolled DNA Origami Assembly by Using Two Photoswitches.

Stimuli-responsive switching molecules have been widely investigated for the purpose of the mechanical control of biomolecules. Recently developed arylazopyrazole (AAP) shows photoisomerization activity, displaying a faster response to light-induced conformational changes and unique absorption spectral properties compared with those of conventionally used azobenzene. Herein, it is demonstrated that AAP can be used as a photoswitching molecule to control photoinduced assembly and disassembly of DNA origami nanostructures. An AAP-modified DNA origami has been designed and constructed. It is observed that the repeated assembly and disassembly of AAP-modified X-shaped DNA origami and hexagonal origami with complementary strands can be achieved by alternating UV and visible-light irradiation. Closed and linear assemblies of AAP-modified X-shaped origami were successfully formed by photoirradiation, and more than 1 µm linear assemblies were formed. Finally, it is shown that the two photoswitches, AAP and azobenzene, can be used in tandem to independently control different assembly configurations by using different irradiation wavelengths. AAP can extend the variety of available wavelengths of photoswitches and stably result in the assembly and disassembly of various DNA origami nanostructures.

Effect of APTES modified TiO2 on antioxidant enzymes activity secreted by Escherichia coli and Staphylococcus epidermidis.

In this work, the impact of APTES-modified TiO2 photocatalysts on antioxidant enzymes (catalase and superoxide dismutase) activity secreted by bacteria was presented. Microbial tests has been examined using Escherichia coli (ATCC 29425) and Staphylococcus epidermidis (ATCC 49461) as model organisms. It was found that APTES-TiO2 affected the activity of antioxidant enzymes. Additionally, obtained APTES-TiO2 photocatalysts were capable of total E. coli and S. epidermidis inactivation under artificial solar light irradiation. The sample modified with the concentration of APTES equals 300 mM (TiO2-4h-120°C-300mM) showed the strongest photocatalytic activity toward both bacteria species. The two-stage photocatalytic mechanism of bacteria response to photocatalysts was proposed.

Light-driven post-translational installation of reactive protein side chains.

Post-translational modifications (PTMs) greatly expand the structures and functions of proteins in nature1,2. Although synthetic protein functionalization strategies allow mimicry of PTMs3,4, as well as formation of unnatural protein variants with diverse potential functions, including drug carrying5, tracking, imaging6 and partner crosslinking7, the range of functional groups that can be introduced remains limited. Here we describe the visible-light-driven installation of side chains at dehydroalanine residues in proteins through the formation of carbon-centred radicals that allow C-C bond formation in water. Control of the reaction redox allows site-selective modification with good conversions and reduced protein damage. In situ generation of boronic acid catechol ester derivatives generates RH2C• radicals that form the native (ß-CH2-γ-CH2) linkage of natural residues and PTMs, whereas in situ potentiation of pyridylsulfonyl derivatives by Fe(II) generates RF2C• radicals that form equivalent ß-CH2-γ-CF2 linkages bearing difluoromethylene labels. These reactions are chemically tolerant and incorporate a wide range of functionalities (more than 50 unique residues/side chains) into diverse protein scaffolds and sites. Initiation can be applied chemoselectively in the presence of sensitive groups in the radical precursors, enabling installation of previously incompatible side chains. The resulting protein function and reactivity are used to install radical precursors for homolytic on-protein radical generation; to study enzyme function with natural, unnatural and CF2-labelled post-translationally modified protein substrates via simultaneous sensing of both chemo- and stereoselectivity; and to create generalized 'alkylator proteins' with a spectrum of heterolytic covalent-bond-forming activity (that is, reacting diversely with small molecules at one extreme or selectively with protein targets through good mimicry at the other). Post-translational access to such reactions and chemical groups on proteins could be useful in both revealing and creating protein function.

A photochemical dehydrogenative strategy for aniline synthesis.

Chemical reactions that reliably join two molecular fragments together (cross-couplings) are essential to the discovery and manufacture of pharmaceuticals and agrochemicals1,2. The introduction of amines onto functionalized aromatics at specific and pre-determined positions (ortho versus meta versus para) is currently achievable only in transition-metal-catalysed processes and requires halogen- or boron-containing substrates3-6. The introduction of these groups around the aromatic unit is dictated by the intrinsic reactivity profile of the method (electrophilic halogenation or C-H borylation) so selective targeting of all positions is often not possible. Here we report a non-canonical cross-coupling approach for the construction of anilines, exploiting saturated cyclohexanones as aryl electrophile surrogates. Condensation between amines and carbonyls, a process that frequently occurs in nature and is often used by (bio-)organic chemists7, enables a predetermined and site-selective carbon-nitrogen (C-N) bond formation, while a photoredox- and cobalt-based catalytic system progressively desaturates the cyclohexene ring en route to the aniline. Given that functionalized cyclohexanones are readily accessible with complete regiocontrol using the well established carbonyl reactivity, this approach bypasses some of the frequent selectivity issues of aromatic chemistry. We demonstrate the utility of this C-N coupling protocol by preparing commercial medicines and by the late-stage amination-aromatization of natural products, steroids and terpene feedstocks.

Photoenzymatic enantioselective intermolecular radical hydroalkylation.

Enzymes are increasingly explored for use in asymmetric synthesis1-3, but their applications are generally limited by the reactions available to naturally occurring enzymes. Recently, interest in photocatalysis4 has spurred the discovery of novel reactivity from known enzymes5. However, so far photoinduced enzymatic catalysis6 has not been used for the cross-coupling of two molecules. For example, the intermolecular coupling of alkenes with α-halo carbonyl compounds through a visible-light-induced radical hydroalkylation, which could provide access to important γ-chiral carbonyl compounds, has not yet been achieved by enzymes. The major challenges are the inherent poor photoreactivity of enzymes and the difficulty in achieving stereochemical control of the remote prochiral radical intermediate7. Here we report a visible-light-induced intermolecular radical hydroalkylation of terminal alkenes that does not occur naturally, catalysed by an 'ene' reductase using readily available α-halo carbonyl compounds as reactants. This method provides an efficient approach to the synthesis of various carbonyl compounds bearing a γ-stereocentre with excellent yields and enantioselectivities (up to 99 per cent yield with 99 per cent enantiomeric excess), which otherwise are difficult to access using chemocatalysis. Mechanistic studies suggest that the formation of the complex of the substrates (α-halo carbonyl compounds) and the 'ene' reductase triggers the enantioselective photoinduced radical reaction. Our work further expands the reactivity repertoire of biocatalytic, synthetically useful asymmetric transformations by the merger of photocatalysis and enzyme catalysis.

Rethinking the Influence of Chloroplast Movements on Non-photochemical Quenching and Photoprotection.

Under blue light, plant chloroplasts relocate to different areas of the cell. The photoreceptor phototropin2 (phot2) mediates the chloroplast movement mechanism under excess blue light alongside the chloroplast unusual positioning1 (chup1) protein. Recently, it has been proposed that leaf transmittance changes associated with chloroplast relocation affect measurements of nonphotochemical quenching (NPQ), resulting in kinetic differences due to these movements (termed "qM"). We evaluated these claims using Arabidopsis (Arabidopsis thaliana) knock-out mutants lacking either phot2 or chup1 and analyzed the kinetics of both the onset and recovery of NPQ under equivalent intensities of both red and blue light. We also evaluated the photoprotective ability of chloroplast movements both during the early onset of photoinhibition and under sustained excess light. We monitored photoinhibition using the chlorophyll fluorescence parameter of photochemical quenching in the dark, which measures the redox state of QA within PSII without any of the complications of traditional F v /F m measurements. While there were noticeable differences between the responses under red and blue light, the chloroplast movement mechanism had no effect on the rate or amplitude of NPQ onset or recovery. Therefore, we were unable to replicate the "qM" component and its corresponding influence on the kinetics of NPQ in Arabidopsis grown under "shade" conditions. Furthermore, chloroplast relocation had no effect on the high-light tolerance of these plants. These data cast doubt upon the existence of a chloroplast movement-dependent component of NPQ Therefore, the influence of chloroplast movements on photoprotection should be thoroughly reevaluated.

Multidimensional assembly of oxygen vacancy-rich amorphous TiO2-BiOBr-sepiolite composite for rapid elimination of formaldehyde and oxytetracycline under visible light.

Herein, a novel oxygen vacancy-rich amorphous TiO2-BiOBr-sepiolite composite was synthesized through a facile one-pot solvothermal method. Under visible light, it exhibited enhanced adsorption and photocatalytic removal activity towards gaseous formaldehyde, whose reaction rate constant is nearly 11.75, 3.44, 1.69, 2.18 and 6.27 times higher than those of amorphous TiO2, BiOBr, TiO2-BiOBr, oxygen vacancy-poor composite and P25, respectively. Moreover, it also displayed significantly improved photodegradation performance towards oxytetracycline under visible light. The improved photocatalytic activity is mainly ascribed to the synergy between the ternary heterogeneous structure and introduced oxygen vacancy, leading to the superior adsorption performance, extended visible-light adsorption scope and faster carriers' separation rate. The photogenerated holes are the dominant active species during the reaction process. Additionally, a plausible photocatalytic degradation pathway for oxytetracycline was also proposed. In general, this work provides a viable strategy of visible-light-driven photocatalyst for practical environmental remediation of indoor volatile organic compounds (VOCs) and pharmaceuticals and personal care products (PPCPs).

Modified MIL-100(Fe) for enhanced photocatalytic degradation of tetracycline under visible-light irradiation.

Iron-based metal-organic frameworks (MOFs) with low cost and excellent photocatalytic potential are extremely attractive in the field of energy utilization and environmental remediation. In this study, a novel In2S3/MIL-100(Fe) photocatalyst was successfully synthesized by a facile solvothermal method for the first time. Several technologies (such as X-ray diffraction, scanning electron microscope, transmission electron microscope, and X-ray photoelectron spectroscopy) were used to characterize the as-obtained samples and demonstrate the successful combination of MIL-100(Fe) and In2S3. Experimental results showed that 18% of tetracycline (TC) was adsorbed under dark condition and another 70% of TC was degraded under visible-light irradiation when treating 100 mL of TC solution (10 mg/L) with 30 mg of In2S3/MIL-100(Fe) composites. The corresponding TC removal efficiency was almost 1.9 and 1.6 times higher than that of pure MIL-100(Fe) and In2S3, respectively. The mechanism investigations revealed that the heterojunction composite exhibited superior charge transfer than either MIL-100(Fe) or In2S3, and this caused more efficient separation of electron-hole pairs. As a result, more radicals and holes were generated in the composite, leading to better photocatalytic performance. This work highlights the powerful combination of MOFs and semiconductor, which is a promising approach to fabricate heterojunction photocatalyst for wastewater purification.

Ultraviolet-Driven Deamination of Cytidine Ribonucleotides Under Planetary Conditions.

A previously proposed synthesis of pyrimidine ribonucleotides makes use of ultraviolet (UV) light to convert ß-d-ribocytidine-2',3'-cyclic phosphate to ß-d-ribouridine-2',3'-cyclic phosphate, while simultaneously selectively degrading synthetic byproducts. Past studies of the photochemical reactions of pyrimidines have employed mercury arc lamps, characterized by narrowband emission centered at 254 nm, which is not representative of the UV environment of the early Earth. To further assess this process under more realistic circumstances, we investigated the wavelength dependence of the UV-driven conversion of ß-d-ribocytidine-2',3'-cyclic phosphate to ß-d-ribouridine-2',3'-cyclic phosphate. We used constraints provided by planetary environments to assess the implications for pyrimidine nucleotides on the early Earth. We found that the wavelengths of light (255-285 nm) that most efficiently drive the deamination of ß-d-ribocytidine-2',3'-cyclic phosphate to ß-d-ribouridine-2',3'-cyclic phosphate are accessible on planetary surfaces such as those of the Hadean-Archaean Earth for CO2-N2-dominated atmospheres. However, continued irradiation could eventually lead to low levels of ribocytidine in a low-temperature, highly irradiated environment, if production rates are slow.

Binding of red form of Orange Carotenoid Protein (OCP) to phycobilisome is not sufficient for quenching.

The Orange Carotenoid Protein (OCP) is responsible for photoprotection in many cyanobacteria. Absorption of blue light drives the conversion of the orange, inactive form (OCPO) to the red, active form (OCPR). Concomitantly, the N-terminal domain (NTD) and the C-terminal domain (CTD) of OCP separate, which ultimately leads to the formation of a quenched OCPR-PBS complex. The details of the photoactivation of OCP have been intensely researched. Binding site(s) of OCPR on the PBS core have also been proposed. However, the post-binding events of the OCPR-PBS complex remain unclear. Here, we demonstrate that PBS-bound OCPR is not sufficient as a PBS excitation energy quencher. Using site-directed mutagenesis, we generated a suite of single point mutations at OCP Leucine 51 (L51) of Synechocystis 6803. Steady-state and time-resolved fluorescence analyses demonstrated that all mutant proteins are unable to quench the PBS fluorescence, owing to either failed OCP binding to PBS, or, if bound, an OCP-PBS quenching state failed to form. The SDS-PAGE and Western blot analysis support that the L51A (Alanine) mutant binds to the PBS and therefore belongs to the second category. We hypothesize that upon binding to PBS, OCPR likely reorganizes and adopts a new conformational state (OCP3rd) different than either OCPO or OCPR to allow energy quenching, depending on the cross-talk between OCPR and its PBS core-binding counterpart.

Implications of dichlorofluorescein photoinstability for detection of UVA-induced oxidative stress in fibroblasts and keratinocyte cells.

Although the dichlorofluorescein (DCF) assay is widely used to detect the production of UVA-induced ROS, the photostability and phototoxicity of the probe after UVA irradiation remains controversial and the experimental conditions often vary across studies, making it difficult to compare results from different studies. This study aimed to evaluate the suitability of the DCF assay for detection of UVA-induced ROS in human cells after UVA irradiation. Human primary fibroblasts (HPF) and HaCaT cells were loaded with 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) (2, 10, and 50 µM) for 10 and 30 min, before and after exposure to UVA radiation (5-50 J cm-2). Fluorescence was recorded immediately or 30 min after irradiation using three different techniques: microplate reading, flow cytometry, and confocal scanning microscopy. Cell viability was assessed by flow cytometry before and after UVA exposure. A UVA-dose-dependent increase in ROS was observed at 5-50 µM DCFDA, and the magnitude of the fluorescent signal was affected by RPMI medium, as well as DCFDA loading concentration and incubation period. However, higher concentrations of DCFDA compromised the viability of both HaCaT and HPF cells after UVA irradiation. The most sensitive and reliable combination for the ROS assay was pre-incubation with 10 µM DCFDA for 30 min in PBS. Reading the fluorescence 30 min after UVA irradiation diminished the emission signal, as did the DCFDA post-incubation. In conclusion, this single-point DCF assay allowed reproducible and sensitive UVA-induced ROS detection in HaCaT and HPF cells without compromising the cell viability or morphology.

Photocatalytic Micromotors Activated by UV to Visible Light for Environmental Remediation, Micropumps, Reversible Assembly, Transportation, and Biomimicry.

Photocatalytic micromotors are light-induced, chemically powered micromachines based on photocatalytic materials, activated by light illumination, and have redox reactions with environmental solutions to produce chemical gradients and bubbles that propel the micromachines through self-diffusiophoresis, self-electrophoresis, and bubble recoil. Due to the fact that excitation light relates largely to the bandgaps of selected materials, the development of photocatalytic micromotors has experienced an evolution from ultraviolet-light-activated to visible-light-activated and potentially biocompatible systems. Furthermore, due to the strong redox capacity and physical effects caused by the products or product gradients, photocatalytic micromotors have applications in environmental remediation, micropumps, reversible assembly, transportation, and biomimicry.

Enhanced photoelectrochemical water splitting activity of carbon nanotubes@TiO2 nanoribbons in different electrolytes.

Hydrogen production from water splitting by a photocatalytic process is one way that can be used to solve global problems related to energy depletion and environmental pollution. This work aims to design and characterize a novel photocatalyst nanohybrid carbon nanotubes@TiO2 nanoribbons (CNTs@TNRs) for enhanced photoelectrochemical (PEC) water splitting in different electrolytes under visible light irradiance. Here, hydrothermal and chemical vapor deposition (HT-CVD) were combined to grow CNTs @ the nanopits of TNRs producing network of nanohybrid CNTs@TNRs. The structural, morphological, optical, and photocatylatic properties of the TNRs and CNTs@TNRs nanohybrid were characterized by different techniques. The crystallite size is increased from 14.86 nm for TNRs to 21.61 nm for CNTs@TNRs nanohybrid. The CNTs@TNRs nanohybrid has well-resolved nanopits on the surface of the TNRs with an average diameter of 10 nm. The absorption edge of CNTs@TNRs relative to TNRs was strongly shifted to the visible light region. The band gap values are 3.78 and 2.07 eV for TNRs and CNTs@TNRs, respectively. The TNRs and CNTs@TNRs were used for the photocatalytic water splitting under visible light irradiance in Na2S2O3, HCl and KOH electrolytes of different concentrations. The calculated incident photon-to-current conversion efficiency (IPCE) was 97% at 510 nm. These values are higher than those previously reported for different photoelectrodes. The number of hydrogen moles was calculated to be 300 µmol h-1 cm-2. Therefore, our work demonstrates a feasible route for efficient PEC water splitting under sunlight irradiation utilizing the novel CNTs@TNRs photocatalyst.

Successes & challenges in the atomistic modeling of light-harvesting and its photoregulation.

Light-harvesting is a crucial step of photosynthesis. Its mechanisms and related energetics have been revealed by a combination of experimental investigations and theoretical modeling. The success of theoretical modeling is largely due to the application of atomistic descriptions combining quantum chemistry, classical models and molecular dynamics techniques. Besides the important achievements obtained so far, a complete and quantitative understanding of how the many different light-harvesting complexes exploit their structural specificity is still missing. Moreover, many questions remain unanswered regarding the mechanisms through which light-harvesting is regulated in response to variable light conditions. Here we show that, in both fields, a major role will be played once more by atomistic descriptions, possibly generalized to tackle the numerous time and space scales on which the regulation takes place: going from the ultrafast electronic excitation of the multichromophoric aggregate, through the subsequent conformational changes in the embedding protein, up to the interaction between proteins.

Photocatalytic Degradation of Pharmaceuticals Carbamazepine, Diclofenac, and Sulfamethoxazole by Semiconductor and Carbon Materials: A Review.

The presence of pharmaceutical compounds in the environment is a reality that calls for more efficient water treatment technologies. Photocatalysis is a powerful technology available but the high energy costs associated with the use of UV irradiation hinder its large scale implementation. More sustainable and cheaper photocatalytic processes can be achieved by improving the sunlight harvesting and the synthesis of semiconductor/carbon composites has proved to be a promising strategy. Carbamazepine, diclofenac, and sulfamethoxazole were selected as target pharmaceuticals due to their recalcitrant behavior during conventional wastewater treatment and persistence in the environment, as properly reviewed. The literature data on the photocatalytic removal of carbamazepine, diclofenac, and sulfamethoxazole by semiconductor/carbon materials was critically revised to highlight the role of the carbon in the enhanced semiconductor performance under solar irradiation. Generally it was demonstrated that carbon materials induce red-shift absorption and they contribute to more effective charge separation, thus improving the composite photoactivity. Carbon was added as a dopant (C-doping) or as support or doping materials (i.e., nanoporous carbons, carbon nanotubes (CNTs), graphene, and derived materials, carbon quantum dots (CQDs), and biochars) and in the large majority of the cases, TiO2 was the semiconductor tested. The specific role of carbon materials is dependent on their properties but even the more amorphous forms, like nanoporous carbons or biochars, allow to prepare composites with improved properties compared to the bare semiconductor. The self-photocatalytic activity of the carbon materials was also reported and should be further explored. The removal and mineralization rates, as well as degradation pathways and toxicity of the treated solutions were also critically analyzed.

Upconversion superballs for programmable photoactivation of therapeutics.

Upconversion nanoparticles (UCNPs) are the preferred choice for deep-tissue photoactivation, owing to their unique capability of converting deep tissue-penetrating near-infrared light to UV/visible light for photoactivation. Programmed photoactivation of multiple molecules is critical for controlling many biological processes. However, syntheses of such UCNPs require epitaxial growth of multiple shells on the core nanocrystals and are highly complex/time-consuming. To overcome this bottleneck, we have modularly assembled two distinct UCNPs which can individually be excited by 980/808 nm light, but not both. These orthogonal photoactivable UCNPs superballs are used for programmed photoactivation of multiple therapeutic processes for enhanced efficacy. These include sequential activation of endosomal escape through photochemical-internalization for enhanced cellular uptake, followed by photocontrolled gene knockdown of superoxide dismutase-1 to increase sensitivity to reactive oxygen species and finally, photodynamic therapy under these favorable conditions. Such programmed activation translated to significantly higher therapeutic efficacy in vitro and in vivo in comparison to conventional, non-programmed activation.

Photoactivation of silicon rhodamines via a light-induced protonation.

Photoactivatable fluorophores are important for single-particle tracking and super-resolution microscopy. Here we present a photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. Using this photoactivatable fluorophore, we create probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophore's outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy.

Photoinduced inhibition of DNA repair enzymes and the possible mechanism of photochemical transformations of the ruthenium nitrosyl complex [RuNO(ß-Pic)2(NO2)2OH].

In this work we have demonstrated that the ruthenium nitrosyl complex [RuNO(ß-Pic)2(NO2)2OH] is suitable for investigation of the inactivation of DNA repair enzymes in vitro. Photoinduced inhibition of DNA glycosylases such as E. coli Endo III, plant NtROS1, mammalian mNEIL1 and hNEIL2 occurs to an extent of ≥90% after irradiation with the ruthenium complex. The photophysical and photochemical processes of [RuNO(ß-Pic)2(NO2)2OH] were investigated using stationary and time-resolved spectroscopy, and mass spectrometry. A possible mechanism of the photo-processes was proposed from the combined spectroscopic study and DTF calculations, which reveal that the photolysis is multistage. The primary and secondary photolysis stages are the photo-induced cleavage of the Ru-NO bond with the formation of a free nitric oxide and RuIII complex followed by ligand exchange with solvent. For E. coli Endo III, covalent interaction with the photolysis product was confirmed by UV-vis and mass spectrometric methods.