Chromophore Structure Research Articles

Characterizing Post‐PKS Modifications of 16‐Demethyl‐rifamycin Revealed Two Dehydrogenases Diverting the Aromatization Mode of Naphthalenic Ring in Ansamycin Biosynthesis

Comprehensive SummaryThe anti‐tuberculous rifamycins belong to naphthalenic ansamycin based on the structure of aromatic chromophore. Herein, we explored the post‐polyketide synthase (PKS) modifications in the biosynthesis of 16‐demethyl‐rifamycins via gene knockout, complementation and in vitro enzyme assays. The collective evidences showed that i) the aromatization of 8‐hydroxyl‐7,8‐dihydronaphtoquinone was accomplished by the combined action of two dehydrogenases, RifS and RifT; ii) the acetylation and methylation of the macrocycle was carried out on naphthoquinone intermediates in preference to naphthol, by Rif‐Orf20 and Rif‐Orf14, respectively; iii) the presence of RifS/T homologs in ansamycin biosynthetic gene clusters corresponds to the dehydrogenation aromatization mode of dihydronaphthalene. These findings cast new insights into the naphthalene formation and post‐PKS modification of ansamycins.

Regulation of Irradiation‐Dependent Long‐Lived Room Temperature Phosphorescence by Controlling Molecular Structures of Chromophores and Matrix

AbstractLong‐lived room temperature phosphorescent (LRTP) materials have attracted widespread attention due to their unique luminescence phenomenon and application prospects in the fields of information encryption and bioimaging. However, achieving intelligent response LRTP from amorphous polymers under atmospheric conditions is fascinating but challenging. Here, a series of irradiation‐dependent LRTP systems with ultralong phosphorescent lifetime and high quantum yields are fabricated by a consuming triplet oxygen strategy, and the LRTP performance exhibits obvious chromophore structure and polymeric molecular weight dependence. By extending the UV irradiation time, the phosphorescence lifetime can be increased by hundreds of times, which can exceed 2 s. Experimental results prove that the molecular weight of the polymer matrix, the doping concentration, and the structure of the chromophore have a crucial influence on the stimulus‐response speed and phosphorescence properties of the materials. This kind of intelligent and irradiation‐dependent LRTP material has excellent application prospects in the field of multi‐level information encryption. This work will effectively promote the development of stimulus‐responsive LRTP materials under atmospheric conditions.

Light- and pH-dependent structural changes in cyanobacteriochrome AnPixJg2

Cyanobacteriochromes (CBCRs) are phytochrome-related photosensory proteins that play an essential role in regulating phototaxis, chromatic acclimation, and cell aggregation in cyanobacteria. Here, we apply solid-state NMR spectroscopy to the red/green GAF2 domain of the CBCR AnPixJ assembled in vitro with a uniformly 13C- and 15N-labeled bilin chromophore, tracking changes in electronic structure, geometry, and structural heterogeneity of the chromophore as well as intimate contacts between the chromophore and protein residues in the photocycle. Our data confirm that the bilin ring D is strongly twisted with respect to the B–C plane in both dark and photoproduct states. We also identify a greater structural heterogeneity of the bilin chromophore in the photoproduct than in the dark state. In addition, the binding pocket is more hydrated in the photoproduct. Observation of interfacial 1H contacts of the photoproduct chromophore, together with quantum mechanics/molecular mechanics (QM/MM)-based structural models for this photoproduct, clearly suggests the presence of a biprotonated (cationic) imidazolium side-chain for a conserved histidine residue (322) at a distance of ~2.7 Å, generalizing the recent theoretical findings that explicitly link the structural heterogeneity of the dark-state chromophore to the protonation of this specific residue. Moreover, we examine pH effects on this in vitro assembled holoprotein, showing a substantially altered electronic structure and protonation of the photoproduct chromophore even with a small pH drop from 7.8 to 7.2. Our studies provide further information regarding the light- and pH-induced changes of the chromophore and the rearrangements of the hydrogen-bonding and electrostatic interaction network around it. Possible correlations between structural heterogeneity of the chromophore, protonation of the histidine residue nearby, and hydration of the pocket in both photostates are discussed.Graphical abstract

Femtosecond multidimensional spectroscopy with multiple repetition-frequency-stabilized lasers: tutorial

Time-resolved multidimensional spectroscopy is an advanced spectroscopic technique that can be used to study the molecular structure and dynamics of chromophores in condensed phases by probing multiple resonances of chemical and biophysical systems. To achieve such a multidimensional measurement, the nonlinear optical response of materials should be measured for varying time delays between multiple optical pulses. Inevitably, the data acquisition time dramatically increases with the number of dimensions and the length of each time-delay scan. Therefore, technical breakthroughs toward improved data acquisition rates and time resolutions have long been sought for more versatile and extensive use of coherent multidimensional spectroscopy. Here, we present a tutorial description of the concepts and methods of coherent multidimensional spectroscopy with multiple repetition-frequency-stabilized lasers. Pulse trains from two lasers with slightly different repetition frequencies enable an asynchronous optical sampling (ASOPS), i.e., an automatic scan of the time delay between the pulses. By combining mechanical delay lines for interferometric pulse-pair generation and signal field detection with an incoherent ASOPS scheme for probing slow population dynamics of chromophores in condensed phases, we show that the dynamic range of time-delay scan and data acquisition speed for coherent multidimensional spectroscopy could be dramatically enhanced. In this tutorial paper, we summarize the current developments in this and related research areas, and provide perspectives on all optically controlled multidimensional spectroscopy with multiple synchronized lasers.

A Hückel Model for the Excited-State Dynamics of a Protein Chromophore Developed Using Photoelectron Imaging.

ConspectusChemistry can be described as the movement of nuclei within molecules and the concomitant instantaneous change in electronic structure. This idea underpins the central chemical concepts of potential energy surfaces and reaction coordinates. To experimentally capture such chemical change therefore requires methods that can probe both the nuclear and electronic structure simultaneously and on the time scale of atomic motion. In this Account, we show how time-resolved photoelectron imaging can do exactly this and how it can be used to build a detailed and intuitive understanding of the electronic structure and excited-state dynamics of chromophores. The chromophore of the photoactive yellow protein (PYP) is used as a case study. This chromophore contains a para-substituted phenolate anion, where the substituent, R, can be viewed as an acrolein derivative. It is shown that the measured photoelectron angular distribution can be directly related to the electronic structure of the para-substituted phenolate anion. By incrementally considering differing R groups, it is also shown that these photoelectron angular distributions are exquisitely sensitive to the conformational flexibility of R and that when R contains a π-system the excited states of the chromophore can be viewed as a linear combination of the π* molecular orbitals on the phenolate (πPh*) and the R substituent (πR*). Such Hückel treatment shows that the S1 state of the PYP chromophore has predominantly πR* character and that it is essentially the same as the chromophore of the green fluorescent protein (GFP). The S1 excited-state dynamics of the PYP chromophore probed by time-resolved photoelectron imaging clearly reveals both structural (nuclear) dynamics through the energy spectrum and electronic dynamics through the photoelectron angular distributions. Both motions can be accurately assigned using quantum chemical calculations, and these are consistent with the intuitive Hückel treatment presented. The photoactive protein chromophores considered here are examples of where a chemists’ intuitive Hückel view for ground-state chemistry appears to be transferable to the prediction of photochemical excited-state reactivity. While elegant and insightful, such models have limitations, including nonadiabatic dynamics, which is present in a related PYP chromophore, where a fraction of the S1 state population forms a nonvalence (dipole-bound) state of the anion.

Cyan fluorescent proteins derived from mNeonGreen.

mNeonGreen, an engineered green fluorescent protein (GFP) derived from lancelet, is one of the most brightly fluorescent homologs of Aequorea victoria jellyfish GFP (avGFP) yet reported. In this work, we investigated whether this bright fluorescence might be retained in homologs of mNeonGreen with modified chromophore structures and altered fluorescent hues. We found mNeonGreen to be generally less tolerant than avGFP to chromophore modification by substitution of the key chromophore-forming tyrosine residue with other aromatic amino acids. However, we were ultimately successful in creating a variant, designated as NeonCyan1, with a tryptophan-derived cyan fluorescent protein (CFP)-type chromophore, and two additional mutants with distinct spectral hues. Structural, computational, and photophysical characterization of NeonCyan1 and its variants provided insight into the factors that control the fluorescence emission color. Though not recommended as replacements for contemporary CFP variants, we demonstrate that NeonCyan1 variants are potentially suitable for live cell imaging applications.

A novel violet fluorescent protein contains a unique oxidized tyrosine as the simplest chromophore ever reported in fluorescent proteins.

We describe an engineered violet fluorescent protein from the lancelet Branchiostoma floridae (bfVFP). This is the first example of a GFP-like fluorescent protein with a stable fluorescent chromophore lacking an imidazolinone ring; instead, it consists of oxidized tyrosine 68 flanked by glycine 67 and alanine 69. bfVFP contains the simplest chromophore reported in fluorescent proteins and was generated from the yellow protein lanFP10A2 by two synergetic mutations, S148H and C166I. The chromophore structure was confirmed crystallographically and by high-resolution mass spectrometry. The photophysical characteristics of bfVFP (323/430 nm, quantum yield 0.33, and Ec 14,300 M-1 cm-1 ) make it potentially useful for multicolor experiments to expand the excitation range of available FP biomarkers and Förster resonance energy transfer with blue and cyan fluorescent protein acceptors.

Photoinduced reaction mechanisms in prototypical and bathy phytochromes.

Phytochromes, found in plants, fungi, and bacteria, exploit light as a source of information to control physiological processes via photoswitching between two states of different physiological activity, i.e. a red-absorbing Pr and a far-red-absorbing Pfr state. Depending on the relative stability in the dark, bacterial phytochromes are divided into prototypical and bathy phytochromes, where the stable state is Pr and Pfr, respectively. In this work we studied representatives of these groups (prototypical Agp1 and bathy Agp2 from Agrobacterium fabrum) together with the bathy-like phytochrome XccBphP from Xanthomonas campestris by resonance Raman and IR difference spectroscopy. In all three phytochromes, the photoinduced conversions display the same mechanistic pattern as reflected by the chromophore structures in the various intermediate states. We also observed in each case the secondary structure transition of the tongue, which is presumably crucial for the function of phytochrome. The three phytochromes differ in details of the chromophore conformation in the various intermediates and the energetic barrier of their respective decay reactions. The specific protein environment in the chromophore pocket, which is most likely the origin for these small differences, also controls the proton transfer processes concomitant to the photoconversions. These proton translocations, which are tightly coupled to the structural transition of the tongue, presumably proceed via the same mechanism along the Pr → Pfr conversion whereas the reverse Pfr → Pr photoconversion includes different proton transfer pathways. Finally, classification of phytochromes in prototypical and bathy (or bathy-like) phytochromes is discussed in terms of molecular structure and mechanistic properties.

Synthesis of alpha-pyrones and chromen-2-ones by transition-metal catalyzed annulations of sulfoxonium and iodonium ylides withcis-stilbene acids

The protocol represents the efficiency of a C–H activation strategy in the construction of pharmacophoric and chromophoric structures employingcis-stilbene acids as directing groups.

Tuning the aromatic backbone twist in dipyrrolonaphthyridinediones.

This communication describes the photophysical behavior of three analogs of cyclophane bearing the dipyrrolonaphthyridinedione (DPND) core. In these molecules, intersystem crossing (ISC) can be successfully induced by distinct changes in the deviation from planarity within the DPND core, allowing at the same time the emission maximum to shift from the green to red region of the visible spectrum without any synthetic modifications of the chromophore structure. This finding may build the foundation for a new paradigm for inducing ISC-type transitions within other centrosymmetric and planar cross-conjugated chromophores.

Ultrafast Aggregation-Induced Tunable Emission Enhancement in a Benzothiadiazole-Based Fluorescent Metal-Organic Framework Linker.

Aggregation-induced emission enhancement (AIEE) is a process recently exploited in solid-state materials and organic luminophores, and it is explained by tight-molecular packaging. However, solution-phase AIEE and its formation mechanism have not been widely explored. This work investigated AIEE phenomena in two donor-acceptor-donor-type benzodiazole-based molecules (the organic building block in metal-organic frameworks) with an acetylene and phenyl π-conjugated backbone tapered with a carboxylic acid group at either end. This was done using time-resolved electronic and vibrational spectroscopy in conjunction with time-dependent density functional theory (TD-DFT) calculations. Fluorescence up-conversion spectroscopy and time-correlated single-photon counting conclusively showed an intramolecular charge transfer-driven aggregate emission enhancement. This is shown by a red spectral shift of the emission spectra as well as an increase in the fluorescence lifetime from 746 ps at 1.0 × 10-11 to 2.48 ns at 2.0 × 10-3 M. The TD-DFT calculations showed that a restricted intramolecular rotation mechanism is responsible for the enhanced emission. The femtosecond infrared (IR) transient absorption results directly revealed the structural dynamics of aggregate formation, as evident from the evolution of the C≡C vibrational marker mode of the acetylene unit upon photoexcitation. Moreover, the IR data clearly indicated that the aggregation process occurred over a time scale of 10 ps, which is consistent with the fluorescence up-conversion results. Interestingly, time-resolved results and DFT calculations clearly demonstrated that both acetylene bonds and the sulfur atom are the key requirements to achieve such a controllable aggregation-induced fluorescence enhancement. The finding of the work not only shows how slight changes in the chemical structure of fluorescent chromophores could make a tremendous change in their optical behavior but also prompts a surge of research into a profound understanding of the mechanistic origins of this phenomenon. This may lead to the discovery of new chemical strategies that aim to synthesize novel chromophores with excellent optical properties for light-harvesting applications.

All-Red-Light Photoswitching of Indirubin Controlled by Supramolecular Interactions.

Red-light responsiveness of photoswitches is a highly desired property for many important application areas such as biology or material sciences. The main approach to elicit this property uses strategic substitution of long-known photoswitch motives such as azobenzenes or diarylethenes. Only very few photoswitches possess inherent red-light absorption of their core chromophore structures. Here, we present a strategy to convert the long-known purple indirubin dye into a prolific red-light-responsive photoswitch. In a supramolecular approach, its photochromism can be changed from a negative to a positive one, while at the same time, significantly higher yields of the metastable E-isomer are obtained upon irradiation. E- to Z-photoisomerization can then also be induced by red light of longer wavelengths. Indirubin therefore represents a unique example of reversible photoswitching using entirely red light for both switching directions.

Strongly Hydrogen-Bonded Schiff Base and Adjoining Polyene Twisting in the Retinal Chromophore of Schizorhodopsins.

A transmembrane proton gradient is generated and maintained by proton pumps in a cell. Metagenomics studies have recently identified a new category of rhodopsin intermediates between type-1 rhodopsins and heliorhodopsins, named schizorhodopsins (SzRs). SzRs are light-driven inward proton pumps. Comprehensive resonance Raman measurements were conducted to characterize the structure of the retinal chromophore in the unphotolyzed state of four SzRs. The spectra of all four SzRs show that the retinal chromophore is in the all-trans and 15-anti configuration and that the Schiff base is protonated. The polyene chain is planar in the center of the retinal chromophore and is twisted in the vicinity of the protonated Schiff base. The protonated Schiff base in the SzRs forms a stronger hydrogen bond than that in outward proton-pumping rhodopsins. We determined that the hydrogen-bonding partner of the protonated Schiff base is not a water molecule but an amino acid residue, presumably an Asp residue in helix G. The present observations provide valuable insights into the inward proton-pumping mechanism of SzRs.

Molecular modeling of methacrylic composite materials doped with nonlinear optical azochromophores with various acceptor fragments

The methacrylate-based composite polymer materials with various weight content of azochromophores-guests – aminoazobenzenes with various acceptor moieties: AAB-NO2, AAB-DCV, AAB-TCV, and AAB-TCP, – are studied by atomistic modeling. Polymer matrices are PMMA, copolymer of methyl methacrylate and methacrylic acid, MMA-MAA, and copolymer MMA-MAZ, MAZ unit containing azochromophore, covalently attached to the side chain via spacer. The realization of various non-covalent interactions (hydrogen and π-π interactions) is considered, special emphasis is given to the role of chromophore structure and polymer matrix nature.It is shown that rather uniform distribution of chromophores in composite materials is retained even at 30 wt% content. In all composites with chromophore content growth both the number of non-covalently bound chromophores and the proportion of chromophores bound inter se increase, while the proportion of chromophores, bound with polymer matrix, decreases.The number of hydrogen bonds (HBs) between chromophores-guests is determined mostly by the nature of the acceptor group of the chromophore, maximum number of HBs being formed by AAB-TCP. The MMA-MAA matrix allows realization of greater number of inter-chain HBs compared to MMA-MAZ matrix, while such bonds are absent in PMMA.Azochromophores in MAZ units of MMA-MAZ matrix form π-π-stacking structures with mostly codirected chromophore dipole moments, besides AAB-TCV guests form such stacking structures with host matrix chromophores. Chromophores in MAZ units more easily interact with guests than with each other via π-π stacking. The studied matrices could be considered promising to be used as hosts at developing composite materials with quadratic nonlinear optical response.

Conformational Analysis of a Retinal Schiff Base Chromophore in Proteorhodopsin by Raman Optical Activity.

Raman optical activity (ROA) spectroscopy was used to study the conformation of the retinal Schiff base chromophore in green-light-absorbing proteorhodopsin, which is a globally distributed light-driven proton pump of aquatic bacteria. The ROA spectrum consisted mostly of the negative vibrational bands of the chromophore, while the hydrogen out-of-plane mode (at 960 cm-1) appeared as the sole positive band. This distinct spectral feature was not explained by the twisted structure of the retinal Schiff base but was reproduced by the structural model in which the polyene chain on the β-ionone ring side was bent out-of-plane. The bent chromophore structure potentially couples with proton pumping through the motion of the sixth helix in contact with the β-ionone ring.

Ligand-based rational design, synthesis and evaluation of novel potential chemical chaperones for opsin

Inherited blinding diseases retinitis pigmentosa (RP) and a subset of Leber's congenital amaurosis (LCA) are caused by the misfolding and mistrafficking of rhodopsin molecules, which aggregate and accumulate in the endoplasmic reticulum (ER), leading to photoreceptor cell death. One potential therapeutic strategy to prevent the loss of photoreceptors in these conditions is to identify opsin-binding compounds that act as chemical chaperones for opsin, aiding its proper folding and trafficking to the outer cell membrane. Aiming to identify novel compounds with such effect, a rational ligand-based approach was applied to the structure of the visual pigment chromophore, 11-cis-retinal, and its locked analogue 11-cis-6mr-retinal. Following molecular docking studies on the main chromophore binding site of rhodopsin, 49 novel compounds were synthesized according to optimized one-to seven-step synthetic routes. These agents were evaluated for their ability to compete for the chromophore binding site of opsin, and their capacity to increase the trafficking of the P23H opsin mutant from the ER to the cell membrane. Different new molecules displayed an effect in at least one assay, acting either as chemical chaperones or as stabilizers of the 9-cis-retinal-rhodopsin complex. These compounds could provide the basis to develop novel therapeutics for RP and LCA.

Quantitative and anatomical imaging of dermal angiopathy by noninvasive photoacoustic microscopic biopsy.

The ability to noninvasively acquire the fine structure of deep tissues is highly valuable but remains a challenge. Here, a photoacoustic microscopic biopsy (PAMB) combined switchable spatial-scale optical excitation with single-element depth-resolved acoustic detection mode was developed, which effectively coordinated the spatial resolution and the penetration depth for visualizations of skin delamination and chromophore structures up to reticular dermis depth, with the lateral resolution from 1.5 to 104 μm and the axial resolution from 34 to 57 μm. The PAMB obtained anatomical imaging of the pigment distribution within the epidermis and the vascular patterns of the deep dermal tissue, enabling quantification of morphological abnormalities of angiopathy without the need for exogenous contrast agents. The features of healthy skin and scar skin, and the abnormal alteration of dermal vasculature in port wine stains (PWS) skin were first precisely displayed by PAMB-shown multi-layered imaging. Moreover, the quantitative vascular parameters evaluation of PWS were carried out by the detailed clinical PAMB data on 174 patients, which reveals distinct differences among different skin types. PAMB captured the PWS changes in capillary-loop depth, diameter, and vascular volume, making it possible to perform an objective clinical evaluation on the severity of PWS. All the results demonstrated the PAMB can provide vascular biopsy and new indexes deep into the dermal skin noninvasively, which should be meaningful to timely evaluate the pathological types and treatment response of skin diseases. This opens up a new perspective for label-free and non-invasive biopsies of dermal angiopathy.

Enhanced photocatalytic degradation of rhodamine 6G (R6G) using ZnO–Ag nanoparticles synthesized by pulsed laser ablation in liquid (PLAL)

Photocatalytic degradation is developing rapidly due to its simple and low-cost process, which results in effective and complete mineralization with no waste disposal problems. In this work, we report the enhancement of Rhodamine 6G (R6G) photocatalytic degradation by applying silver (Ag) nanoparticles on a zinc oxide (ZnO) nanoparticle structure using a pulsed laser ablation in liquid (PLAL) technique. The unique structure was obtained without applying any chemical substance and the Ag concentration was varied by changing the Ag target ablation time from 1 to 5 min. Generally, the inclusion of Ag increased the photocatalytic ability of ZnO and facilitated a higher degradation rate of R6G than pure ZnO. The generation of hydroxyl radical (OH•) was found as the main oxidative pathway, which led to R6G degradation by cleaving the chromophore structure. The kinetics of the photocatalytic degradation reaction was also carried out in this work. This research showed the great potential of a hybrid nanomaterial, developed through a laser ablation technique, for environmental protections.

Resonance Raman Determination of Chromophore Structures of Heliorhodopsin Photointermediates.

Light is utilized as energy or information by rhodopsins (membrane proteins that contain a retinal chromophore). Heliorhodopsins (HeRs) are a new class of rhodopsins with low sequence identity (<15%) to microbial and animal rhodopsins. Their physiological roles remain unknown, although the involvement of a long-lived intermediate in the photocycle suggests a light-sensor function. Characterization of the molecular structures of the intermediates is essential to an understanding of the roles and mechanisms of HeRs. We determined the chromophore structures of the intermediates in HeR 48C12 by time-resolved resonance Raman spectroscopy and observed that the hydrogen bond of the protonated Schiff base strengthened prior to deprotonation. The chromophore is photoisomerized from the all-trans to the 13-cis form and is reisomerized in the transition from the O intermediate to the unphotolyzed state. Our results demonstrate that the chromophore structure evolves similarly to microbial rhodopsins, despite the dissimilarity in amino acid residues surrounding the chromophore.

A sensitive chemiluminescence detection approach for determination of 2,4-dinitrophenylhydrazine derivatized aldehydes using online UV irradiation – luminol CL reaction. Application to the HPLC analysis of aldehydes in oil samples

Aldehydes are toxic carbonyl compounds that are identified in various matrices surrounding us. For instance, aldehydes could be formed during the cooking and frying of foods which affects the food quality and safety. Derivatization is a must for the determination of aldehydes as they lack intrinsic chromophoric groups. 2,4-Dinitrophenyl hydrazine (DNPH) is the most used derivatizing reagent for aldehydes and the formed hydrazones could be determined by either HPLC-UV or LC-MS. However, UV detection is non-sensitive, and the MS equipment is expensive and not widely available. Thus, herein we report a smart chemiluminescence (CL) detection method for the DNPH aldehydes derivatives. These derivatives are supposed to possess photosensitization ability due to the presence of strong chromophoric structures; nitrobenzene and phenyl hydrazone. Upon their UV irradiation, singlet oxygen is found to be produced which then converts the DNPH-aldehyde derivative into hydroperoxide. Next, the hydroperoxide reacts with luminol in an alkaline medium producing a strong CL. An HPLC system with online UV irradiation and online reaction with luminol followed by CL detection was constructed and used for the determination of aldehydes after their derivatization with DNPH. The developed method showed excellent sensitivity with detection limits down to 1.5–18.5 nM. The achieved sensitivity is superior to that obtained by HPLC-UV and LC-MS detection methods for DNPH-aldehydes derivatives. Additionally, our approach is an chemiluminogenic where the DNPH reagent itself does not produce CL which is an excellent advantage. The method was applied successfully for the determination of aldehydes in canola oil samples using simple liquid-liquid extraction showing good recovery (87.0–106.0%), accuracy (87.2–106.6), and precision (RSD≤10.2%). After analysis of fresh and heated oil samples, it was demonstrated that heating of oil, even for short time, strongly elevated the level of their aldehydes' content. At last, it was found that the results of the analysis of aldehydes in oil samples using the proposed method perfectly matched those obtained by a reference LC-MS method.