Natural and non-natural antenna chromophores in the DNA photolyase from Thermus thermophilus

X-ray crystallographic and functional analysis of the class I DNA photolyase from Thermus thermophilus revealed the binding of flavin mononucleotide (FMN) as an antenna chromophore. The binding mode of FMN closely coincides with the binding of a deazaflavin-like chromophore in the related class I DNA photolyase from Anacystis nidulans. Compared to the R46E mutant, which lacks a conserved arginine in the binding site for the antenna chromophore, the FMN-comprising holophotolyase exhibits an eightfold higher activity at 450 nm. The facile incorporation of the flavin cofactors 8-hydroxy-deazariboflavin and 8-iodo-8-demethyl-riboflavin into the binding site for the antenna chromophore paves the way for wavelength-tuning of the activity spectra of DNA photolyases by using synthetic flavins.     

Synthesis and nonlinear optical properties of soluble fluorinated polyimides containing hetarylazo chromophores with large hyperpolarizability

The novel fluorinated polyimides with side-chain nonlinear optical (NLO) chromophores were synthesized from hydroxyl polyimides, followed by the Mitsunobu reaction with NLO chromophores. Molecular structural characterization for the resulting polymers was achieved by ¹H NMR, FT-IR, UV-Vis spectra, elemental analysis and gel permeation chromatography (GPC). The polymers exhibit excellent solubility in common organic solvents, good film-forming properties, high glass transition temperature (T_g) in the range from 193 to 200 °C and thermal stability up to 290 °C. The polyimides P1 and P2 containing hetarylazo chromophores with large hyperpolarizability possess a high electro-optic (EO) coefficient (r₃₃), which is larger than that of the polyimide P3 attached DR1. Excellent temporal stability and low optical losses in the range of 1.9-2.1 dB/cm at 1.55 μm were observed for these polymers. Such new NLO fluorinated polyimides are distinguished by an excellent combination of NLO activity, temporal stability, and optical loss.     

Photochemistry and photophysics of copolymers of (−)menthyl acrylate with 4-hydroxystilbene acrylate

Copolymers of trans-4-hydroxystilbene with (—)menthyl acrylate exhibit upon irradiation transåcis isomerization of the side-chain stilbene chromophores analogously to trans-4-hydroxystilbene-2-methylpropanoate, chosen as low molecular weight model compound. U.v. measurements on polymer samples indicate a deviation from first-order kinetics. In the fluorescence spectra of all-trans polymer samples both excimer and monomer emissions are observed. On irradiation the excimeric component is quenched much faster than the monomeric one. Fluorescence polarization experiments indicate a higher conformational rigidity of the stilbene chromophore when inserted in polymeric systems. Comparison of chiroptical properties of both unirradiated and irradiated copolymers suggests that on irradiation a local conformational rearrangement of macromolecules takes place.     

Reconstruction of the absorption spectrum of an object spot from the colour values of the corresponding pixel(s) in its digital image: the challenge of algal colours

A novel procedure for deriving the absorption spectrum of an object spot from the colour values of the corresponding pixel(s) in its image is presented. Any digital image acquired by a microscope can be used; typical applications are the analysis of cellular/subcellular metabolic processes under physiological conditions and in response to environmental stressors (e.g. heavy metals), and the measurement of chromophore composition, distribution and concentration in cells. In this paper, we challenged the procedure with images of algae, acquired by means of a CCD camera mounted onto a microscope. The many colours algae display result from the combinations of chromophores whose spectroscopic information is limited to organic solvents extracts that suffers from displacements, amplifications, and contraction/dilatation respect to spectra recorded inside the cell. Hence, preliminary processing is necessary, which consists of in vivo measurement of the absorption spectra of photosynthetic compartments of algal cells and determination of spectra of the single chromophores inside the cell. The final step of the procedure consists in the reconstruction of the absorption spectrum of the cell spot from the colour values of the corresponding pixel(s) in its digital image by minimization of a system of transcendental equations based on the absorption spectra of the chromophores under physiological conditions.     

Chromophore exchange in the blue light-sensitive photoreceptor YtvA from Bacillus subtilis

YtvA from Bacillus subtilis was found as the first prokaryotic phototropin-like blue-light-responsive photoreceptor. It is composed of two domains, the photoactive LOV (light, oxygen, voltage) domain, which binds a flavin mononucleotide (FMN) as a chromophore and a STAS (sulfate transporter/anti-sigma-factor antagonist) domain, which generates a physiological signal. Here we present a routine chromophore-exchange protocol that allows chemically synthesized, structurally modified chromophores instead of the naturally present flavin mononucleotide (FMN) chromophore to be introduced. FMN was exchanged for riboflavin (RF), flavin adenine dinucleotide (FAD), 7,8-didemethyl flavin mononucleotide (DMFMN), and 8-isopropyl flavin mononucleotide (iprFMN). LOV domains reconstituted with new flavins undergo the same photocycle as native YtvA LOV, consisting of triplet formation and covalent binding of the chromophore followed by a thermal recovery of the parent state, albeit with different kinetics and photophysical properties. Interestingly, the iprFMN chromophore, inducing steric hindrances to the protein, exhibits a very fast light-to-dark-conversion and shows a high fluorescence quantum yield (0.4). Incorporation of FAD causes an increase of its fluorescence quantum yield from 0.04 (H(2)O) to 0.2.     

Green-Emitting Rhodamine Dyes for Vital Labeling of Cell Organelles Using STED Super-Resolution Microscopy

Fluorescence microscopy reveals the localization, spatial distribution, and temporal dynamics of the specifically labeled organelles in living cells. Labeling with exogenous conjugates prepared from fluorescent dyes and small molecules (ligands) is an attractive alternative to the use of fluorescent proteins, but proved to be challenging due to insufficient cell-permeability of the probes, unspecific staining, or low dye brightness. We evaluated four green-emitting rhodamine dyes and their conjugates intended for the specific labeling of lysosomes, mitochondria, tubulin, and actin in living cells. The imaging performance of the probes in living human fibroblasts has been studied by using confocal and stimulated emission depletion (STED) super-resolution microscopy with a commercial 595 nm STED laser. Two bright and photostable dyes (LIVE 510 and LIVE 515) provide specific and versatile staining.     

The unique optical behaviour of bio‐related materials with organic chromophores

Molecularly designed materials based on macromolecules and organic dyes offer unique opportunities in connection with the possibility of preparing optically responsive ‘smart’ materials. Indeed macromolecules are able to transmit and amplify small signals reaching sites at interacting distance through the involvement of the whole chain. The corresponding materials can then acquire stimuli‐responsive properties in relation to specific features connected to primary structure and conformation. As a first approach to benefit from the above features for preparing eco‐compatible smart materials, bio‐related polypeptides, polysaccharides and polyesters can be used as the macromolecular partner in combination with a selected dye following different interaction methodologies. Two distinct routes were used to prepare optically responsive products from the above bio‐related polymers, respectively based either on the covalent bonding to the original macromolecules of photochromic molecular species, such as azobenzene and spiropyran, or on the morphology‐modulated dispersion of highly conjugated dyes in the polymer bulk. Examples related to the two different routes have been investigated in our laboratory and are presented and discussed also with reference to selected recent cases from the literature. Copyright © 2012 Society of Chemical Industry     

Mutagenic stabilization of the photocycle intermediate of green fluorescent protein (GFP)

The optical spectra of the Aequorea victoria green fluorescent protein (GFP) are governed by an equilibrium between three different chromophore states. Mutants that predominantly show either the protonated (A) or the deprotonated (B) form of the chromophore have previously been described. In contrast, the I form, which is formed by rapid excited-state deprotonation of the A form of the chromophore, has only been described as an obligatory photochemical intermediate. We report the design of a new GFP mutant with a stabilized I form. For this purpose, we introduced two isosteric point mutations, Thr203Val and Glu222Gln, that selectively raise the potential energy of both the A and the B form. Knowledge of the absorption spectrum of the I form at room temperature allows the detailed analysis of concentration dependent changes in bulk wild-type(wt)-GFP spectra, as well as the determination of the dimerization constant of GFP. This information expands the use of GFP to that of a spectral probe for protein concentration. We determined energy differences between the chromophore ground states in the monomer and the dimer and reconstructed part of the potential energy surface.     

Colorimetric Determination of Adenylation Domain Activity in Nonribosomal Peptide Synthetases by Using Chrome Azurol S

Adenylation domains are the main contributor to structural complexity among nonribosomal peptides due to their varied but stringent substrate selection. Several in vitro assays to determine the substrate specificity of these dedicated biocatalysts have been implemented, but high sensitivity is often accompanied by the cost of laborious procedures, expensive reagents or the requirement for auxiliary enzymes. Here, we describe a simple protocol that is based on the removal of ferric iron from a preformed chromogenic complex between ferric iron and Chrome Azurol S. Adenylation activity can be rapidly followed by a decrease in absorbance at 630 nm, visualized by a prominent color change from blue to orange.