A new protein conformation indicator based on biarsenical fluorescein with an extended benzoic acid moiety.

We demonstrate herein a new protein conformation indicator based on biarsenical fluorescein with an extended benzoic acid moiety. The present indicator is reactive to a genetically introduced tetracysteine motif (Cys-Cys-Xaa-Xaa-Cys-Cys, where Xaa is a noncysteine amino acid) of proteins. Compared to the original biarsenical fluorescein (FlAsH) and the biarsenical Nile red analogue (BArNile), the present indicator exhibited larger fluorescence intensity changes in response to Ca(2+)-induced conformational rearrangements of calmodulin. A calculation of the highest occupied molecular orbital (HOMO) level of the benzoic acid moiety of the indicator molecule supports possible involvement of a photoinduced electron transfer (PET) process. These results indicate that the present indicator is useful for sensitive detection of protein conformational changes.     

Cross-strand split tetra-Cys motifs as structure sensors in a beta-sheet protein

We have designed "split tetra-Cys motifs" that bind the biarsenical fluorescein dye 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH) across strands of a model beta-rich protein. Our strategy was to divide the linear FlAsH binding tetra-Cys sequence such that dye could be fully liganded only when the strands were arranged in space correctly by native protein conformational proximities. We introduced pairs of alternating cysteines on adjacent beta strands of cellular retinoic acid binding protein to create FlAsH binding sites in the native structure. Selective labeling occurred both in vitro and in vivo relative to sites with fewer than four Cys or with inappropriate geometry. Interestingly, two of the split tetra-Cys motif-carrying proteins bound FlAsH whether native or urea unfolded, while one was capable of binding FlAsH only when native. This latter design exemplifies the potential of split motifs as structure sensors.     

Synthesis, screening, and sequencing of cysteine-rich one-bead one-compound peptide libraries

Cysteine-rich peptides are valued as tags for biarsenical fluorophores and as environmentally important reagents for binding toxic heavy metals. Due to the inherent difficulties created by cysteine, the power of one-bead one-compound (OBOC) libraries has never been applied to the discovery of short cysteine-rich peptides. We have developed the first method for the synthesis, screening, and sequencing of cysteine-rich OBOC peptide libraries. First, we synthesized a heavily biased cysteine-rich OBOC library, incorporating 50% cysteine at each position (Ac-X8-KM-TentaGel). Then, we developed conditions for cysteine alkylation, cyanogen bromide cleavage, and direct MS/MS sequencing of that library at the single bead level. The sequencing efficiency of this library was comparable to a traditional cysteine-free library. To validate screening of cysteine-rich OBOC libraries, we reacted a library with the biarsenical FlAsH and identified beads bearing the known biarsenical-binding motif (CCXXCC). These results enable OBOC libraries to be used in high-throughput discovery of cysteine-rich peptides for protein tagging, environmental remediation of metal contaminants, or cysteine-rich pharmaceuticals.     

Photostability and Spectral Properties of Fluorinated Fluoresceins and their Biarsenical Derivatives: A Combined Experimental and Theoretical Study

New fluorinated biarsenical derivatives with improved optical properties based on highly photostable analogs of fluorescein were recently introduced. The photophysical parameters of the triplet excited states as well as photosensitized oxidation reactions of these dyes were determined in order to investigate the influence of molecular structure on the exceptional photostability of these fluorophores. The lack of correspondence between triplet quantum yields and lifetimes with the photobleaching rates of some of the fluorophores of the series suggests that differential reactivities of the excited states with ground state oxygen accounts for the different photodegradation resistances. The UV–visible absorption and emission spectra of the fluorinated fluoresceins and their biarsenical derivatives were evaluated using a TD-DFT/BP86/6-31G** approach, taking bulk solvent effects into account by means of the polarizable continuum model. The calculated properties are in good agreement with experimental data. The S₀→S₁ vertical excitation energies in the gas phase and in water were obtained with the optimized geometries of the excited states. This type of calculation could be used in the rational design of new dyes.     

A Double‐Clicking Bis‐Azide Fluorogenic Dye for Bioorthogonal Self‐Labeling Peptide Tags

Herein, we give the very first example for the development of a fluorogenic molecular probe that combines the two‐point binding specificity of biarsenical‐based dyes with the robustness of bioorthogonal click‐chemistry. This proof‐of‐principle study reports on the synthesis and fluorogenic characterization of a new, double‐quenched, bis‐azide fluorogenic probe suitable for bioorthogonal two‐point tagging of small peptide tags by double strain‐promoted azide–alkyne cycloaddition. The presented probe exhibits remarkable increase in fluorescence intensity when reacted with bis‐cyclooctynylated peptide sequences, which could also serve as possible self‐labeling small peptide tag motifs.     

New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications

We recently introduced a method (Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269-272 and Griffin, B. A.; Adams, S. R.; Jones, J.; Tsien, R. Y. Methods Enzymol. 2000, 327, 565-578) for site-specific fluorescent labeling of recombinant proteins in living cells. The sequence Cys-Cys-Xaa-Xaa-Cys-Cys, where Xaa is an noncysteine amino acid, is genetically fused to or inserted within the protein, where it can be specifically recognized by a membrane-permeant fluorescein derivative with two As(III) substituents, FlAsH, which fluoresces only after the arsenics bind to the cysteine thiols. We now report kinetics and dissociation constants ( approximately 10(-11) M) for FlAsH binding to model tetracysteine peptides. Affinities in vitro and detection limits in living cells are optimized with Xaa-Xaa = Pro-Gly, suggesting that the preferred peptide conformation is a hairpin rather than the previously proposed alpha-helix. Many analogues of FlAsH have been synthesized, including ReAsH, a resorufin derivative excitable at 590 nm and fluorescing in the red. Analogous biarsenicals enable affinity chromatography, fluorescence anisotropy measurements, and electron-microscopic localization of tetracysteine-tagged proteins.     

Tuning the Photophysical Properties of Symmetric Squarylium Dyes: Investigation on the Halogen Modulation Effects

A series of symmetric squarylium dyes (SQDPA-X) with different halogen (X=F, Cl, Br, I) substituents have been developed. The photophysical properties could be facilely tuned by the halogen modulation effects. The strategy of incorporating different halogen substitutions into AIE active luminogens enables a facile approach for exploring new intriguing organic fluorescent dyes.     

Isoform-specific PKA dynamics revealed by dye-triggered aggregation and DAKAP1alpha-mediated localization in living cells

The tetracysteine sequence YRECCPGCCMWR fused to the N terminus of green fluorescent protein (GFP) self-aggregates upon biarsenical labeling in living cells or in vitro. Such dye-triggered aggregates form temperature-dependent morphologies and are dispersed by photobleaching. Fusion of the biarsenical aggregating GFP to the regulatory (R) or catalytic (C) subunit of PKA traps intact holoenzyme in compact fluorescent puncta upon biarsenical labeling. Contrary to the classical model of PKA activation, elevated cAMP does not allow RIalpha and Calpha to diffuse far apart unless the pseudosubstrate inhibitor PKI or locally concentrated substrate is coexpressed. However, RIIalpha releases Calpha upon elevated cAMP alone, dependent on autophosphorylation of the RIIalpha inhibitory domain. DAKAP1alpha overexpression induced R and C outer mitochondrial colocalization and showed similar regulation. Overall, effective separation of type I PKA is substrate dependent, whereas type II PKA dissociation relies on autophosphorylation.     

Rhodamines with a Chloronicotinic Acid Fragment for Live Cell Superresolution STED Microscopy**

Formylation of 2,6-dichloro-5-R-nicotinic acids at C-4 followed by condensation with 3-hydroxy-N,N-dimethylaniline gave analogs of the popular TAMRA fluorescent dye with a 2,6-dichloro-5-R-nicotinic acid residues (R=H, F). The following reaction with thioglycolic acid is selective, involves only one chlorine atom at the carbon between pyridine nitrogen and the carboxylic acid group and affords new rhodamine dyes absorbing at 564/ 573 nm and emitting at 584/ 597 nm (R=H/ F, in aq. PBS). Conjugates of the dyes with “small molecules” provided specific labeling (covalent and non-covalent) of organelles as well as of components of the cytoskeleton in living cells and were combined with fluorescent probes prepared from 610CP and SiR dyes and applied in two-color STED microscopy with a 775 nm STED laser.     

Fluorescent DNA nanotags: supramolecular fluorescent labels based on intercalating dye arrays assembled on nanostructured DNA templates

Fluorescence detection and imaging are vital technologies in the life sciences and clinical diagnostics. The key to obtaining high-resolution images and sensitive detection is to use fluorescent molecules or particles that absorb and emit visible light with high efficiency. We have synthesized supramolecular complexes consisting of a branched DNA template and fluorogenic intercalating dyes. Because dyes can intercalate up to every other base pair, high densities of fluorophores are assembled yet the DNA template keeps them far enough away from each other to prevent self-quenching. The efficiency with which these noncovalent assemblies absorb light is more than 10-fold greater than that of the individual dye molecules. F?rster resonance energy transfer from the intercalated dyes to covalently attached acceptor dyes is very efficient, allowing for wavelength shifting of the emission spectrum. Simple biotinylation of the DNA template allows for labeling of streptavidin-coated synthetic microspheres and mouse T-cells.     

pH-induced changes in electronic absorption and fluorescence spectra of phenazine derivatives

The visible electronic absorption and fluorescence spectra as well as fluorescence polarization degrees of imidazo-[4,5-d]-phenazine (F1), 2-methylimidazo-[4,5-d]-phenazine (F2), 2-trifluoridemethylimidazo-[4,5-d]-phenazine (F3), 1,2,3-triazole-[4,5-d]-phenazine (F4) and their glycosides, imidazo-[4,5-d]-phenazine-N1-beta-D-ribofuranoside (F1rib), 1,2,3-triazole-[4,5-d]-phenazine-N1-beta-D-glucopyranoside (F4gl), were investigated in aqueous buffered solutions over the pH range of 0-12, where the spectral transformations were found to be reversible. The effects of protonation and deprotonation on spectral properties of these dyes were studied. We have determined the ranges of pH, where individual ionic species are predominant. In aqueous buffered solutions the fluorescence was found only for neutral species of F1, F1rib, F2, and F4gl dyes, whereas for the ionic forms of these dyes, as well as for F3 and F4 ones, the fluorescence has not been detected. The concentrational deprotonation pKa values were evaluated from experimental data. It was shown that donor-acceptor properties of the substituent group in the second position of the pentagonal ring substantially affect the values of the deprotonation constants and the character of protonation for chromophore. The substitution of a hydrogen atom in the NH-group by the sugar residue blocks the formation of the anionic species, and results in enhancement of the dye emission intensity. The steep emission dependence for F1 and F1rib over pH range of 0-7 with intensities ratio of IpH 7/IpH 1=60 allows us to propose them as possible indicator dyes in luminescence based pH sensors for investigation of processes accompanied by acidification, e.g. as gastric pH-sensors. A comparative analysis of the studied dyes has shown that F4gl is the most promising compound to be used as a fluorescent probe for investigation of molecular hybridization of nucleic acids.     

Synthesis and Evaluation of [18F]‐Ammonium BODIPY Dyes as Potential Positron Emission Tomography Agents for Myocardial Perfusion Imaging

Recently, we demonstrated the potential of a [¹⁸F]‐trimethylammonium BODIPY dye for cardiac imaging. This is the first example of the use of the [¹⁸F]‐ammonium BODIPY dye for positron emission tomography (PET) myocardial perfusion imaging (MPI). In this report, we extend our study to other ammonium BODIPY dyes with different nitrogen substituents. These novel ammonium BODIPY dyes were successfully prepared and radiolabeled by the SnCl₄‐assisted ¹⁸F–¹⁹F isotopic exchange method. The microPET results and the biodistribution data reveal that nitrogen substituent changes have a significant effect on the in vivo and pharmacological properties of the tracers. Of the novel [¹⁸F]‐ammonium BODIPY dyes prepared in this work, the [¹⁸F]‐dimethylethylammonium BODIPY is superior in terms of myocardium uptake and PET imaging contrast. These results support our hypothesis that the ammonium BODIPY dyes have a great potential for use as PET/optical dual‐modality MPI probes.     

Synthesis, characterization and colour determination using CIELAB colour space of stilbene dyes

The synthesis of six new symmetrical disazo direct dyes containing 4,4′-diaminostilbene-2,2′-disulphonic acid as middle component and N-(2-chlorophenyl)-2-hydroxybenzamide, N-(3-chlorophenyl)-2-hydroxybenzamide, N-(4-chlorophenyl)-2-hydroxybenzamide, N-(2-bromophenyl)-2-hydroxybenzamide, N-(3-bromophenyl)-2-hydroxybenzamide, N-(4-bromophenyl)-2-hydroxybenzamide as coupling components is presented. The synthesized dyes were analyzed by thin layer chromatography, electronic spectra and HPLC technique. Their structures were elucidated by FT/IR and 13C-NMR spectroscopy. The CIELAB (1976) colour space was used in all the colour measurements for the six disazo stilbene dyes under the CIE recommended illuminants: D65 (natural day light), A (tungsten light), F2 (fluorescent light) and the standard 10° observer, respectively. The colour differences: ΔEab* and ΔECMC were calculated against one standard. The results reveal a good colouring power of the new azo-stilbene dyes.     

Fluorescence properties of systems with multiple Förster transfer pairs

A mathematical model has been developed to compute the spectroscopic properties of fluorescence systems with multiple F?rster transfer pairs in a homogeneous 3-dimensional matrix. This model is based on F?rster energy transfer theory and needs only a limited number of parameters which depend only on the properties of the individual dyes and their pair-wise interactions. Yet, the model allows the accurate prediction on the fluorescence properties of systems comprising mutual F?rster transfer between three dyes. The model is compared to an experimental system composed of reverse micelles and water soluble dyes. Although the experimental system might include additional effects that may influence the fluorescence properties (e.g. adsorption to the micelle walls, aggregation of the dyes) the agreement between the mathematical model and the experimental system is reasonably good.     

Homodimeric monomethine cyanine dyes as fluorescent probes of biopolymers

The fluorescence properties of newly synthesized homodimeric monomethine cyanine dyes in the presence of biopolymers are investigated. They do not fluoresce in TE buffer and bidistilled water but become strongly fluorescent (Q(F)=0.3-0.9) in the region 530-650 nm when bound to dsDNA and ssDNA. The detection limit of dsDNA is about 1.7 ng/ml. Some of dyes studied are able to distinguish between dsDNA and ssDNA, RNA, BSA in solution and gel electrophoresis. The influence of different factors (temperature, pH and viscosity of the medium, presence of histone) on the formation of the dye-biopolymer complexes is investigated. The results of steady-state and dynamic fluorescence measurements concerning the different types of binding between dyes and biopolymers show that the new dyes are applicable in molecular biology as highly sensitive fluorescence labels.     

苯并吲哚半菁、二次甲基菁染料的微波合成、光谱性能及其与生物分子的相互作用

采用微波技术,分别以N-烷基-2,3,3-三甲基-4,5-苯并吲哚六氟合磷酸盐与二甲氨基苯甲醛或3-吲哚甲醛为原料,在哌啶的催化下快速合成了4种苯并吲哚半菁染料H1～H4和4种苯并吲哚二次甲基菁染料F1～F4,并采用UV-Vis,1H NMR,IR,元素分析确证了产物的结构.研究了染料在不同溶剂中的吸收光谱性质,结果显示:两类染料在质子性溶剂中随着溶剂极性的增加最大吸收波长出现蓝移;而在非质子性溶剂中随着溶剂极性的减小最大吸收波长发生红移.同时,研究了染料H3和F2在生理条件下与鲑鱼精DNA、牛血清白蛋白、溶菌酶、淀粉酶和糜蛋白酶的相互作用,发现染料H3的荧光强度随着DNA浓度的增加而增强.     

Cyanide sensing with organic dyes: studies in solution and on nanostructured Al2O3 surfaces

The synthesis of two new azo phenyl thiourea compounds and their optical response to different anions is reported herein. Solution studies in methanol indicate that cyanide induces a colour change in these dyes (whereas no changes are observed in the presence of other anions, such as F(-), Cl(-), Br(-), CH(3)COO(-), H(2)PO(4) (-), HSO(4) (-)). Interestingly, in DMSO these dyes are responsive not only to cyanide, but also to fluoride, acetate and dihydrogen phosphate. Each of these anions induces a different colour change. In the second part of the paper, we report the attachment of one of these dyes onto nanostructured TiO(2) and Al(2)O(3) films. The stability of these sensitised films to pH was studied and we concluded that the sensitised Al(2)O(3) films are more robust, and hence, better than the TiO(2) for anion sensing. The dye-sensitised Al(2)O(3) films were immersed in solutions of different anions and their response studied. The films can detect cyanide down to 3 ppm in aqueous solution with relatively good selectivity over other anions.     

Triazine dye binding of human alpha-fetoprotein and albumin

Column chromatography was used to investigate the interaction of human alpha-fetoprotein and albumin with different immobilized dyes. Binding of alpha-fetoprotein to the dye conjugates was studied at pH 7.0. Between 56 and 93% of the total alpha-fetoprotein applied to the column was recovered in the break-through fractions of the respective runs. Of all the dyes, Cibacron Blue F3G-A adsorbs alpha-fetoprotein most strongly. This interaction clearly depends on the degree of dye substitution of the gel. A relatively weak interaction exists between alpha-fetoprotein and immobilized Procion Red HE-3B. This is used in the purification of alpha-fetoprotein by negative chromatography resulting in a 16.6-fold enrichment of this protein. Human albumin binds tightly to immobilized Cibacron Blue F3G-A as well as to Cibacron Brilliant Blue FBR-P and shows a lower affinity to Procion Blue MX-R. Procion Red dyes, which are structurally different from Cibacron Blue F3G-A are also capable of interacting with serum albumin. The results obtained are discussed in terms of the present theoretical conceptions about dye-protein interactions.     

Revealing two-state protein-protein interactions of calmodulin by single-molecule spectroscopy

We report a single-molecule fluorescence resonance energy transfer (FRET) and polarization study of conformational dynamics of calmodulin (CaM) interacting with a target peptide, C28W of a 28 amino acid oligomer. The C28W peptide represents the essential binding sequence domain of the Ca-ATPase protein interacting with CaM, which is important in cellular signaling for the regulation of energy in metabolism. However, the mechanism of the CaM/C28W recognition complex formation is still unclear. The amino-terminal (N-terminal) domain of the CaM was labeled with a fluorescein-based arsenical hairpin binder (FlAsH) that enables our unambiguous probing of the CaM N-terminal target-binding domain motions on a millisecond time scale without convolution of the probe-dye random motions. By analyzing the distribution of FRET efficiency between FlAsH labeled CaM and Texas Red labeled C28W and the polarization fluctuation dynamics and distributions of the CaM N-terminal domain, we reveal binding-unbinding motions of the N-terminal domain of the CaM in CaM/C28W complexes, which is strong evidence of a two-state binding interaction of CaM-mediated cell signaling.     

Protein purification using immobilised triazine dyes.

This review attempts to identify proteins which selectively interact with immobilised triazine dyes such as Cibacron blue F3GA and Procion red HE 3B. Different support matrices are compared by examining the capacities of these dyes for proteins. Various approaches to the immobilisation of triazine dyes are considered together with the use of spacers. Some theories of the mechanism of protein retardation by immobilised dyes are discussed. A number of methods are suggested for the measurement of dye concentrations and for the modification of the binding of proteins to dye columns. The variety of elution methods is compared with a view to optimizing purifications. The scope of applications is reviewed as well as the choice of dye. Some advantages of triazine dyes over other affinity ligands are given. It is concluded that although no satisfactory mechanism for the binding of triazine dyes to proteins has yet been proposed, these dyes possess considerable potential for protein purification, particularly when applied on the large scale.