Hydrophobic Fluorophore Research Articles

Conformational Variability of the Glycine Receptor M2 Domain in Response to Activation by Different Agonists

Models describing the structural changes mediating Cys loop receptor activation generally give little attention to the possibility that different agonists may promote activation via distinct M2 pore-lining domain structural rearrangements. We investigated this question by comparing the effects of different ligands on the conformation of the external portion of the homomeric alpha1 glycine receptor M2 domain. Conformational flexibility was assessed by tethering a rhodamine fluorophore to cysteines introduced at the 19' or 22' positions and monitoring fluorescence and current changes during channel activation. During glycine activation, fluorescence of the label attached to R19'C increased by approximately 20%, and the emission peak shifted to lower wavelengths, consistent with a more hydrophobic fluorophore environment. In contrast, ivermectin activated the receptors without producing a fluorescence change. Although taurine and beta-alanine were weak partial agonists at the alpha1R19'C glycine receptor, they induced large fluorescence changes. Propofol, which drastically enhanced these currents, did not induce a glycine-like blue shift in the spectral emission peak. The inhibitors strychnine and picrotoxin elicited fluorescence and current changes as expected for a competitive antagonist and an open channel blocker, respectively. Glycine and taurine (or beta-alanine) also produced an increase and a decrease, respectively, in the fluorescence of a label attached to the nearby L22'C residue. Thus, results from two separate labeled residues support the conclusion that the glycine receptor M2 domain responds with distinct conformational changes to activation by different agonists.

Micelle‐Induced Versatile Performance of Amphiphilic Intramolecular Charge‐Transfer Fluorescent Molecular Sensors

A series of amphiphilic intramolecular charge-transfer fluorescent molecular sensors AS1-3, equipped with a rod-shaped hydrophobic 2-phenylbenzoxazole fluorophore and a hydrophilic tetraamide Hg(2+)-ion receptor, have been prepared. These sensor molecules could be incorporated into the hydrophobic sodium dodecyl sulfate (SDS) micelle, which is confirmed by the clear spectral blue shift and emission enhancement observed at the critical micelle concentration of SDS. Systematic examination of the sensor-Hg(2+) complexation, by using both UV/visible and fluorescence spectroscopy, indicates that SDS significantly modulates both the binding event and signal transformation of these sensor molecules. The potential advantages are fourfold: 1) SDS substantially increases the Hg(2+)-ion association constant and results in an amplified sensitivity. 2) SDS initiates spectral features which facilitate Hg(2+)-ion analysis, for example, in addition to the strengthened fluorescence of the free sensors AS1-3, the original "on-off" response of AS2 toward the Hg(2+) ion is transformed into a self-calibrated two-wavelength ratiometric signal, while for AS3, Hg(2+)-ion complexation in the presence of SDS results in a 180 nm blue shift, which is preferred to the 51 nm spectral shift obtained without SDS. 3) Thermoreversible tuning of the dynamic detection range is realized. 4) Highly specific Hg(2+)-ion identification could be achieved by using the SDS-induced fingerprint emission (358 nm) of the AS2-Hg(2+) complex. Altogether, this work demonstrates a convenient and powerful strategy that remarkably elevates the performance of a given fluorescent molecular sensor. It also implies that for a specific utilization, much attention should be paid to the microenvironment in which the sensor resides, as the behavior of the sensor might be different from that in the bulk solution.

Fluorescently labeled inhibitor for profiling cytoplasmic peptide:N-glycanase

Cytoplasmic peptide:N-glycanase (PNGase) is an enzyme that removes N-glycans from misfolded glycoproteins. The function of cytoplasmic PNGase plays a significant role in the degradation of misfolded glycoproteins, which is critical for cell viability. Recently, we reported that haloacetoamidyl derivatives of high-mannose-type oligosaccharides selectively modify the catalytic cysteine of cytoplasmic PNGase and serve as its specific inhibitor. Interestingly, a drastically simplified chloroacetamidyl chitobiose derivative [(GlcNAc)(2)-ClAc] was also reactive to PNGase. In our work, it was conjugated to a hydrophobic fluorophore in order to render (GlcNAc)(2)-ClAc cells permeable. We demonstrated that this compound [BODIPY-(GlcNAc)(2)-ClAc] specifically binds to cytoplasmic PNGase from budding yeast (Png1). To date, only Z-VAD-fmk is known as an inhibitor of PNGase. BODIPY-(GlcNAc)(2)-ClAc and Z-VAD-fmk share the same binding site on Png1, while BODIPY-(GlcNAc)(2)-ClAc has markedly stronger inhibitory activity. The functional analysis of PNGase using Z-VAD-fmk should be carefully interpreted because of its intrinsic property as a caspase inhibitor. In sharp contrast, chloroacetamidyl chitobiose was not reactive to caspase. In addition, BODIPY-(GlcNAc)(2)-ClAc did not bind either chitobiose-binding lectins or PNGase from other sources. Moreover, fluorescent microscopy clearly showed that BODIPY-(GlcNAc)(2)-ClAc was efficiently introduced into cells. These results suggest that this compound could be an in vivo inhibitor of cytoplasmic PNGase.

Development of fluorescent materials for Diffuse Fluorescence Tomography standards and phantoms

The availability of fluorescence standards is necessary in the development of systems and methods for fluorescence imaging. In this study, two approaches for developing diffuse fluorescence materials to be used as standards or phantoms in diffuse fluorescent tomography applications were investigated. Specifically, silicone rubber and polyester casting resin were used as base materials, and silicone pigments or TiO(2) / India Ink were added respectively to vary the optical properties. Characterization of the optical properties achieved was performed using time-resolved methods. Subsequently, different near-infrared fluorochromes were examined for imparting controlled and stable fluorescence properties. It was determined that hydrophobic fluorophores (IR 676 and IR 780 Iodide) suspended in dichloromethane and hydrophilic fluorophores (Cy5.5 and AF 750) suspended in methanol produced diffusive silicone and resin fluorescent materials, respectively. However only the hydrophobic fluorophores embedded within silicone resulted in the construction of a material with the characteristics of a standard, i.e. stability of fluorescence intensity with time and a linear dependence of normalized fluorescence intensity to fluorophore concentration.

Micromodel study on repartitioning phenomena of a strongly hydrophobic fluorophore at a colloid/1‐octanol interface

Colloids are considered as potential carriers of otherwise immobile contaminants. In this framework the contaminant‐colloid interaction is assumed to be permanent, or the contaminant release from the colloids is not explicitly assessed. In this study, we report on micromodel experiments to visualize and quantify the behavior of colloids in a chemically reactive environment. Fluorescent polystyrene latex beads were injected into a micromodel pore network containing water, air, and 1‐octanol. The colloids were found to attach to the hydrophobic 1‐octanol phase with slightly higher attachment rates compared to the solid interface. Colloids attached to the 1‐octanol sometimes disintegrated because of a swelling of the polystyrene polymer. The hydrophobic fluorescent dye was then found to partition into the 1‐octanol. While the 1‐octanol droplets were slowly dissolving, the fluorescent dye was staying in the droplet at first. At high concentrations and very small droplets the dye was dissolving with 1‐octanol. Although one might be tempted to neglect the repartitioning of contaminants from colloids to other phases because of a limited mass of colloid associated contaminants, this effect has to be included into colloid transport models at contaminated sites with nonaqueous phase liquids, which are more attractive to the contaminant than the colloid or which change the stability of the colloid.

PH‐dependent aggregation of cutinase is efficiently suppressed by 1,8‐ANS

We have studied the thermal stability of the triglyceride-hydrolyzing enzyme cutinase from F. solani pisi at pH values straddling the pI (pH 8.0). At the pI, increasing the protein concentration from 5 to 80 microM decreases the apparent melting temperature by 19 degrees C. This effect vanishes at pH values more than one unit away from pI. In contrast to additives such as detergents and osmolytes, the hydrophobic fluorophore 1,8-ANS completely and saturably suppresses this effect, restoring 70% of enzymatic activity upon cooling. ANS binds strongly to native cutinase as a noncompetitive inhibitor with up to 5 ANS per cutinase molecule. Only the first ANS molecule stabilizes cutinase; however, the last 4 ANS molecules decrease Tm by up to 7 degrees C. Similar pI-dependent aggregation and suppression by ANS is observed for T. lanuginosus lipase, but not for lysozyme or porcine alpha-amylase, suggesting that this behavior is most prevalent for proteins with affinity for hydrophobic substrates and consequent exposure of hydrophobic patches. Aggregation may be promoted by a fluctuating ensemble of native-like states associating via intermolecular beta-sheet rich structures unless blocked by ANS. Our data highlight the chaperone activity of small molecules with affinity for hydrophobic surfaces and their potential application as stabilizers at appropriate stoichiometries.

Water‐Soluble Dendrimeric Two‐Photon Tracers for In Vivo Imaging

In a (nut)shell: Covalently wrapped in a protecting and solubilizing dendrimeric shell (see picture), an initially hydrophobic two-photon fluorophore becomes a biocompatible contrast agent with strong emission for imaging in vivo by multiphoton microscopy.

Spiropyran-based photochromic polymer nanoparticles with optically switchable luminescence.

Polymer nanoparticles of 40-400 nm diameter with spiropyran-merocyanine dyes incorporated into their hydrophobic cavities have been prepared; in contrast to their virtually nonfluorescent character in most environments, the merocyanine forms of the encapsulated dyes are highly fluorescent. Spiro-mero photoisomerization is reversible, allowing the fluorescence to be switched "on" and "off" by alternating UV and visible light. Immobilizing the dye inside hydrophobic pockets of nanoparticles also improves its photostability, rendering it more resistant than the same dyes in solution to fatigue effects arising from photochemical switching. The photophysical characteristics of the encapsulated fluorophores differ dramatically from those of the same species in solution, making nanoparticle-protected hydrophobic fluorophores attractive materials for potential applications such as optical data storage and switching and biological fluorescent labeling. To evaluate the potential for biological tagging, these optically addressable nanoparticles have been delivered into living cells and imaged with a liquid nitrogen-cooled CCD.

Fluorescent nanoparticles as selective Cu(ii) sensors

Latex nanoparticles functionalized with cyclam (1,4,8,11-tetraazacyclotetradecane), a copper chelator, have been doped with a fluorescent dye (BODIPY derivative: 4,4-difluoro-8-(2',4',6'-trimethyl)phenyl-2,6-diethyl-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene). The bulky, hydrophobic fluorophore dissolves within the nanoparticles' polymer core up to a concentration of about 88.4 micromol g(-1). At this concentration the fluorescence yield is about 0.80. Adding Cu2+ ions to the solution decreases the fluorescence because of the energy transfer between the dye and the violet copper cyclam complexes. The response is fast: 90% of the quenching occurs within 1 s. The Cu2+ detection threshold is of 1 nanomolar. No interferences were observed with zinc and nickel ions.

Composite Phospholipid−Calcium Carbonate Microparticles: Influence of Anionic Phospholipids on the Crystallization of Calcium Carbonate

The synthesis and characterization of calcium carbonate microparticles by reaction of calcium chloride and ammonium bicarbonate in the presence of negatively charged phospholipid mixtures of negative and zwitterionic phospholipids has been reported. Negatively charged phospholipids influence the crystal morphology of calcium carbonate and induce the formation of thermodynamically less stable veterite polymorph as opposed to calcite polymorph. The phospholipids are entrapped in the calcium carbonate microparticles during the crystallization process, with a uniform distribution of phospholipids in the interior of the microparticles. This phenomenon was exploited to encapsulate a model hydrophobic fluorophore, the tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dichloride complex, to simulate encapsulation of hydrophobic drug molecules. Thermogravimetric analysis reveals that, in these microparticles, the calcium carbonate and the phospholipid exhibit strong interactions.

Energy Transfer from a Fluorescent Hydrogel to a Hosted Fluorophore

The fluorescent properties of a new 1,3,5-cyclohexyltricarboxamide-based low-molecular-weight hydrogelator (1) derivatized with one hydrophobic fluorophore and two hydrophilic substituents have been investigated. Gels of 1 are composed of long, nonbranched fibers of uniform diameter, as shown by cryo-transmission electron microscopy (cryo-TEM). The aggregation of the naphthalene fluorophore moieties of the gelator molecules in the gel fibers favors the occurrence of a fast energy migration process that allows a very efficient sensitization of the fluorescence of a hosted fluorophore. Such processes have been investigated by the addition of propyldansylamide (PDNS), at two different concentrations, to gels of 1. Around 30% of the total PDNS added to the gels was found to be incorporated in the gel fibers, as confirmed by deconvolution of the fluorescence spectrum, excited-state lifetime measurements, and steady-state and time-resolved fluorescence anisotropy measurements. Moreover, anisotropy measurements show that the fluorophore that is incorporated within the gel fibers is almost completely immobilized, indicating that the interactions of PDNS with the gelator moieties are very strong. This particular configuration of donor (1) and acceptor (PDNS) molecules leads to a very efficient antenna effect, where 50% of the absorbed photons are funneled through to the dansyl derivative when one PDNS molecule is incorporated in the gel fibers for every 100 gelator molecules. A 5-fold higher concentration of PDNS increases the percentage of funneled photons to 75%.

Quantitative membrane loading of polymer vesicles.

We utilize a series of structurally homologous, multi-porphyrin-based, fluorophores (PBFs) in order to explore the capacity of polymer vesicles (polymersomes) to stably incorporate large hydrophobic molecules, non-covalently within their thick lamellar membranes. Through aqueous hydration of dry, uniform thin-films of amphiphilic polymer and PBF species deposited on Teflon, self-assembled polymersomes are readily generated incorporating the hydrophobic fluorophores in prescribed molar ratios within their membranes. The size-dependent spectral properties of the PBFs allow for ready optical verification ( steady-state absorption and emission spectroscopy) of the extent of vesicle membrane loading and enable delineation of intermembranous molecular interactions. The resultant effects of PBF membrane-loading on polymersome thermodynamic and mechanical stability are further assessed by cryogenic transmission electron microscopy (cryo-TEM) and micropipet aspiration, respectively. We demonstrate that polymersomes can be loaded at up to 10 mol/wt% concentrations, with hydrophobic molecules that possess sizes comparable to those of large pharmaceutical conjugates ( ranging 1.4-5.4 nm in length and = 0.7-5.4 kg mol), without significantly compromising the robust thermodynamic and mechanical stabilities of these synthetic vesicle assemblies. Due to membrane incorporation, hydrophobic encapsulants are effectively prevented from self-aggregation, able to be highly concentrated in aqueous solution, and successfully shielded from deleterious environmental interactions. Together, these studies present a generalized paradigm for the generation of complex multi-functional materials that combine both hydrophilic and hydrophobic agents, in mesoscopic dimensions, through cooperative self-assembly.

Supramolecular Fluorophores for Biological Studies: Phenylene Vinylene-Amino Acid Amphiphiles

We report here on a family of self-assembling fluorescent organic amphiphiles with a biomolecular L-lysine hydrophile and a photonically active phenylene vinylene hydrophobe. Unlike conventional amphiphiles, these segmented dendrimers feature a rigid, branched hydrophobe, and have packing characteristics controlled by the ratio of cross-sectional areas of the hydrophobe and hydrophile. In dilute solution, the amphiphiles form supramolecular aggregates, which are easily taken in by cells through an endocytic pathway, and have no discernible effect on cell proliferation or morphology. An analogous pyrene-based amphiphile was cytotoxic, suggesting that cell survival may be linked either to the self-assembling nature of the amphiphiles, or to the specific properties of the phenylene vinylene segment. The combination of photonic and biological components in these amphiphiles provides great potential for applications in sensing or delivery of molecules to intracellular targets.

Normal-phase nanoscale liquid chromatography-mass spectrometry of underivatized oligosaccharides at low-femtomole sensitivity.

We here describe the online liquid chromatography (LC) electrospray ionization mass spectrometry (MS) of underivatized glycans using a nanoscale normal-phase amide column at a flow rate of 300 nL/min. Retention on the amide column is based on polar interactions of the oligosaccharide hydroxyl groups with the stationary phase, and thus, the retention time predictably increases with elongation of the oligosaccharide chain. The system is characterized by its high chromatographic resolution, which routinely allows the separation of isobaric structures. Separation of oligosaccharide mixtures over a 1-h range permits the detailed characterization of the different species by multiple ion selection and fragmentation steps using ion trap MS. The here presented miniaturization of the online-LC system to the nanoscale in combination with ion trap MS allows the detection of oligosaccharide species in a mixture at low-femtomole sensitivity. Online normal-phase nano-LC-MS of complex oligosaccharide mixtures further facilitates the sensitive and detailed structural analysis of oligosaccharides by overcoming the need for cumbersome and time-consuming derivatization procedures such as reductive amination for labeling with hydrophobic fluorophores or labeling with tritium. The method should be useful for the sensitive and quick analysis of glycosylation patterns and individual oligosaccharides from biotechnologically produced glycoproteins as well as scarcely available biological samples.

Three conformational states of the p300 CH1 domain define its functional properties.

Numerous transcription factors interact with the basal transcriptional machinery through the transcriptional co-activators p300 and CREB-binding protein (CBP). The Zn(2+)-binding cysteine/histidine-rich 1 (CH1) domain of p300/CBP binds many of these transcription factors, including hypoxia-inducible factor (HIF). We studied the structural and biophysical properties of the p300 CH1 domain alone and bound to the HIF-1 alpha C-terminal transactivation domain (TAD) to understand the diverse binding properties of CH1. The Zn(2+)-bound CH1 domain (CH1-Zn(2+)) and the HIF-1 alpha TAD-CH1 complex (CH1-Zn(2+)-HIF-1 alpha) are similarly helical, whereas metal-free CH1 is mostly random coil. CH1-Zn(2+) undergoes noncooperative thermal denaturation, does not have a near-UV elliptical signal, and binds the hydrophobic fluorophore ANS. In contrast, the CH1-Zn(2+)-HIF-1 alpha complex undergoes cooperative thermal denaturation, does produce a near-UV signal, and does not bind ANS. Addition of Zn(2+) ions to metal-free CH1 produced one conformational change, and subsequent addition of a HIF-1 alpha TAD peptide induced a second conformational change as detected by intrinsic tryptophan fluorescence spectroscopy. The NMR (1)H-(15)N HSQC spectrum of CH1-Zn(2+) exhibits few poorly dispersed peaks with broad line widths. Removal of metal ions produces more poorly dispersed peaks with sharper line widths. Addition of a HIF-1 alpha TAD peptide to CH1-Zn(2+) produces many well-dispersed peaks with sharp line widths. Taken together, these data support three conformational states for CH1, including an unstructured metal-free domain, a partially structured Zn(2+)-bound domain with molten globule characteristics, and a stable, well-ordered HIF-1 alpha TAD-CH1 complex.

Microscale analysis of mucin‐type O‐glycans by a coordinated fluorophore‐assisted carbohydrate electrophoresis and mass spectrometry approach

The total glycan moiety was released in a single step from native glycoproteins by a nonreductive beta-elimination procedure. The generated oligosaccharides were further derivatized either with the hydrophobic fluorophore 2-aminoacridone (AMAC) or the charged 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) fluorophore, and the resulting fluorescent derivatives were separated according to their hydrodynamic size or charge with high-resolution gel electrophoresis. Both N- and O-glycans released by this beta-elimination procedure might be analyzed simultaneously. AMAC derivatization allows a rapid separation of neutral and charged oligosaccharides without prior fractionation. Derivatized oligosaccharide species were then eluted from the gel slices and analyzed by mass spectrometry. This methodology allowed the rapid structural characterization of each glycan in term of monosaccharide composition and sequence. Using this technique we were able to screen several heterogeneous O-glycan mixtures isolated at the picomolar range from reference glycoproteins or mucins.

Alpha-crystallin and ATP facilitate the in vitro renaturation of xylanase: enhancement of refolding by metal ions.

Alpha-crystallin is a multimeric protein that functions as a molecular chaperone and shares extensive structural homology to small heat shock proteins. For the functional in vitro analysis of alpha-crystallin, the xylanase Xyl II from alkalophilic thermophilic Bacillus was used as a model system. The mechanism of chaperone action of alpha-crystallin is less investigated. Here we studied the refolding of Gdn HCl-denatured Xyl II in the presence and absence of alpha-crystallin to elucidate the molecular mechanism of chaperone-mediated in vitro folding. Our results, based on intrinsic tryptophan fluorescence and hydrophobic fluorophore 8-anilino-1-naphthalene sulfonate binding studies, suggest that alpha-crystallin formed a complex with a putative molten globule-like intermediate in the refolding pathway of Xyl II. The alpha-crystallin.Xyl II complex exhibited no functional activity. Addition of ATP to the complex initiated the renaturation of Xyl II with 30%-35% recovery of activity. The nonhydrolyzable analog 5'-adenylyl imidodiphosphate (AMP-PNP) was capable of reconstitution of active Xyl II to a lesser extent than ATP. Although the presence of Ca(2+) was not required for the in vitro refolding of Xyl II, the renaturation yield was enhanced in its presence. Experimental evidence indicated that the binding of ATP to the alpha-crystallin.Xyl II complex brought about conformational changes in alpha-crystallin facilitating the dissociation of xylanase molecules. This is the first report of the enhancement of alpha-crystallin chaperone functions by metal ions.

Chaperone-like activity and surface hydrophobicity of 70S ribosome

Ribosomes have been shown to mediate refolding of proteins in vitro. In order to understand the mechanism of action, we have explored the 70S ribosome surface for hydrophobicity, one of the important aspects in chaperone–target protein interaction. We find that the 70S ribosome displays significant hydrophobicity on its surface when probed with the hydrophobic fluorophore 8-anilino-1-naphthalene sulfonate. To understand the functional significance of this hydrophobicity we investigated the ability of the ribosome to prevent aggregation of insulin B chain and α-lactalbumin induced by reducing the interchain and intrachain disulfide bond respectively with dithiothreitol (DTT) and photo aggregation of γ-crystallin at 37°C. The 70S ribosome offers complete protection towards light-induced aggregation of γ-crystallin (at 1:2 (w/w) ratio of crystallin:ribosome) and DTT-induced aggregation of α-lactalbumin (at 1:3) and there is appreciable protection (at 1:3) against the aggregation of insulin B chain. We also investigated the role of 70S ribosome in refolding of bovine carbonic anhydrase. Ribosomes improved the folding yield in a concentration-dependent manner. These results clearly demonstrate a general chaperone-like activity of 70S ribosome and implicate its surface hydrophobicity.

Ciliate epibiont effects on feeding, energy reserves, and sensitivity to hydrocarbon contaminants in an estuarine harpacticoid copepod

Although epibiotic protozoans are commonly observed on the chitinous exoskeleton of aquatic crustaceans, relatively little is known about their ecological significance. The significance of protozoan epibionts on benthic copepods has never been examined.Coullana sp., a meiobenthic harpacticoid copepod, is abundant in Louisiana salt marshes and has high incidence (∼50%) of ciliate epibionts. Field and laboratory grazing experiments indicated that ciliate epibionts did not hinderCoullana feeding on benthic or planktonic algae. Contrary to expectations,Coullana with high levels of ciliate epibionts (>8 ind−1) grazed at a significantly higher rate on14C-labeled benthic diatoms added to intact sediment cores than didCoullana with no epibionts.Coullana neutral lipids (examined using Nile Red, a hydrophobic fluorophore) were not significantly influenced by the presence of ciliate epibionts, suggesting that copepods are able to compensate for any additional energetic demands imposed by epibionts. Epibiont effects onCoullana susceptibility to hydrocarbon contaminants were measured by examining survivorship in diesel-spiked sediments. The presence of ciliate epibionts significantly decreased survivorship at relatively low PAH concentrations (12.8 ppm). While ciliate epibionts onCoullana do not dramatically alter total food acquisition or energy storage, they may cause stress, which in turn makesCoullana more susceptible to contaminants and possibly other natural stressors such as food limitation.

Fluorescent pigments of the retinal pigment epithelium and age-related macular degeneration

The major hydrophobic fluorophore of the retinal pigment epithelium (RPE) is A2E, a pyridinium bis-retinoid derived from all- trans-retinal and phosphatidyl-ethanolamine. The accumulation of fluorophores such as A2E is implicated in the pathogenesis of age-related macular degeneration (AMD), a disease associated with the deterioration of central vision and a leading cause of blindness in the elderly. Recent chemical and biological studies have provided insight into the synthesis and biosynthesis of A2E, the spectroscopic properties of this pigment, and the role of A2E and RPE cell death.