Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein.

The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIF pocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small-molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1.

Determination of association equilibrium constant from single molecule fluorescence localization microscopy.

Single molecule fluorescence localization microscopy provides molecular localization with a precision in the tens of nanometer range in the plane perpendicular to the light propagation. This opens the possibility to count molecules and correlate their locations, starting from a map of the actual positions in a single molecule super resolution image. Considering molecular pair correlation as an indication of interaction, and a way to discern them from free molecules, we describe a method to calculate thermodynamic equilibrium constants. In this work, we use as a test system two complementary homo-oligonucleotides, one strand marked with Cyanine 3.5 and the other with Alexa Fluor 647. Hybridization is controlled by the amount of each strand, temperature, and the ionic force, and measured in steady state emission. The same samples are examined in Stochastic Optical Reconstruction Microscopy (STORM) experiments with split-field simultaneous two-colour detection. The effect of multiblinking, labelling-detection efficiency, and determination of the critical distance for association are discussed. We consistently determine values in STORM coincident with those of the bulk experiment.

Analysis of sparse molecular distributions in fibrous arrangements based on the distance to the first neighbor in single molecule localization microscopy.

Single Molecule Localization Microscopy (SMLM) currently attains a lateral resolution of around 10 nm approaching molecular size. Together with increasingly specific fluorescent labeling, it opens the possibility to quantitatively analyze molecular organization. When the labeling density is high enough, SMLM provides clear images of the molecular organization. However, either due to limited labeling efficiency or due to intrinsically low molecular abundance, SMLM delivers a small set of sparse and highly precise localizations. In this work, we introduce a correlation analysis of molecular locations based on the functional dependence of the complementary cumulative distribution function (CCDF) of the distance to the first neighbor (r1). We demonstrate that the log(-log(CCDF(r1))) vs. log(r1) is characterized by a scaling exponent n that takes extreme values of 2 for a random 2D distribution and 1 for a strictly linear arrangement, and find that n is a robust and sensitive metric to distinguish characteristics of the underlying structure responsible for the molecular distribution, even at a very low labeling density. The method enables the detection of fibrillary organization and the estimation of the diameter of host fibers under conditions where a visual inspection provides no clue.

A fluorescence nanoscopy marker for corticotropin-releasing hormone type 1 receptor: computer design, synthesis, signaling effects, super-resolved fluorescence imaging, and in situ affinity constant in cells.

Class B G protein-coupled receptors (GPCRs) are involved in a variety of human pathophysiological states. These groups of membrane receptors are less studied than class A GPCRs due to the lack of structural information, delayed small molecule drug discovery, and scarce fluorescence detection tools available. The class B corticotropin-releasing hormone type 1 receptor (CRHR1) is a key player in the stress response whose dysregulation is critically involved in stress-related disorders: psychiatric conditions (i.e. depression, anxiety, and addictions), neuroendocrinological alterations, and neurodegenerative diseases. Here, we present a strategy to label GPCRs with a small fluorescent antagonist that permits the observation of the receptor in live cells through stochastic optical reconstruction microscopy (STORM) with 23 nm resolution. The marker, an aza-BODIPY derivative, was designed based on computational docking studies, then synthesized, and finally tested in biological cells. Experiments on hippocampal neurons demonstrate antagonist effects in similar concentrations as the well-established antagonist CP-376395. A quantitative analysis of two color STORM images enabled the determination of the binding affinity of the new marker in the cellular environment.

Temperature dependent spectroscopic and excited state dynamics of 3-hydroxychromones with electron donor and acceptor substituents.

We have studied the photophysical and photochemical behavior of three compounds derived from 3-hydroxychromone (3-HC), capable of undergoing excited state proton transfer (ESIPT). The compounds have two substituents, located in positions 2 and 7, one on each ring of the 3-HC heterocycle. The substituent pattern shows different electron donating and acceptor features. The compounds were studied by absorption and emission spectroscopy, steady state anisotropy, and time resolved emission spectroscopy (TRES) as a function of temperature. Results were interpreted using time dependent density functional theory calculations. Compared to reference compounds of 3-HC substituted only in the 2 position, the compounds show similar absorption and emission spectra, shifted 20-30 nm to higher wavelengths due to extended conjugation. TRES shows the existence of ESIPT in the thermodynamic equilibrium regime. This process is endothermic in all three compounds. The different behavior compared to monosubstituted 3-HC is attributed to the extended conjugation and to the electron donor acceptor character of the substituents, which has a more pronounced effect when the electron acceptor is located in position 2.

Two-Photon Excitation of a Plasmonic Nanoswitch Monitored by Single-Molecule Fluorescence Microscopy.

Visible-light excitation of the surface plasmon band of silver nanoplates can effectively localize and concentrate the incident electromagnetic field enhancing the photochemical performance of organic molecules. Herein, the first single-molecule study of the plasmon-assisted isomerization of a photochrome-fluorophore dyad, designed to switch between a nonfluorescent and a fluorescent state in response to the photochromic transformation, is reported. The photochemistry of the switchable assembly, consisting of a photochromic benzooxazine chemically conjugated to a coumarin moiety, is examined in real time with total internal reflection fluorescence microscopy in the presence of silver nanoplates excited with a 633 nm laser. The metallic nanostructures significantly enhance the visible light-induced performance of the photoconversion, which normally requires ultraviolet excitation. The resulting ring-open isomer is strongly fluorescent and can also be excited at 633 nm. These stochastic emission events are used to monitor photochromic activation and show quadratic dependence on incident power. The utilization of a single laser wavelength for both photochromic activation and excitation effectively mimics a pseudo two-colours system.

Thermoplasmonic ssDNA Dynamic Release from Gold Nanoparticles Examined with Advanced Fluorescence Microscopy.

Plasmon excitation of spherical gold nanoparticles carrying a fluorescent labeled 30 bp dsDNA cargo, with one chain covalently attached through two S-Au bonds to the surface, results in release of the complementary strand as ssDNA that can be examined in situ using high-resolution fluorescence microscopy. The release is dependent on the total energy delivered, but not the rate of delivery, an important property for plasmonic applications in medicine, sensors, and plasmon-induced PCR.

Chiral power change upon photoisomerization in twisted nematic liquid crystals.

In this work, we use the photoisomerization of azobenzenes, a phenanthrospirooxazine, and a fulgide in a twisted nematic liquid crystalline phase to change the chiral twisting power of the system. The changes are probed by the rotatory power of linearly polarized light. Time resolved and steady state experiments are carried out. The chiral change and the photoisomerization process have similar characteristic recovery times and activation energy, thus probing that the change is induced by the modification in the chemical composition of the photochromic dopant system. The amplitude of the light twisting power change correlates with the order change in the liquid crystal (LC) but not with the modification in the absorption characteristics of the system. This indicates that the driving force of the chiral change is the microscopic order modification in the LC phase that affects the helical pitch of the phase.

Influence of the glass transition on rotational dynamics of dyes in thin polymer films: single-molecule and ensemble experiments.

We performed polarized fluorescence emission studies of Nile Red (NR) in poly(methyl methacrylate) (PMMA), poly(ethyl methacrylate) (PEMA), and poly(butyl methacrylate) (PBMA) at the single molecule (SM) and at the ensemble level to study the in cage movements of the ground-state molecule in polymer films of nanometric thickness at room temperature. Experiments were performed with wide field irradiation. At the ensemble level, the linearly polarized irradiation was used to induce a photoselection by bleaching, which is compensated by rotational diffusion. Both results show an appreciable difference in mobility of NR in the films that is correlated with the different glass-transition temperatures of the films, particularly in PEMA, which displays a clearly distinct behavior between the 200 nm films, representing a rigid environment, and the 25 nm ones, showing much higher mobility. We developed a model of broad application for polarized photobleaching that allows obtaining rotational diffusion coefficients and photobleaching quantum yields in an easy way from ensemble experiments. The parameters obtained from ensemble measurements correlate well with the results from SM experiments.

Mapping the fluorescence performance of a photochromic-fluorescent system coupled with gold nanoparticles at the single-molecule-single-particle level.

Single-molecule (SM) fluorescence microscopy was used to investigate the photochromic fluorescent system spiropyran-merocyanine (SP ↔ MC) interacting with gold nanoparticles (AuNPs). We observe a significant increase in the brightness of the emissive MC form, in the duration of its ON time, and in the total number of emitted photons. The spatial distribution of SMs with improved photophysical performance was obtained with 40 nm precision relative to the nearest AuNP. We demonstrate that even photochromic systems with poor photochemical performance for SM can become suitable for long time monitoring and high performance microscopy by interaction with metallic NP.

Fluorescent polymer coatings with tuneable sensitive range for remote temperature sensing.

Polymer films of poly(vinyl alcohol) containing the fluorescent dyes 4-aminophthalimide (AP) or 6-propionyl-2-dimethylamino-naphthalene (Prodan) are used as temperature-sensitive fluorescent coatings for remote temperature sensing. Temperature can be obtained by a two-wavelength ratiometric-based emission intensity measurement. The coatings are sensitive in a 100K temperature range that can be tuned by polymer-solute interactions. The usable range is 200-300 K for AP and 280-380 K for Prodan.

The mechanism of the photochromic transformation of spirorhodamines.

We investigate the equilibrium, kinetics, and mechanism of the photochromic transformation of a series of amido spirorhodamine compounds-differing in the nature of the substituents of the amido group and in the rhodamine chromophore-in ethanol at room temperature in the presence of trifluoroacetic acid. A proton participates in the equilibrium between the spiro form and the open rhodamine form. The relaxation times in the dark or under continuous irradiation show a linear dependence on the proton concentration. The slopes of these plots show a linear free energy relation with the equilibrium constant of the transformation. A mechanism involving reversible reaction steps between four states: the two thermodynamically stable isomers, a protonated spiro form, and a deprotonated open form, can account for the kinetic observations in the dark and under irradiation.

Detection of low quantum yield fluorophores and improved imaging times using metallic nanoparticles.

The behavior of a fluorophore near a gold nanoparticle is rationalized by a theoretical description of the parameters that modify the fluorescence emission: nanoparticle-fluorophore distance, fluorescence quantum yield (φ(0)), and fluorophore absorption and emission spectra, to find optimum conditions for designing fluorophore-nanoparticle probes. The theoretical maximum gain in brightness of the nanoparticle-fluorophore system with respect to the isolated molecule increases almost inversely proportional to φ(0). The brightness enhancement in imaging experiments in vitro was assessed by using Au-SiO(2) core-shell nanoparticles deposited on glass. A ~13-fold emission brightness enhancement for weakly fluorescent molecules was observed. A significant increase in fluorophore photostability, rendering longer imaging times, was obtained for fluorophores interacting with gold nanoparticles incorporated by endocytosis in cells. Our results illustrate a way to increase imaging times and to study molecules in the vicinity of a metallic nanoparticle after photobleaching of background fluorescence.

Photoisomerization of alfa calcidol by a sensitized quantum chain reaction.

The production of vitamin D3 is a pharmaceutically relevant process, producing high added-value products. Precursors are extracts from vegetal origin but bearing mainly an E geometry in the 5,6 double bond. The synthesis of vitamin D3 (5-E-α-calcidol) with the correct Z stereochemistry in the 5,6 double bond from the E isomer using anthracene and triethylamine (TEA) as the sensitizer system was studied from the kinetic and mechanistic point of view. The sensitized isomerization of E-calcidol by irradiation of anthracene takes place only in deoxygenated solution and yields the Z isomer in ca 5% yield in the photostationary state. When TEA is added to the system, the E-Z reaction is not inhibited by oxygen any more, the quantum yield of photoisomerization to the Z isomer grows linearly with the concentration of E-calcidol, while conversions higher than 95% to the Z isomer are reached in the photostationary state and E-Z quantum yields as high as 45 at [E-calcidol] = 25 mM are reached. If TEA is replaced by 1,4-diazabicyclo[2.2.2]octane, the reaction rate drops to one-third at the same amine concentration. The observations can be explained by a quantum chain reaction mechanism. The high conversion achieved eliminates the need of isomer separation.

Critical effects on attractive solutes in binary liquid mixtures close to their consolute point: a new experimental strategy.

The effect of near-criticality upon the properties of dilute solutions of attractive solutes has been previously studied only using pure solvents close to the vapor-liquid critical point. The experimental difficulties that plague this thermodynamic region have somewhat obscured the interpretations of the results. Consequently, the coupling of long-range critical fluctuations with short-range intermolecular interactions is still a matter of debate. We developed a new strategy consisting of studying the changes in the solvation shell of probe molecules dissolved in a binary solvent mixture close to its lower consolute critical point. The study is based on UV-vis absorption and fluorescent emission measurements of the solvatochromic and thermochromic effects of two dyes, Reichardt's Dye and Nile Red, dissolved in mixtures of lutidine-water. The results show unambiguously the existence of a subtle change in the composition of the near-critical solvent surrounding the probe molecules with respect to the bulk composition, thus we conclude that the coupling of long-range fluctuations with short-range interactions is now firmly established.

Proton transfer from 2-naphthol to aliphatic amines in supercritical CO2.

The proton transfer from 2-naphthol to aliphatic amines was studied in supercritical CO(2) (scCO(2)) and in cyclohexane as reference solvent, by absorption and fluorescence spectroscopy and by time-resolved emission. Irradiation of 2-naphthol in scCO(2) in the presence of ethyldiisopropylamine shows dynamic fluorescence quenching of the acidic form of 2-naphthol and emission from the basic form. Fluorescence excitation spectra show that the emission of the basic form is originated upon excitation of the acidic form. The interaction between 2-naphthol and the amines is described by the formation of a complex with proton donor-acceptor character in the ground and excited states of 2-naphthol. The acidity increase of 2-naphthol upon electronic excitation to the first excited singlet in scCO(2) is as high as in water. Proton transfer quantum yields of 0.6 can be easily achieved in scCO(2). The results have implications for carrying out acid-base catalyzed reactions in scCO(2).

Freezing single molecule dynamics on interfaces and in polymers.

Heterogeneous line broadening and spectral diffusion of the fluorescence emission spectra of perylene diimide molecules have been investigated by means of time dependent single molecule spectroscopy. The influence of temperature and environment has been studied and reveals strong correlation to spectral diffusion processes. We followed the freezing of the molecular mobility of quasi free molecules on the surface upon temperature lowering and by embedding into a poly(methyl methacrylate) (PMMA) polymer. Thereby changes of optical transition energies as a result of both intramolecular changes of conformation and external induced dynamics by the surrounding polymer matrix could be observed. Simulations of spectral fluctuations within a two-level system (TLS) model showed good agreement with the experimental findings.

Single photon fluorescent microlithography for live-cell imaging.

Using fluorescent dyes to trigger the polymerization of a commercial polyurethane resin allows a rapid fabrication of micrometer and submicrometer sized fluorescent structures by one-photon absorption. Here, we show that standard He-Ne lasers emitting at 632.8 nm can be used to start the photopolymerization and that very low laser power is required. This procedure allows the fabrication of fiduciary fluorescent references on standard glass coverslips, mica sheets, or gold-coated coverslips for laser scanning or standard fluorescent microscopy. The biocompatibility of the polymerized resin with cells in culture was tested by growing Xenopus melanophores and a standard laser scanning microscope was used to demonstrate that it is possible to use equipment readily available in several laboratories. We show that fluorescent structure with less than 10 nm in height may be used as references in fluorescence microscopy allowing a smooth environment for cell growth. Different dyes were tested and the conditions for one-photon polymerization were outlined.

Cage effect in supercritical fluids and compressed gases in the photolysis of an asymmetrically substituted diazene.

We studied the photolysis of (1-biphenyl-4-yl-1-methyl-ethyl)-tert-butyl diazene in supercritical CO(2) and Xe, as well as in compressed Kr. The compound has good solubility in the mentioned fluids, allowing the photolysis measurements to be performed in CO(2) at 1.4 K above T(c) and at pressures as low as 70 bar. We monitored relative cage effect after nanosecond laser pulses by measuring the absorbance at 320 nm (DeltaA(t-->0)) corresponding to the total amount of out-of-cage 1-biphenyl-4-yl-1-methyl-ethyl radical (BME.) produced after nitrogen loss of the diazene. In supercritical CO(2) and Xe, isothermal values of DeltaA(t-->0) showed an increase-decrease behavior with increasing pressure at constant temperature, a typical feature of the transition from the solvent energy transfer to the friction controlled regimes. The comparison of the behavior of DeltaA(t-->0) in CO(2) at reduced temperatures between 1.004 and 1.027, in Xe, and in Kr points to an absence of enhanced cage effect near the critical point. Compatibility with spectroscopic data is analyzed.

Photophysics and photochemistry of an asymmetrically substituted diazene: a suitable cage effect probe.

The photophysics and photochemistry of (1-biphenyl-4-yl-1-methyl-ethyl)-tert-butyl diazene were thoroughly studied by laser flash photolysis from the picosecond to the microsecond time domain. The compound has favorable features as a radical photoinitiator and as a probe for cage effect studies in liquids, supercritical fluids, and compressed gases. The biphenyl moiety acts as an antenna efficiently transferring electronic energy to the dissociative (1)n,pi* state centered on the azo moiety. By picosecond experiments irradiating at the biphenyl- and at the azo-centered transitions, we were able to demonstrate this fact as well as determine a lifetime of 0.7 ps for the buildup of 1-biphenyl-4-yl-1-methyl-ethyl radicals (BME*). The sum of in-cage reaction rate constants of BME* radicals by combination and disproportionation is 5 x 10(10) s(-1). The free radical quantum yield in solution is 0.21 (phi(BME*)) in n-hexane at room temperature, whereas the dissociation quantum yield approaches 50%. The symmetric ketone, 2,4-bis-biphenyl-4-yl-2,4-dimethyl-pentan-2-one, was used as a reference compound for the production and reaction of BME* radicals. Transient IR measurements show CO stretching bands of the excited (3)pi,pi* and (1)n,pi* states but no dissociation up to 0.5 ns. A fluorescence lifetime of 1 ns for this ketone is consistent with this observation. By transient actinometry and kinetic decays in the microsecond time range, we measured epsilon(BME*) = (2.3 +/- 0.2) x 10(4) M(-1) cm(-1) at 325 nm and a second-order rate constant of 5.8 x 10(9) M(-1) s(-1) for the consumption of BME* radicals.