Time-resolved Fluorescence Lifetime Measurements Research Articles

Abnormal push-pull benzo[4,5]imidazo[1,2-a][1,2,3]triazolo[4,5-e]pyrimidine fluorophores in planarized intramolecular charge transfer (PLICT) state: Synthesis, photophysical studies and theoretical calculations

The combination of excellent luminescence with high solvent polarity effect and aggregation induced emission (AIE) is an ideal combination for creating fluorophores/probes with high microenvironmental sensitivity. However, many push-pull chromophores of the D−A type in common intramolecular charge transfer (ICT) state with a significant solvatochromic effect and AIE activity, have poor luminescent properties. Herein, to overcome this problem by using reactions of nucleophilic aromatic hydrogen substitution (SNH), we have designed a series of novel 4-heteroaryl-substituted 2-aryl-2H-benzo[4,5]imidazo[1,2-a][1,2,3]triazolo[4,5-e]pyrimidine fluorophores possessing a planarized intramolecular charge transfer (PLICT) state. All these fluorophores exhibited high luminescence quantum yields (up to 60%) and large Stokes shift values of up to 7459 cm−1. Among them, the fluorophore 4h was found to exhibit the most pronounced positive solvatochromic effect and the probe 4f exhibited the most pronounced aggregation induced emission characteristics. This AIE behavior was further confirmed by means of time-resolved fluorescence lifetime measurements as well as DFT-assisted geometry optimization studies. In the presence of trifluoroacetic acid (TFA) compound 4h exhibited a well-pronounced acidochromism via visible color change from yellow-green to orange which returned to the original yellow-green solution after the addition of triethylamine (TEA). The Stern-Volmer constant for the probe 4h towards TFA was 38 M−1. Finally, for the compounds 4f, g, h theoretical calculations in the ground and excited states in different solvents were carried out to confirm the PLICT process. Based on all above the herein reported PLICT fluorophores 4a-h can be successfully applied as biological probes and optical switches.

FRET-based fluorescent probe for drug assay from amino acid@gold-carbon nanoparticles.

Biocompatible and luminescent nanostructures synthesized by capping gold-carbon nanoparticles (HOOC-4-C6H4-AuNPs) with amino acids tyrosine, tryptophan, and cysteine were used for the quantitative estimation of ranitidine (RNH), a peptic ulcer and gastroesophageal reflux drug. We applied a fluorescence quenching mechanism to investigate the viability of the energy transfer based on gold-carbon nanosensors. Förster resonance energy transfer (FRET) calculations showed a donor-acceptor distance of 1.69nm (Tyr@AuNPs), 2.27nm (Trp@AuNPs), and 2.32nm (Cys@AuNPs). The constant time-resolved fluorescence lifetime measurements supported the static quenching nature. This method was successfully utilized in the detection and quantification of RNH, with a limit of detection (LOD) of 0.174, 0.56, and 0.332μM for Tyr@AuNP, Trp@AuNP, and Cys@AuNP bioconjugates, respectively. This approach was also successful in the quantification of RNH in spiked serum samples.

Luminescence and nonlinear optical properties of Er3+- doped ZnWO4 nanostructures

Monoclinic ZnWO4 nanostructures with various concentration of Er were prepared by hydrothermal approach. The synthesized nanostructures were characterized by UV–vis spectra, X-ray diffraction (XRD) technique, steady-state and time-resolved fluorescence lifetime measurements. XRD results confirmed that Er3+ ions were successfully incorporated into ZnWO4 lattice without any secondary phase formation. The incorporation and energy transfer of Er into ZnWO4 were revealed by emission spectra and lifetime decay curves. The nonlinear optical properties of the nanostructures were studied by continuous wave Z-scan technique at 532 nm using Nd: YAG laser. From the open aperture and closed aperture Z-scan traces, the extracted nonlinear absorption and refraction data are attributed to reverse saturable absorption and self-defocusing nature of the samples. The optical nonlinearity of the nanostructures was found to be increasing with increased Er3+ dopant concentration. The observed nonlinearity suggests that the synthesized nanostructures could be explored for its applications in advanced photonic devices.

Cooperative effect between BaTiO3 and CaFe2O4 in a cocatalyst-free heterojunction composite for improved photochemical H2 generation

A series of novel BaTiO3/CaFe2O4 heterojunction composites with different weight ratios of CaFe2O4 vs BaTiO3 was successfully fabricated by sonication-calcination method using the pre-prepared BaTiO3 and CaFe2O4 powders synthesized in hydrothermal and sol-gel methods, respectively. The composites were well characterized using XRD, UV–vis DRS, SEM, TEM, EDS and XPS to substantiate that BaTiO3 and CaFe2O4 coexist in the heterojunction composite. The highest photocatalytic hydrogen generation rate was obtained for BaTiO3/CaFe2O4 (40 wt%) compared to either of its individual counterparts and this improvement indicated the existence of a cooperative effect between BaTiO3 and CaFe2O4 in the heterojunction. Based on UV-vis-DRS, photoluminescence and time-resolved fluorescence lifetime measurements, the cooperative effect between BaTiO3 and CaFe2O4 originated from the improved photoresponse in the visible light region and efficient separation of the photogenerated electron–hole pairs augmenting their availability for the photocatalytic reaction. A plausible photocatalytic mechanism was also deduced using electrochemical impedance spectroscopy measurements, describing the migration direction of the separated charge carriers. Moreover, the best composite BaTiO3/CaFe2O4 (40 wt%) exhibited fairly stable photoactivity for H2 production using the sacrificial agent (Na2S and Na2SO3) without the assistance of any noble metals as cocatalysts.

AIEE-active blue-emitting molecules derived from methoxyl-decorated triarylcyclopentadienes: Synthesis, crystal structures, photophysical and electroluminescence properties

Five highly efficient blue-emitting triarylcyclopentadiene derivatives 1–5 were designed and synthesized by introducing phenyl, methoxyphenyl, methoxybiphenyl and methoxynaphthyl groups to cyclopentadiene backbone, respectively. These compounds possess good thermal stability with decomposition temperatures of 264–340 °C and exhibit intense blue fluorescence with maximum emission peaked at 457–470 nm. All compounds emit weak fluorescence in solution, but they become strong emitters in the aggregated state indicating typical aggregation-induced emission enhancement (AIEE) properties. AIEE behaviour was further confirmed by fluorescent changes depended on temperature and solution viscosity as well as time-resolved fluorescence lifetime measurements. Unexpected substituent effect on aggregation structures and fluorescent properties of 1–5 was also observed. Single-crystal structure analysis revealed that highly twisted conformations and bulky substituents on cyclopentadiene core could dramatically suppress the intermolecular π-π interactions leading to intense blue emission of these compounds in aggregates. Furthermore, the non-doped and doped organic light-emitting diodes (OLEDs) using compounds 2 and 5 as the emitting layers have been fabricated, respectively. A non-doped OLED with compound 5 displays good electroluminescent performance with a light-blue emission and a maximum luminance of 3886 cd/m2.

Lifetime measurements using two-step laser excitation for high-lying even-parity levels and improved theoretical oscillator strengths in Y ii

We report new time-resolved laser-induced fluorescence lifetime measurements for 22 highly excited even-parity levels in singly ionized yttrium (Y II). To populate these levels belonging to the configurations 4d6s, 5s6s 4d5d, 5p2, 4d7s and 4d6d, a two-step laser excitation technique was used. Our previous pseudo-relativistic Hartree-Fock model (Biemont et al. 2011) was improved by extending the configuration interaction up to n = 10 to reproduce the new experimental lifetimes. A set of semi-empirical oscillator strengths extended to transitions falling in the spectral range λλ194-3995 nm, depopulating these 22 even-parity levels in Y II, is presented and compared to the values found in the Kurucz's data base (Kurucz 2011). (Less)

Fluorescence lifetime microscopy of NADH distinguishes alterations in cerebral metabolism in vivo.

Evaluating cerebral energy metabolism at microscopic resolution is important for comprehensively understanding healthy brain function and its pathological alterations. Here, we resolve specific alterations in cerebral metabolism in vivo in Sprague Dawley rats utilizing minimally-invasive 2-photon fluorescence lifetime imaging (2P-FLIM) measurements of reduced nicotinamide adenine dinucleotide (NADH) fluorescence. Time-resolved fluorescence lifetime measurements enable distinction of different components contributing to NADH autofluorescence. Ostensibly, these components indicate different enzyme-bound formulations of NADH. We observed distinct variations in the relative proportions of these components before and after pharmacological-induced impairments to several reactions involved in glycolytic and oxidative metabolism. Classification models were developed with the experimental data and used to predict the metabolic impairments induced during separate experiments involving bicuculline-induced seizures. The models consistently predicted that prolonged focal seizure activity results in impaired activity in the electron transport chain, likely the consequence of inadequate oxygen supply. 2P-FLIM observations of cerebral NADH will help advance our understanding of cerebral energetics at a microscopic scale. Such knowledge will aid in our evaluation of healthy and diseased cerebral physiology and guide diagnostic and therapeutic strategies that target cerebral energetics.

Photophysical studies of a food hydrocolloid, Gum Arabic with resorcinol based acridinedione dyes in water

Abstract Photophysical and electrochemical studies of photoinduced electron transfer (PET) based acridinedione dye with water soluble food hydrocolloid, Gum Arabic (GA) were carried out in aqueous solution. Addition of GA to resorcinol based acridinedione dye (ADDR) which is a PET dye, results in a fluorescence quenching accompanied with a blue shift in the emission maximum (6 nm). The fluorescence quenching is attributed to the promotion of the PET process through space between the donor moiety (OCH 3 ) and the acceptor moiety of basic acridinedione dye ring structure (C O). The fluorescence quenching of ADDR is found to be dynamic in nature. The fluorescence decay of ADDR in the presence of GA shows tri-exponential decay, which implies that there exists a heterogeneous micro environment around the vicinity of the dye with variation in the fluorescence lifetime and their relative distribution. The interaction of ADDR with GA in water is found to be largely hydrophobic, but GA containing several hydrogen-bonding functional groups (sugar moieties and amino acids) also influences the excited state spectral properties of ADDR dye. GA serves as an excellent promoter of electron transfer (ET) through space between the donor and acceptor moiety of ADDR. The variation in oxidation potential of ADDR on the addition of GA is elucidated from cyclic voltammetry studies and the existence of multi-environment of ADDR in GA is elucidated from time resolved fluorescence lifetime measurements.

Differential Effect of Oxicam Non-Steroidal Anti-Inflammatory Drugs on Membranes and Their Consequence on Membrane Fusion

Non-steroidal anti-inflammatory drugs (NSAIDs) are the most commonly used analgesics and antipyretics, which form an interesting drug group because of their new and alternate functions. The ability of the NSAIDs belonging to the oxicam chemical group to induce membrane fusion at low physiologically relevant concentrations is a new function that has drawn considerable attention. Membrane fusion is dependent on the interplay of physicochemical properties of both drugs and membranes. Here, we have elucidated the effects of different oxicam drugs, Meloxicam, Piroxicam, Tenoxicam, Lornoxicam, and Isoxicam, on an identical membrane-mimetic system. This highlights only the differential effects of the drugs on drug-membrane interactions, which in turn modulate their role as membrane fusogens. The partitioning behavior and the location of the drugs in dimyristoylphosphatidylcholine vesicles have been studied using second-derivative absorption spectroscopy, fluorescence quenching, steady-state fluorescence anisotropy, and time-resolved fluorescence lifetime measurements. Fusion kinetics has been monitored by fluorescence assays and dynamic light scattering was used to provide a snapshot of the vesicle diameter distribution at different time points. The differential perturbing effect of the drugs on the membrane is dependent both on their partitioning and location. Although partitioning governs the extent of fusion, the location modulates the rates of each step.

Humidity sensing mechanism based on the distance dependent interactions between BODIPY dye molecules and gold thin films

Radiative decay rate of the Boradiazaindacene (BODIPY) dye molecules in the presence of a gold thin film is analyzed using a conventional time correlated single photon counting technique. The metal thin film and the spacer thickness effects on the fluorescence lifetime of BODIPY molecules are investigated. When the thickness of the gold layer is reduced, the reflectivity of the thin film decreases; thus, the emission field penetrates the gold and this causes further quenching. As compared to free BODIPY dye molecules, time-resolved experiments show that the fluorescence lifetime of BODIPY which is brought into the proximity of the metal surface exhibit an oscillatory behavior as a function of the thickness spacer between fluorescent dye and the metal surface. This interference effect is employed to build a humidity sensor based on time resolved fluorescence lifetime measurements using a relative humidity sensitive polyethylene glycol (PEG) polymer coating as a spacer material. The swelling characteristic of PEG coating provides nanometer accuracy to control the emitter–metal surface distance. Humidity induced change in the thickness of the PEG coating is investigated for a range from 13 to 87% using a single wavelength ellipsometry. It is envisaged that humidity induced changes in the fluorescence lifetime of the dye molecules represent a clear example of the Drexhage interference model.

Single-molecule enzymatic conformational dynamics: spilling out the product molecules.

Product releasing is an essential step of an enzymatic reaction, and a mechanistic understanding primarily depends on the active-site conformational changes and molecular interactions that are involved in this step of the enzymatic reaction. Here we report our work on the enzymatic product releasing dynamics and mechanism of an enzyme, horseradish peroxidase (HRP), using combined single-molecule time-resolved fluorescence intensity, anisotropy, and lifetime measurements. Our results have shown a wide distribution of the multiple conformational states involved in active-site interacting with the product molecules during the product releasing. We have identified that there is a significant pathway in which the product molecules are spilled out from the enzymatic active site, driven by a squeezing effect from a tight active-site conformational state, although the conventional pathway of releasing a product molecule from an open active-site conformational state is still a primary pathway. Our study provides new insight into the enzymatic reaction dynamics and mechanism, and the information is uniquely obtainable from our combined time-resolved single-molecule spectroscopic measurements and analyses.

Design and evaluation of a device for fast multispectral time-resolved fluorescence spectroscopy and imaging

The application of time-resolved fluorescence spectroscopy (TRFS) to in vivo tissue diagnosis requires a method for fast acquisition of fluorescence decay profiles in multiple spectral bands. This study focusses on development of a clinically compatible fiber-optic based multispectral TRFS (ms-TRFS) system together with validation of its accuracy and precision for fluorescence lifetime measurements. It also presents the expansion of this technique into an imaging spectroscopy method. A tandem array of dichroic beamsplitters and filters was used to record TRFS decay profiles at four distinct spectral bands where biological tissue typically presents fluorescence emission maxima, namely, 390, 452, 542, and 629 nm. Each emission channel was temporally separated by using transmission delays through 200 μm diameter multimode optical fibers of 1, 10, 19, and 28 m lengths. A Laguerre-expansion deconvolution algorithm was used to compensate for modal dispersion inherent to large diameter optical fibers and the finite bandwidth of detectors and digitizers. The system was found to be highly efficient and fast requiring a few nano-Joule of laser pulse energy and <1 ms per point measurement, respectively, for the detection of tissue autofluorescent components. Organic and biological chromophores with lifetimes that spanned a 0.8-7 ns range were used for system validation, and the measured lifetimes from the organic fluorophores deviated by less than 10% from values reported in the literature. Multi-spectral lifetime images of organic dye solutions contained in glass capillary tubes were recorded by raster scanning the single fiber probe in a 2D plane to validate the system as an imaging tool. The lifetime measurement variability was measured indicating that the system provides reproducible results with a standard deviation smaller than 50 ps. The ms-TRFS is a compact apparatus that makes possible the fast, accurate, and precise multispectral time-resolved fluorescence lifetime measurements of low quantum efficiency sub-nanosecond fluorophores.

Folding and Unfolding Pathways of the Human Telomeric G-Quadruplex

Sequence analogs of human telomeric DNA such as d[AGGG(TTAGGG)3] (Tel22) fold into monomeric quadruplex structures in the presence of a suitable cation. To investigate the pathway for unimolecular quadruplex formation, we monitored the kinetics of K+-induced folding of Tel22 by circular dichroism (CD), intrinsic 2-aminopurine fluorescence, and fluorescence resonance energy transfer (FRET). The results are consistent with a four-step pathway U ↔ I1 ↔ I2 ↔ I3 ↔ F where U and F represent unfolded and folded conformational ensembles and I1, I2, and I3 are intermediates. Previous kinetic studies have shown that I1 is formed in a rapid pre-equilibrium and may consist of an ensemble of “prefolded” hairpin structures brought about by cation-induced electrostatic collapse of the DNA. The current study shows that I1 converts to I2 with a relaxation time τ1=0.1s at 25°C in 25mM KCl. The CD spectrum of I2 is characteristic of an antiparallel quadruplex that could form as a result of intramolecular fold-over of the I1 hairpins. I3 is relatively slowly formed (τ2≈3700s) and has CD and FRET properties consistent with those expected of a triplex structure as previously observed in equilibrium melting studies. I3 converts to F with τ3≈750s. Identical pathways with different kinetic constants involving a rapidly formed antiparallel intermediate were observed with oligonucleotides forming mixed parallel/antiparallel hybrid-1 and hybrid-2 topologies {e.g. d[TTGGG(TTAGGG)3A] and d[TAGGG(TTAGGG)3TT]}. Aspects of the kinetics of unfolding were also monitored by the spectroscopic methods listed above and by time-resolved fluorescence lifetime measurements using a complementary strand trap assay. These experiments reveal a slow, rate-limiting step along the unfolding pathway.

Designing a Thermostable Switch-Based Biosensor

There is a critical need for developing new diagnostic chemical reporter tools that are stable, cost effective, and capable of sensitive detection. Fluorescent protein biosensors relay changes in fluorescence based on the physical interaction between a protein and the ligand. The thermostable arginine binding protein (ArgBP) from the Gram-negative, hyperthermophilic bacterium, Thermotoga maritima, has been modified to create a reagentless fluorescent biosensor. Typically, single Cys mutants are constructed and covalently modified with environmentally-sensitive fluorescent probes. However, no theoretical framework exists to accurately model placement of the probe to the protein sensor. In this work, site-directed mutations were engineered whereby a significant fluorescence quenching (>80%) is observed based on a photo-induced electron transfer (PET) mechanism, which occurs after TmArgBP binds with increasing concentrations of arginine. This method gave significantly better fluorescence response to arginine and responded in a predictable distance-dependent manner when compared with construction of single Cys mutants of TmArgBP. Using the PET method, a dissociation constant of 0.86 microMolar was determined to be consistent with previously published data for this protein. Time-resolved fluorescence lifetime measurements indicated that fluorescence deactivation of the TmArgBP biosensor is a result of static quenching, which is consistent with the formation of ground state complexes between the fluorescent probe and an internal quencher. In addition to the development of a thermostable fluorescent protein sensor, this investigation provides a generalizable spectroscopic tool for the study of conformational movements within a single polypeptide chain.

Excited-state dynamics in diketopyrrolopyrrole-based copolymer for organic photovoltaics investigated by transient optical spectroscopy

We investigate the photoexcited state dynamics in a donor–acceptor copolymer, poly{3,6-dithiophene-2-yl-2,5-di(2-octyldodecyl)-pyrrolo[3,4-c]pyrrole-1,4-dione-alt-naphthalene} (pDPP-TNT), by picosecond fluorescence and femtosecond transient absorption spectroscopies. Time-resolved fluorescence lifetime measurements of pDPP-TNT thin films reveal that the lifetime of the singlet excited state is 185 ± 5 ps and that singlet–singlet annihilation occurs at excitation photon densities above 6 × 1017 photons/cm3. From the results of singlet–singlet annihilation analysis, we estimate that the single-singlet annihilation rate constant is (6.0 ± 0.2) × 10−9 cm3 s−1 and the singlet diffusion length is ~7 nm. From the comparison of femtosecond transient absorption measurements and picosecond fluorescence measurements, it is found that the time profile of the photobleaching signal in the charge-transfer (CT) absorption band coincides with that of the fluorescence intensity and there is no indication of long-lived species, which clearly suggests that charged species, such as polaron pairs and triplet excitons, are not effectively photogenerated in the neat pDPP-TNT polymer.

Ultrafast Excited-State Dynamics and Dispersion Studies of Third-Order Optical Nonlinearities in Novel Corroles

Ultrafast nonlinear optical (NLO) properties of four novel Corroles in the visible spectral range (660–800 nm) were evaluated using picosecond Z-scan technique. Ultrafast excited state dynamics have also been appraised with picosecond (ps) and femtosecond (fs) degenerate pump–probe techniques using excitation wavelengths of 800 and 600 nm, respectively. The excitation by 800 nm photons resulted in two-photon absorption at adequately high peak intensities, thereby facilitating the access to higher excited states (Sn). The nonradiative relaxation mechanisms from these states, reflected in the pump–probe data, consisted of double-exponential decay with a slow component in the range of 54–277 ps and faster component in the range of 2.0 to 2.5 ps. When excited with 600 nm photons (unfocused), photoinduced absorption was observed with the first excited state S1 being populated, and as a consequence single decay was observed in the data of all molecules studied. These retrieved lifetimes were analogous to those obtained with picosecond pump–probe data. The long lifetime is attributed to nonradiative decay from the S1 state with possible contribution from triplet states, whereas the shorter lifetime is attributed to the internal conversion (S2 to S1*), followed by vibrational relaxation (S1* to S1) processes. Time-resolved fluorescence lifetime measurements revealed the magnitude of radiative lifetimes to be in the nanosecond regime. NLO coefficients were evaluated from the Z-scan data at wavelengths of 660, 680, 700, 740, and 800 nm. Large two-photon absorption coefficients (β)/cross-sections (σ2) at 740 nm/680 nm were recorded for these molecules, making them apposite for applications such as two-photon induced photodynamic therapy and lithography. Figure of merit, T, was <1 for all molecules at 740 and 800 nm, suggesting that these molecules find use in photonic device applications.

Recognition-Mediated Light-Up of Thiazole Orange with Cucurbit[8]uril: Exchange and Release by Chemical Stimuli

This article reports a convenient supramolecular strategy to construct fluorescent photoswitchable molecular assemblies between a macrocyclic host, cucurbit[8]uril (CB8), and a fluorogenic dye, thiazole orange (TO). The interaction mechanism and the stable stoichiometric host-guest arrangements have been claimed on the basis of the optical absorption, steady-state and time-resolved fluorescence lifetime and anisotropy measurements, and also the geometry optimization studies. The CB8 recognized TO in its 2:2 stoichiometry exhibited spectacular fluorescence enhancement of the order of 1700 fold, which is the largest directly determined value so far reported for a dye in an organic macrocyclic system. This prospective 2CB8:2TO assembly responded to selected chemical stimuli such as metal ions, adamantylamine, and tryptophan, providing different dissociation mechanisms and demonstrating a controlled exchange and release action desired with such noncovalently linked assemblies. Positively, considering the aqueous solubility and biocompatibility of the host-guest constituents, this methodology can evolve into a general approach to deliver and operate intracellularly functional molecular components under chemical/thermal/optical trigger control, especially for therapeutic applications.

Photophysical studies of PET based acridinedione dyes with globular protein: Bovine serum albumin

Interaction of acridinedione dyes with model transport proteins, bovine serum albumin (BSA) in aqueous solution were investigated by fluorescence spectral studies. A fluorescence enhancement was observed on the addition of BSA to photoinduced electron transfer (PET) based acridinedione dyes, which posses C 6H 4(p-OCH 3) in the 9th position of the basic acridinedione ring. On the contrary, the addition of BSA to non-PET based acridinedione dyes with methyl or phenyl substitution in the 9th position does not result in any fluorescence enhancement. The enhancement in the fluorescence intensity is attributed to the suppression of PET process through space between –OCH 3 group and the acridinedione moiety is elucidated by steady state fluorescence measurements. The fluorescence anisotropy value ( r) of 0.40 reveals that the motion of the dye molecule is highly constrained and is largely confined to the rigid microenvironment of the protein molecule. The binding constant ( K) was found to be in the order of 6.0×10 3 [M] −1, which implies the existence of hydrophobic interaction between the PET based dye and BSA. Time resolved fluorescence lifetime measurements reveal that the PET based acridinedione dye preferably binds in the hydrophobic interior of BSA.

Identification and Characterization of Binding Sites on S100A7, a Participant in Cancer and Inflammation Pathways

S100A7 (psoriasin) is a member of the S100 family of signaling proteins. It is implicated in and considered a therapeutic target for inflammation and cancer, yet no small molecule ligands for S100A7 have been identified. To begin the development of specific small molecule inhibitors of S100A7 function, we have used a series of surface binding fluorescent dyes to probe the surface hydrophobic sites. Two naphthalene-based dyes (2,6-ANS and 1,8-ANS) were found to bind S100A7 in a distinct cleft. We characterized the binding interaction by determining both the structure of S100A7 bound to 2,6-ANS and the structure of S100A7 bound to 1,8-ANS to 1.6 A. In both cases, two molecules of dye were docked such that the naphthalene groups were positioned in two symmetry-related grooves that are formed by the N-terminal helices of each monomer. We observed that Met12 acts as a gatekeeper to the binding cleft, adopting an "open" conformation for the more elongated 2,6-ANS while remaining in a "closed" conformation for the more compact 1,8-ANS. Steady-state fluorescence experiments revealed that S100A7 binds two copies of 2,6-ANS, each with a K(d) of 125 muM. Time-resolved fluorescence lifetime measurements indicated that the two molecules of 2,6-ANS bind in two independent binding sites with different fluorescence lifetimes, suggesting that the S100A7 homodimer is not perfectly symmetric in solution. Isothermal titration calorimetry studies demonstrate that S100A7 has a higher affinity for 2,6-ANS than 1,8-ANS. Yeast two-hybrid studies were also used to probe contributions of individual residues of an S100A7 triple mutant with respect to Jab1 binding. Mutation of Leu78, which forms part of the Met12 cleft occupied by 2,6-ANS, reduced the level of Jab1 binding, suggesting a potentially important role for the Met12 hydrophobic pocket in defining a Jab1 interface. Additional Y2H studies also delineate contributions of Gln88 and in particular Asp56 that shows the most significant abrogated binding to Jab1. Collectively, these data suggest a complex interaction between S100A7 and the much larger Jab1. These studies form the basis for the development of small molecule reporters and modifiers of S100A7 form and function.

Hydrogen-Bonding Effects on Film Structure and Photoelectrochemical Properties of Porphyrin and Fullerene Composites on Nanostructured TiO2 Electrodes

Hydrogen-bonding effects on film structures and photophysical, photoelectrochemical, and photovoltaic properties have been examined in mixed films of porphyrin and fullerene composites with and without hydrogen bonding on nanostructured TiO2 electrodes. The nanostructured TiO2 electrodes modified with the mixed films of porphyrin and fullerene composites with hydrogen bonding exhibited efficient photocurrent generation compared to the reference systems without hydrogen bonding. Atomic force microscopy, infrared reflection absorption and ultraviolet−visible absorption spectroscopies, and time-resolved fluorescence lifetime and transient absorption spectroscopic measurements disclosed the relationship between the film structures and optical and photoelectrochemical properties relating to the formation of hydrogen bonding between the porphyrins and/or the C60 moieties in the films on the electrode surface. These results show that hydrogen bonding is a potential methodology for the fabrication of donor and ac...