Side-chain dynamics of an alpha-helical polypeptide monitored by fluorescence

The "blob" model, developed to analyze the fluorescence decays of polymers randomly labeled with pyrene, has been applied to a series of pyrene-labeled poly(glutamic acid)s (PyPGA) in DMF and carbonated buffer solutions at pH 9. Poly(glutamic acid) (PGA) exists in the ionized form in the buffer solutions as poly(sodium glutamate) (PGNa). PGA adopts an alpha-helical conformation in DMF, whereas in aqueous solution PGNa is a random coil. Fluorescence, UV-vis absorption, and circular dichroism measurements indicate that in our studies pyrene pendants attached themselves along PGA in a clustered manner. Simulations were carried out to establish that the geometry of the PGA alpha-helix induces the high level of pyrene clustering. Since the level of pyrene clustering decreased with lower pyrene content, information about naked PGA was retrieved by extrapolating the trends obtained by fluorescence to zero pyrene content. Analysis of the fluorescence decays demonstrated that during its lifetime an excited pyrene probes a 32 amino acid section of the PGA alpha-helix. This result was supported by molecular mechanics optimizations. This study establishes that the blob model, originally used to monitor the encounters between pyrenes attached randomly onto a polymer adopting a random coil conformation, can also be applied to study the dynamics of the side chains of structured proteins. Since the blob model helps in monitoring the encounters between amino acids in the initial state (i.e., random coil) and in the final state (i.e., structured protein) of the folding pathway of a protein, it could be applicable to the study of protein folding.     

Comparison of the association level of a pyrene-labeled associative polymer obtained from an analysis based on two different models

A hydrophobically modified alkali swellable emulsion copolymer (HASE) was labeled with pyrene and its fluorescence behavior was monitored by steady-state and time-resolved fluorescence as increasing amounts of the surfactant sodium dodecyl sulfate (SDS) were added to the solution. In aqueous solution, the pyrene pendants are aggregated. As SDS is added, the surfactant binds to the pyrene aggregates, which leads to their breakup at an onset SDS concentration of 1.25 x 10(-3) mol/L. The breakup of the pyrene aggregates is complete at 4.25 x 10(-3) mol/L, which is slightly larger than the critical micellar concentration of SDS in 0.01 M Na(2)CO(3) aqueous solution at pH 9 found to equal 3.5 x 10(-3) mol/L by surface tension measurements. The pyrene pendants were present as different species in solution, and the fractions representative of all emissive pyrene species were determined from the global analysis of the monomer and excimer fluorescence decays. Two analyses were applied to the decays. In the first analysis, the diffusional encounters between pyrene pendants were described by the blob model. In the second analysis, no assumptions were made on how the pyrene pendants encountered each other. Both analyses yielded identical results which demonstrate that the determination of the fractions of the different emissive pyrene species of a solution of a pyrene-labeled associative polymer does not depend on the model chosen to account for the diffusional encounters taking place between pyrene pendants.     

Effect of side-chain length on the side-chain dynamics of alpha-helical poly(L-glutamic acid) as probed by a fluorescence blob model

Two series of pyrene-labeled poly(glutamic acid) (Py-PGA) were synthesized utilizing two different linkers for pyrene attachment, namely 1-pyrenemethylamine (PMA) and 1-pyrenebutylamine (PBA). Several Py-PGAs were synthesized for each series with pyrene contents ranging from 4 to 15 mol %. Py-PGA forms a rigid alpha-helix in DMF that effectively locks the backbone in place, thus enabling only side-chain or linker motions to be monitored by time-resolved fluorescence. Time-resolved fluorescence decays were acquired for the pyrene monomer of the Py-PGA constructs and the fluorescence blob model (FBM) was used to quantify the dynamics of the different linkers connecting pyrene to the backbone. Nitromethane was used to shorten the lifetime of the pyrene monomer, in effect controlling the probing time of the pyrene group, from 50 to 155 ns for PGA-PBA and from 50 to 215 ns for PGA-PMA. The FBM analysis of the fluorescence decays led to the conclusion that excimer formation around the rigid alpha-helix backbone takes place in a compact environment. The number of glutamic acid units within a blob, N blob, decreased only slightly with decreasing probing time for both Py-PGA constructs as a result of the compact distribution of the chromophores around the alpha-helix. The PGA alpha-helix was modeled using Hyperchem software and the ability of two pyrene groups to encounter was evaluated as they were separated by increasing numbers of amino acids along the alpha-helix. The number of amino acids required for two pyrenes to lose their ability to overlap and form excimer matched closely the N blob values retrieved using the FBM.     

Dynamics of short as compared with long poly(acrylic acid) chains hydrophobically modified with pyrene, as followed by fluorescence techniques

New low and high molecular weight poly(acrylic acid), PAA, 2000 g mol(-1) and 450,000 g mol(-1), respectively, were tagged with pyrene (low and high contents of probe) and its behaviour in solution was investigated using absorption and fluorescence (steady-state and time-resolved) techniques. Fluorescence data shows that the degree and level of intramolecular association strongly depends on the molecular weight. With the short pyrene-labeled PAA chains in aqueous solution, the excimer-to-monomer fluorescence ratio I(E)/I(M) decreases with the increase of pH, oppositely to the increase in the I(E)/I(M) ratio with the increase in pH previously observed with the long chain PAA. Time-resolved data suggest that excimer formation with the short pyrene-labeled PAA polymers (ca. 28 acrylic acid monomers per chain) in water is largely due to excitation of Ground State Dimers, GSD. The increment of pH, and the consequent gradual ionization of the carboxylic groups in the chain, initially increases the fraction of GSD, possibly due to the occurrence of special micelle-like chain conformations, inside which the pyrene units are accommodated. A further increase of the pH above the pK(a) values, resulting in the full ionization of carboxylic groups, apparently destabilizes such chain conformations, which leads to a pH effect on the photophysical properties identical to that of the long chain polymers. In water, the dynamic data shows the existence of two excimers coexisting with two monomer classes. In methanol and dioxane (good solvents for the pyrene probe) at room temperature, where one excimer and two monomers are present, all rate constants could be obtained, as well as the fractions of ground-state species. It is thus shown that different types of interactions are produced with small- and long-sized PAA polymers, i.e., the size of the polymer matters.     

Circularly polarized luminescence from pyrene excimers

Recently, many reports have emerged about circularly polarized luminescence (CPL) based on excimer emission of pyrenes. The intense CPL was observed from various pyrene derivatives such as pyrenes having chiral side chains, chiral oligomers bearing multiple pyrene rings, and pyrenes encapsulated by γ-cyclodextrins. The luminescence dissymmetry factor was found to be obviously higher than the absorption one in those pyrenes. In addition, several pyrenes revealed reversible “on-off” CPL switching upon the complexation/decomplexation of metal ions.     

Pyrene-stacked nanostructures constructed in the recombinant tobacco mosaic virus rod scaffold

The effect of pyrenes introduced into a tobacco mosaic virus (TMV) coat protein monomer on the formation and stability of the TMV assembly was investigated. The possible arrangement of the pyrenes in the inner cavity of the TMV rod was also estimated. The pyrene derivative was introduced to four specific amino acids in the cavity of the TMV rod structure. Rod-structure formation was examined by atomic force microscopy (AFM). Two pyrene-attached mutants (positions 99 and 100) assembled to increase the length of the rod structures by 2.5 microm at pH 5.5. The interaction of the pyrene moieties in the TMV cavity was investigated by steady-state and time-resolved spectroscopic analysis. Strong excimer emission with significantly short wavelength (465 nm) was observed from the two mutants mentioned above. Excitation and UV-visible spectra indicate that the pyrene moieties form pi-stacked structures in the TMV cavity. Details of the pyrene interaction were investigated by analyzing the fluorescence lifetime of the excimer. Results suggest that the pyrenes formed preassociated rigid structures with partially overlapped geometry in the restricted space of the TMV cavity. The pyrenes effectively stabilize the TMV rod through a pi-stacking interaction in a well-ordered way, and the single pyrene moiety introduced into the monomer affects the overall formation of the TMV rod structure.     

Study of the semidilute solutions of poly(N,N-dimethylacrylamide) by fluorescence and its implications to the kinetics of coil-to-globule transitions

The large scale motions of poly(N,N-dimethylacrylamide) chains randomly labeled with pyrene (Py-PDMA) were monitored by steady-state and time-resolved fluorescence in semidilute solutions of naked PDMA in acetone and DMF for polymer concentrations ranging from 0 to 550 g/L. Although increasing the polymer concentration of the solution led to a decrease of the mobility of the chromophore attached onto the PDMA backbone, this reduction was rather modest when compared to the large increase of the macroscopic viscosity. This result indicated that locally, the monomer constituting the chains experienced freedom of movement despite the high solution viscosity. The restricted mobility of the chromophore was characterized by the number of monomers occupying the volume probed by the excited chromophore during its lifetime, referred to as a fluorescence "blob". The number of monomers constituting a fluorescence blob, N(F)(-)(blob), and the volume of a fluorescence blob, V(F)(-)(blob), were found to decrease as the polymer concentration of the solution increased, reflecting the decreased mobility experienced by the chromophore. In DMF, the radius of an F-blob was found to scale as N(nu)(F)(-)blob, where nu equaled 0.66 +/- 0.03, very close to the expected value of the Flory exponent of 0.6 for a polymer in a good solvent. The combined knowledge of how N(F)(-)(blob) varies with the fluorescence lifetime of the chromophore and the coil density of the polymer was used to propose a new means of studying coil-to-globule transitions with potential implications for predicting the rate of protein folding.     

A case for using randomly labeled polymers to study long-range polymer chain dynamics by fluorescence

The process of excimer formation was studied for a series of pyrene end-labeled polystyrenes (PS(X)-Py 2 where X is the polymer molecular weight equal to 3, 4.5, 8, 12.7, and 14.6 K) and two series of polystyrenes randomly labeled with pyrene (CoE-PS and CoA-PS) in seven different solvents. The solvent viscosities ranged from 0.41 to 1.92 mPa x s, while the solvent quality ranged from good to poor solvents for polystyrene, as determined by intrinsic viscosity measurements. Steady-state fluorescence spectra of the pyrene-labeled polymers were acquired, and the excimer to monomer ratios showed that excimer formation increased strongly with a decrease in solvent viscosity. The monomer and excimer time-resolved fluorescence decays were also acquired and fitted globally to either the Birks' scheme or the fluorescence blob model (FBM) for the end- or randomly labeled polymers, respectively. All parameters reporting on the long-range polymer chain dynamics (LRPCD) obtained from the analysis of the fluorescence data acquired with the PS(X)-Py 2, CoE-PS, and CoA-PS series yielded virtually identical trends, demonstrating that these fluorescence experiments yield results that are internally consistent with one another. Considering the substantial advantages associated with the preparation and study of randomly labeled polymers, this report presents an appealing case for the use of randomly labeled polymers in the study of LRPCD.     

Study of maleated ethylene-propylene copolymers by fluorescence: Evidence for succinimide induced polar associations in an apolar solvent

A maleated ethylene-propylene copolymer (EP-MAH) was labelled with 1-naphthalene- and/or 1-pyrenemethylamine to yield an EP copolymer bearing succinimide pendants all labelled with a chromophore. The labelled EPs were reacted with LiAlH₄ so that the polar succinimide linker group between the EP backbone and the chromophore was converted into apolar pyrrolidine units. The resulting products were purified through a gel permeation chromatography column to remove the cleaved off chromophores. FT-IR spectroscopy revealed that after reduction, the peak assigned to the succinimide carbonyls was strongly diminished. UV-vis absorption and steady-state and time-resolved fluorescence measurements were performed in hexane and THF. The reduction of the succinimide carbonyls was found to have a significant effect on the luminescence properties of the labelled EPs. The polar associations taking place between the succinimide moieties in hexane were found to be dramatically decreased after reduction as shown by UV-vis absorption, steady-state excitation and emission fluorescence, time-resolved fluorescence, and fluorescence resonance energy transfer. These results demonstrate that the presence of pyrene aggregates for EP-MAH labelled with 1-pyrenemethylamine is due primarily to the polar succinimide moieties rather than the aromatic pyrene.     

The effects of photochemical and mechanical damage on the excited state dynamics of charge-transfer molecular crystals composed of tetracyanobenzene and aromatic donor molecules

Charge-transfer molecular crystals are structurally well-defined systems whose electron transfer dynamics can be studied using time-resolved spectroscopy. In this paper, five 1:1 complexes, consisting of 1,2,4,5-tetracyanobenzene as the electron acceptor and durene, 9-methylanthracene, naphthalene, phenanthrene, and pyrene as electron donors, are studied using time-resolved fluorescence and transient absorption in the diffuse reflectance geometry. Two different sample morphologies were studied: single crystals and powders prepared by pulverizing the crystals and diluting them with barium sulfate microparticles. Fluorescence lifetime and transient absorption measurements performed on the crystals and the powders yielded different results. The crystals typically exhibited long-lived monoexponential fluorescence decays, while the powders had shorter multiexponential decays. Exposure of both types of samples to high laser fluence was also shown to induce faster excited state decay dynamics as observed using fluorescence and diffuse reflectance. In addition to the more rapid decays, these molecular crystals exhibited relatively high photobleaching quantum yields on the order of 10(-4). Previous work that interpreted picosecond decays in the transient absorption as evidence for rapid recombination and charge dissociation should be re-evaluated based on the susceptibility of this class of compounds to mechanical and photochemical damage.     

Synthesis and characterization of novel pyrene-dendronized porphyrins exhibiting efficient fluorescence resonance energy transfer: optical and photophysical properties

A novel series of pyrene dendronized porphyrins bearing two and four pyrenyl groups (Py(2)-TMEG1 and Py(4)-TMEG2) were successfully synthesized. First and second generation Fréchet type dendrons (Py(2)-G1OH and Py(4)-G2OH) were prepared from 1-pyrenylbutanol and 3,5-dihydroxybenzyl alcohol. These compounds were further linked to a trimesitylphenylporphyrin containing a butyric acid spacer via an esterification reaction to obtain the desired products. Dendrons and dendronized porphyrins were fully characterized by FTIR and (1)H NMR spectroscopy and their molecular weights were determined by matrix-assisted laser desorption ionization time of flight mass spectrometry. Their optical and photophysical properties were studied by absorption and fluorescence spectroscopies. The formation of dynamic excimers was detected in the pyrene-labeled dendrons, with more excimer being produced in the higher generation dendron. The fluorescence spectra of the pyrene dendronized porphyrins exhibited a significant decrease in the amount of pyrene monomer and excimer emission, jointly with the appearance of a new emission band at 661 nm characteristic of porphyrin emission, an indication that fluorescence resonance energy transfer (FRET) occurred from one of the excited pyrene species to the porphyrin. The FRET efficiency was found to be almost quantitative ranging between 97% and 99% depending on the construct. Model Free analysis of the fluorescence decays acquired with the pyrene monomer, excimer, and porphyrin core established that only residual pyrene excimer formation in the dendrons could occur before FRET from the excited pyrene monomer to the ground-state porphyrin core.     

Tripodal pyrene chromophores for semiconductor sensitization: new footprint design

Large footprint tripodal linkers for metal oxide nanoparticle sensitization, substituted with pyrene as the dye, and three COOR binding groups in para or meta position, were prepared to study the effect of the anchoring group position and of the footprint size on the sensitization processes. Two tripods based on tetraphenylsilane, (1-pyrenyl-4-ethynyl-phenyl)-tris(4-carbomethoxyphenyl-4-ethynyl-phenyl)silane, and (1-pyrenyl-4-ethynyl-phenyl)-tris(4-(4-methoxybenzyloxycarbonyl)phenyl-4-ethynyl-phenyl)silane, decomposed during hydrolysis, while the tetraphenyladamantane derivative, 1-(1-pyrenyl-4-ethynyl-phenyl)-3,5,7-(3-carbomethoxyphenyl-4-ethynyl-phenyl)adamantane, was chemically stable and was readily converted into the corresponding acid and bound to TiO₂ films. The FT-IR-ATR spectrum of 1-(1-pyrenyl-p-ethynyl-phenyl)-3,5,7-(3-carboxyphenyl-4-ethynyl-phenyl)adamantane bound to TiO₂ nanoparticles showed bands characteristic of carboxylate bidentate bonds. The UV-vis absorption and fluorescence emission in THF solution at room temperature were typical of pyrenes substituted with oligophenyleneethynylene linkers.     

Intriguing H-aggregate and H-dimer formation of coumarin-481 dye in aqueous solution as evidenced from photophysical studies

Photophysical properties of coumarin-481 (C481) dye in aqueous solution show intriguing presence of multiple emitting species. Concentration and wavelength dependent fluorescence decays and time-resolved emission spectra and area-normalized emission spectra suggest the coexistence of dye monomers, dimers, and higher aggregates (mostly trimers) in the solution. Because of the efficient intramolecular charge transfer (ICT) state to twisted intramolecular charge transfer (TICT) state conversion, the dye monomers show very short fluorescence lifetime of ~0.2 ns. Fluorescence lifetimes of dimers (~4.1 ns) and higher aggregates (~1.4 ns) are relatively longer due to steric constrain toward ICT to TICT conversion. Observed results indicate that the emission spectra of the aggregates are substantially blue-shifted compared to monomers, suggesting H-aggregation of the dye in the solution. Temperature-dependent fluorescence decays in water and time-resolved fluorescence results in water-acetonitrile solvent mixtures are also in support of the dye aggregation in the solution. Though dynamic light scattering studies could not recognize the dye aggregates in the solution due to their small size and low concentration, fluorescence up-conversion measurements show a relatively higher decay tail in water than in water-acetonitrile solvent mixture, in agreement with higher dye aggregation in aqueous solution. Time-resolved fluorescence results with structurally related non-TICT dyes, especially those of coumarin-153 dye, are also in accordance with the aggregation behavior of these dyes in aqueous solution. To the best of our knowledge, this is the first report on the aggregation of coumarin dyes in aqueous solution. Present results are important because coumarin dyes are widely used as fluorescent probes in various microheterogeneous systems where water is always a solvent component, and the dye aggregation in these systems, if overlooked, can easily lead to a misinterpretation of the observed results.     

荧光淬灭法研究疏水缔合共聚物(PAM-b-PPOEA)_n自组装聚集数

运用荧光淬灭技术,包括稳态荧光淬灭法(SSFQ)和时间分辨荧光淬灭法(TRFQ),研究了疏水缔合水溶性丙烯酰胺2苯氧基丙烯酸酯多嵌段共聚物[P(AM POEA)]在水溶液中自组装的聚集数.这类聚合物在水溶液中易形成胶束状聚集体,探针芘分子和淬灭剂二苯酮增溶于疏水微区,荧光测定结果很好地符合Poisson淬灭模型.实验结果表明聚合物链结构、聚合物浓度和无机盐对聚集体的尺寸具有重要影响.聚合物自组装聚集数NA随疏水单体含量的增加和疏水嵌段长度的减小而增大,同时也随聚合物浓度和NaCl浓度增加而增大.另外对聚合物链结构、聚集数和溶液粘度的相互关系进行了讨论.     

Molecular Packing and Solid-State Photophysical Properties of 1,3,6,8-Tetraalkylpyrenes

The relationship between the photophysical properties and molecular orientation of 1,3,6,8-tetraalkylpyrenes in the solid state is described herein. The introduction of alkyl groups with different chain structures (in terms of length and branching) did not affect the photophysical properties in solution, but significantly shifted the emission wavelengths and fluorescence quantum yields in the solid state for some samples. Pyrenes bearing ethyl, isobutyl, or neopentyl groups at the 1-, 3-, 6-, and 8-positions showed similar emission profiles in both the solution and solid states. In contrast, pyrenes bearing other alkyl groups exhibited an excimer emission in the solid state, similar to that of the parent pyrene. On studying the photophysical properties in the solid state with respect to the obtained crystal structures, the observed solid-state photophysical properties were found to depend on the relative position of the pyrene chromophores. The solid-state photophysical properties can be controlled by the alkyl groups, which provide changing crystal packing. Among the pyrenes tested, 1,3,6,8-tetraethylpyrene showed the highest fluorescence quantum yield of 0.88 in the solid state.     

A label-free fluorescence sensor for probing the interaction of oligonucleotides with target molecules

This paper describes a universal, label-free fluorescence sensor that gauges the interaction of oligonucleotides with their targets. The sensor is based on supramolecular assemblies formed by oligonucleotides (polyanions) and small molecule cation surfactant in aqueous solution. The environmentally dependent dye pyrene, encapsulated in the apolar interiors of the assemblies via hydrophobic interactions works as the fluorescence probe. Target binding causes the conformational change of the oligonucleotides, which results in disorganization of supramolecular assemblies, release of pyrene into the aqueous solution and subsequent quenching of its fluorescence. The kinetic processes (including the formation of supramolecular assemblies and the release of pyrenes after adding the targets) were investigated. The fluorescence decreases of pyrenes are proportional to the concentrations of targets within the linear ranges. This label-free fluorescence system is simple, convenient, low cost, and can serve as an alternative tool for interaction studies of oligonucleotides with their targets, especially with small molecular targets.     

Fluorescence studies on binding of pyrene and its derivatives to humic acid

Binding of pyrene (PyH) and its derivatives to humic acid (HA) has been studied by fluorescence spectroscopy. The nature of the interaction between HA and pyrene derivatives are extensively investigated by employing three derivatives ranging from anionic to cationic compounds: 1-pyrenebutylic acid (PyA), 1-pyrenemethanol (PyM), and 1-pyrenebutyltrimethylammonium bromide (PyB). Binding constants between HA and PyX (X=H, A, M, B) are obtained by steady-state fluorescence quenching techniques, and it is found that PyB has a markedly large binding constant among the pyrene family. This is attributed to a strong electrostatic interaction between cationic PyB and anionic HA. The result suggests that an electrostatic interaction plays a dominant role in binding of pyrenes to humic acid. The importance of electrostatic interaction was also confirmed by a salt effect on the binding constant. Influence of collisional quenching on the binding constant, which causes overestimation of the binding constant, was examined by time-resolved fluorescence spectroscopy as well as temperature effect in steady-state fluorescence measurements. It is elucidated that collisional quenching does not much bring overestimation into the binding constants.     

Global and target analysis of time-resolved fluorescence spectra of Di-9H-fluoren-9-yldimethylsilane: dynamics and energetics for intramolecular excimer formation

Dynamics and energetics for intramolecular excimer formation of a diarylsilane, di-9H-fluoren-9-yldimethylsilane (DFYDMS) have been investigated by means of ps time-resolved fluorescence spectroscopy and ab initio calculation. Multiple fluorescence decay curves were globally deconvolved to generate time-resolved fluorescence spectra and decay-associated spectra (DAS), from which species-associated spectra (SAS) were obtained. It is shown in the global analysis that there are at least three excited states: Two states are the locally excited (LE) states (lambda(max) approximately 320 nm) having lifetimes of 0.70 +/- 0.04 and 1.75 +/- 0.02 ns, and another is the excimer state (lambda(max) approximately 400 nm) having a lifetime of 7.34 +/- 0.02 ns. The species which decays with 0.70 ns evolves into a species with a red-shifted spectrum, which in turn decays in 7.34 ns. The experimental and ab initio results indicate that the rise time of 0.70 ns corresponds to the conversion of the initial S(1) LE state having a near sandwich geometry to the S(1) excimer state adopting a true sandwich geometry.     

Pyrene aromatic arrays on RNA duplexes as helical templates

RNA oligomers having multiple (2 to 4) pyrenylmethyl substituents at the 2'-O-sugar residues were synthesized. UV-melting studies showed that the pyrene-modified RNAs could form duplexes with complementary RNA sequences without loss of thermal stability. Absorption, fluorescence, and circular dichroism (CD) spectra revealed that the incorporated pyrenes projected toward the outside of A-form RNA duplexes and assembled in helical aromatic arrays along the minor grooves of the RNA duplexes. Results of computer simulations agreed with the assembled structures of the pyrenes. The helical pyrene arrays exhibited remarkably strong excimer fluorescence, which was dependent on the sequence contexts of RNA duplexes.