Fluorescence Anisotropy Decay Research Articles

Photoinduced Excited-State Energy-Transfer Dynamics of a Nitrogen-Cored Symmetric Dendrimer: From the Perspective of the Jahn–Teller Effect

We report an interesting view to understand the ultrafast excited-state energy-transfer (EET) process in the D3-symmetric dendrimer tris(4-ethynylphenyl)amine (TEPA) from the perspective of the well-known E⊗e Jahn–Teller (JT) effect. Upon excitation to two lowest excited states (S1 and S2) with doubly degenerate E symmetry, two sets of e vibrational modes, dihedral angle twist and strong pyramidalization near the nitrogen core, lead to the JT distortion and symmetry lowering. Through the excited-state dynamics simulation with the on-the-fly surface-hopping approach at the TDDFT level, we find that the system may either travel three equivalent minima of S1 state or undergo the nonadiabatic transitions between S1 and S2 states. These motions induce the ultrafast EET among different branches and the reorientation of the transition dipole moments, finally leading to the ultrafast fluorescence anisotropy decay. This energy-transfer mechanism can provide some interesting insights on the excited-state dynamics o...

The effect of local dynamics of Atto 390-labeled lysozyme on fluorescence anisotropy modeling.

Fluorescence anisotropy decay is a popular optical technique to study the structure, size, shape, and even functions of biomolecules. The method measures the time dependence of the depolarization of a fluorophore and is therefore sensitive to the changes in the rotational motion (e.g., aggregation and binding) or changes in the mobility of segments of biopolymers (such as the ones associated with tertiary structure changes). Fluorescence anisotropy decay often requires the use of fluorescent dyes that need to be covalently attached to the biomolecule. The location of the attachment on the biomolecule (e.g., a protein) and the linker used, affect the mobility of the dye and its anisotropy decay. With this study we have combined the experimental data with molecular dynamic simulations to offer a more correct interpretation of the fluorescence anisotropy decay of a popular fluorescent dye (Atto 390) attached to the N-terminus of Hen Egg White Lysozyme (HEWL). Our model showed how the use of relatively simple molecular dynamics computation to simulate the motion of the dye, provide a model to interpret the experimental fluorescence anisotropy decay that yields a better estimate of the hydrodynamic radius of HEWL. The improvement is provided by a more detailed description of the segmental motion of the dye attached to the protein.

Resolution of sub-nanosecond motions in botulinum neurotoxin endopeptidase: An evidence of internal flexibility

Botulinum neurotoxins (BoNTs) are the most poisonous substances known to mankind, which act on the peripheral nervous system leading to flaccid paralysis. Although co-crystal structure of BoNT/A light chain (LC) reveals some unique features of the biological function of this molecule, structural characteristics in solution reveal its dynamic features, not available through the published crystal structures. In this study, we have examined internal flexibility of this molecule by measuring rotational correlation time as a function of viscosity, using frequency domain fluorescence anisotropy decay technique. Fluorescence anisotropy decay of BoNT/A LC resolved sub-nanosecond local motion (faster component), interpreted as internal flexibility of the molecule was affected significantly with viscosity. Both local and global movements were affected by viscosity, which indicates the accessibility of protein core and flexibility of overall structure. In conclusion, this work demonstrates the presence of flexibility in the internal peptide segments, which appears to play a significant role in BoNT/A LC biological function.

Photoexcited Energy Transfer in a Weakly Coupled Dimer.

Nonadiabatic excited-state molecular dynamics (NA-ESMD) simulations have been performed in order to study the time-dependent exciton localization during energy transfer between two chromophore units of the weakly coupled anthracene dimer dithia-anthracenophane (DTA). Simulations are done at both low temperature (10 K) and room temperature (300 K). The initial photoexcitation creates an exciton which is primarily localized on a single monomer unit. Subsequently, the exciton experiences an ultrafast energy transfer becoming localized on either one monomer unit or the other, whereas delocalization between both monomers never occurs. In half of the trajectories, the electronic transition density becomes completely localized on the same monomer as the initial excitation, while in the other half, it becomes completely localized on the opposite monomer. In this article, we present an analysis of the energy transfer dynamics and the effect of thermally induced geometry distortions on the exciton localization. Finally, simulated fluorescence anisotropy decay curves for both DTA and the monomer unit dimethyl anthracene (DMA) are compared. Our analysis reveals that changes in the transition density localization caused by energy transfer between two monomers in DTA is not the only source of depolarization and exciton relaxation within a single DTA monomer unit can also cause reorientation of the transition dipole.

Ground and excited state proton transfer of the bioactive plant flavonol robinetin in a protein environment: spectroscopic and molecular modeling studies.

We performed spectroscopic and molecular modeling studies to explore the interaction of the bioactive plant flavonol robinetin (3,7,3',4',5'-OH flavone), with the carrier protein human serum albumin (HSA). Multiparametric fluorescence sensing, exploiting the intrinsic "two color" fluorescence of robinetin (comprising excited state intramolecular proton transfer (ESIPT) and charge transfer (CT) emissions) reveals that binding to HSA significantly affects the emission and excitation profiles, with strongly blue-shifted (∼29 nm) normal fluorescence and remarkable increase in the ESIPT fluorescence anisotropy (r) and lifetime (τ). Flavonol-induced HSA (tryptophan) fluorescence quenching data yield the dynamic quenching constant (KD) as 5.42 × 10(3) M(-1) and the association constant (Ks) as 5.59 × 10(4) M(-1). Time-resolved fluorescence anisotropy decay studies show dramatic (∼170 times) increase in the rotational correlation time (τ(rot)), reflecting greatly enhanced restrictions in motion of robinetin in the protein matrix. Furthermore, prominent induced circular dichroism (ICD) bands appear, indicating that the chiral environment of HSA strongly perturbs the electronic transitions of the intrinsically achiral robinetin molecule. Molecular docking calculations suggest that robinetin binds in subdomain IIA of HSA, where specific interactions with basic residues promote ground state proton abstraction and stabilize an anionic species, which is consistent with spectroscopic observations.

Folding and Hydrodynamics of a DNA i-Motif from the c-MYC Promoter Determined by Fluorescent Cytidine Analogs

The four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemiprotonation of cytosine results in a stable C-C+ basepair. Here, we performed fundamental studies to examine iM formation from a C-rich strand from the promoter of the human c-MYC gene. We used a number of biophysical techniques to characterize both the hydrodynamic properties and folding kinetics of a folded iM. Our hydrodynamic studies using fluorescence anisotropy decay and analytical ultracentrifugation show that the iM structure has a compact size in solution and displays the rigidity of a double strand. By studying the rates of circular dichroism spectral changes and quenching of fluorescent cytidine analogs, we also established a mechanism for the folding of a random coil oligo into the iM. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and to determine the pKa of an iM. We established that the C-C+ hydrogen bonding of certain bases initiates the folding of the iM structure. We also showed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding compared with bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded forms. Taken together, our results lay the foundation for using fluorescent dC analogs to follow structural changes during iM formation. Our technique may also be useful for examining folding and structural changes in more complex iMs.

Detection and characterization of liquid|solid and liquid|liquid|solid interfacial gradients of water nanodroplets in wet N-octyl-2-pyrrolidone.

We report on the rotational diffusion dynamics and fluorescence lifetime of lissamine rhodamine B sulfonyl chloride (LRSC) in two thin-film experimental configurations. These are liquid|solid interfaces, where N-octyl-2-pyrrolidone (NOP) containing water and ethylene glycol (EG) thin films are each supported on glass, and a liquid|liquid|solid interface where thin films of water and NOP, both supported on glass, are in contact with one another, forming an NOP|water interface. The reorientation dynamics and fluorescence lifetime of LRSC are measured as a function of distance from the NOP|glass and EG|glass interfaces and from the NOP|water and NOP|glass interfaces in the liquid|liquid|solid experimental configuration. Fluorescence anisotropy decay data from the liquid|solid systems reveal a liquid film depth-dependent gradient spanning tens of micrometers from the NOP|glass interface into the wet NOP phase, while this gradient is absent in EG. We interpret these findings in the context of a compositional gradient in the NOP phase. The spatially resolved fluorescence lifetime and anisotropy decay data for an NOP|water|glass interfacial structure exhibits the absence of a gradient in the anisotropy decay profile normal to the NOP|water interface and the presence of a fluorescence lifetime gradient as a function of distance from the NOP|water interface. The compositional heterogeneity for both interfacial systems is in the form of water nanodroplets in the NOP phase. We understand this compositional gradient in the context of the relative surface energies of the water, NOP, and glass components.

Fluorescence Anisotropy Uncovers Changes in Protein Packing with Inclusion Growth in a Cellular Model of Polyglutamine Aggregation

The aggregation of polyglutamine-rich proteins is closely linked with numerous neurodegenerative disorders. In pathological and cellular models, the appearance of protein-rich inclusions in cells acts as a read out of protein aggregation. The precise organization of aggregated protein in these inclusions and their mode of growth are still poorly understood. Here, fluorescence anisotropy-based measurements have been used to probe protein packing across inclusions of varying brightness, formed by an monomeric enhanced green fluorescent protein (mEGFP)-tagged polyglutamine model peptide in cells. High-resolution, confocal-based steady-state anisotropy measurements report a large depolarization, consistent with extensive homo-Förster (fluorescence) resonance energy transfer (FRET) between the sequestered mEGFP-tagged protein molecules. An inverse correlation of fluorescence anisotropy with intensity is seen across inclusions, which becomes emphasized when the observed fluorescence anisotropy values of inclusions are corrected for the fluorescence contribution of the diffusible protein, present within and around smaller inclusions. Homo-FRET becomes enhanced as inclusion size increases. This enhancement is confirmed by two-photon excitation-based time-resolved fluorescence anisotropy decay measurements, which also suggest that the mEGFP-tagged protein molecules are arranged in multiple ways within inclusions. Bright inclusions display faster FRET rates with a larger number of mEGFP moieties participating in homo-FRET than faint inclusions do. These results are consistent with a model in which the protein is more closely packed in the brighter inclusions. In such a possible mechanism, the higher packing density of protein molecules in brighter inclusions would suggest that inclusion growth could involve an intermolecular compaction event within the inclusion, as more monomers and aggregates are recruited into the growing inclusion.

Sugar Binding Effects on the Enzymatic Reaction and Conformation Near the Active Site of Pokeweed Antiviral Protein Revealed by Fluorescence Spectroscopy

In various trials for elucidating the physiological function of pokeweed antiviral protein (PAP), studies on the interaction with sugar are essential. The fluorescence titration curves showed that PAP retained the strong affinity against N-acetylglucosamine (NAG) and two sites in one PAP molecule co-operatively participated in the binding. In the complex of PAP with NAG, Trp208 located at the entrance lid site of substrate came closer to Tyr72 about 0.3 Å. Furthermore, the fluorescence anisotropy decay measurement demonstrated that the segmental rotation of Trp208 was enlarged by the binding of PAP with NAG. Such conformational changes around the active site closely correlate with the enzymatic activity of PAP. The N-glycosidase activity of PAP was enhanced more than two times in the presence of NAG. The obtained results consistently suggested the enzymatic activity of PAP would be regulated through the conformation change near the active site induced by the binding with NAG.

Structural Disruption of Phospholipid Bilayers over a Range of Length Scales by n-Butanol

We report on the exposure of planar multicomponent lipid bilayers supported on mica to n-butanol. The bilayer contains 49 mol % 1,2-dioleoyl-sn-phosphatidylcholine (DOPC), 10 mol % cholesterol, 40 mol % sphingomyelin, and 1 mol % sulforhodamine-tagged 1,2-dioleoyl-sn-phosphatidylethanolamine (SR-DOPE). Phase separation of the cholesterol domains is seen within the bilayer structure, and exposure of this supported bilayer to controlled amounts of n-butanol in the aqueous overlayer produces morphological changes over a range of length scales. We report steady state fluorescence imaging, fluorescence lifetime imaging, and fluorescence anisotropy decay imaging for these bilayers. These data are consistent with literature reports on the interactions of lipid bilayers with n-butanol and provide molecular-scale insight relative to bilayer organization that has not been available to date. The exposure of these bilayers to n-butanol leads to more extensive disruption of the bilayer than is seen for their exposure to ethanol.

Influence of Solute Charge and Pyrrolidinium Ionic Liquid Alkyl Chain Length on Probe Rotational Reorientation Dynamics

In recent years, the effect of molecular charge on the rotational dynamics of probe solutes in room-temperature ionic liquids (RTILs) has been a subject of growing interest. For the purpose of extending our understanding of charged solute behavior within RTILs, we have studied the rotational dynamics of three illustrative xanthene fluorescent probes within a series of N-alkylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([Cnmpyr][Tf2N]) RTILs with different n-alkyl chain lengths (n = 3, 4, 6, 8, or 10) using time-resolved fluorescence anisotropy decay. The rotational dynamics of the neutral probe rhodamine B (RhB) dye lies between the stick and slip boundary conditions due to the influence of specific hydrogen bonding interactions. The rotation of the negatively charged sulforhodamine 640 (SR640) is slower than that of its positively charged counterpart rhodamine 6G (R6G). An analysis based upon Stokes-Einstein-Debye hydrodynamics indicates that SR640 adheres to stick boundary conditions due to specific interactions, whereas the faster rotation of R6G is attributed to weaker electrostatic interactions. No significant dependence of the rotational dynamics on the solvent alkyl chain length was observed for any of the three dyes, suggesting that the specific interactions between dyes and RTILs are relatively independent of this solvent parameter.

Ethanol-Induced Perturbations to Planar Lipid Bilayer Structures

We report on the formation of planar lipid bilayer structures on mica where the bilayer contains the phosphocholine 1,2-dioleoyl-sn-phosphatidylcholine (DOPC), cholesterol, sphingomyelin and sulforhodamine-tagged-1,2-dioleoyl-sn-phosphatidylethanolamine (SR-DOPE). Phase separation is seen for the cholesterol domains within the bilayer structure, and exposure of this supported bilayer to controlled concentrations of ethanol reveals organizational changes on both the micrometer- and molecular-length scales. We report steady state fluorescence imaging, fluorescence lifetime imaging, and fluorescence anisotropy decay imaging for these bilayers. These data are complementary to existing information on the interactions of lipid bilayers with ethanol and point to subtle but important changes in the molecular-scale organization of these structures.

Molecular dynamics simulation of energy migration between tryptophan residues in apoflavodoxin

By performing molecular dynamics (MD) simulations of apoflavodoxin over the same timescale as fluorescence anisotropy decay measurements, the anisotropy model of two unidirectional FRET steps from two tryptophan residues to a third one can be reproduced from the tryptophan atomic coordinates in the MD trajectory.

The Transportation Potential of Human Serum Albumin for miR106A

Genetic therapy using microRNA in sequence knock-downs can benefit from a vector delivery system to effectively deliver targeted therapy in vivo. RNAi (RNA interference) mechanisms use splitting of a double-stranded miRNA to a single-strand that down-regulates genetic expression. Human serum albumin (HSA), a blood plasma transport protein, was selected for investigation as a possible transport of single- or double-strand miRNA. The binding effects of a single or double-stranded mimic miRNA, miR-106-A, with HSA was studied through the use of time-resolved fluorescence anisotropy decay and FRET, using covalent linking of the dye Atto 390 to the 3' OH ends of the mimic RNA and Atto 488 to the N-terminus of HSA. Single-stranded RNA was achieved by heat denaturation and subsequent rapid annealing in the HSA solution to prevent double-stranding re-association. The results show that the ionic strength of the buffer has a strong effect on binding and that at physiological conditions using PBS buffer and 37 °C incubation, HSA does weakly bind the double-stranded RNA, but not the single-stranded form.

Unfolding of a Small Protein Proceeds via Dry and Wet Globules and a Solvated Transition State

Dissecting a protein unfolding process into individual steps can provide valuable information on the forces that maintain the integrity of the folded structure. Solvation of the protein core determines stability, but it is not clear when such solvation occurs during unfolding. In this study, far-UV circular dichroism measurements suggest a simplistic two-state view of the unfolding of barstar, but the use of multiple other probes brings out the complexity of the unfolding reaction. Near-UV circular dichroism measurements show that unfolding commences with the loosening of tertiary interactions in a native-like intermediate, N∗. Fluorescence resonance energy transfer measurements show that N∗ then expands rapidly but partially to form an early unfolding intermediate IE. Fluorescence spectral measurements indicate that both N∗ and IE have retained native-like solvent accessibility of the core, suggesting that they are dry molten globules. Dynamic quenching measurements at the single tryptophan buried in the core suggest that the core becomes solvated only later in a late wet molten globule, IL, which precedes the unfolded form. Fluorescence anisotropy decay measurements show that tight packing around the core tryptophan is lost when IL forms. Of importance, the slowest step is unfolding of the wet molten globule and involves a solvated transition state.

Solvent Controlled Energy Transfer Processes in Triarylamine-Triazole Based Dendrimers

Fluorescence upconversion measurements of three different dendrimers G1–G3 based on triarylamines connected by triazole linkers show a strong and fast initial decay of fluorescence anisotropy for t...

Detailed kinetic analysis of the interaction between the FOXO4–DNA-binding domain and DNA

The FOXO forkhead transcription factors are potent transcriptional activators involved in a wide range of key biological processes. In this work, the real-time kinetics of the interaction between the FOXO4–DNA binding domain (FOXO4–DBD) and the DNA was studied by using surface plasmon resonance (SPR). SPR analysis revealed that the interaction between FOXO4–DBD and the double stranded DNA containing either the insulin-responsive or the Daf-16 family member-binding element is preferably described by using a conformational change model which suggests a structural change of FOXO4–DBD upon binding to the DNA. This was further confirmed by using the time-resolved tryptophan fluorescence anisotropy decay measurements which revealed profound reduction of segmental dynamics of FOXO4–DBD upon the complex formation. Alanine scanning of amino acid residues engaged in polar contacts with the DNA showed that certain non-specific contacts with the DNA backbone are very important for both the binding affinity and the binding specificity of FOXO4–DBD.

A 1,3‐Phenylene‐Bridged Hexameric Porphyrin Wheel and Efficient Excitation Energy Transfer along the Wheel

A 1,3-phenylene-bridged hexameric Zn(II) porphyrin wheel was synthesized by a Suzuki-Miyaura coupling reaction through a one-pot or a stepwise route. The hexameric wheel structure was revealed by using X-ray diffraction analysis. The porphyrin wheel exhibits a split Soret band due to effective exciton coupling and displays efficient excitation energy transfer along the wheel. Measurements of fluorescence anisotropy decay and pump-power-dependent decay reveal a rapid excitation energy hopping along the wheel with a rate of 1.4 ps.

Photophysical Behavior of 8-Anilino-1-Naphthalenesulfonate in Vesicles of Pulmonary Surfactant Dipalmitoylphosphatidylcholine (DPPC) and Its Sensitivity toward the Bile Salt–Vesicle Interaction

The photophysical behavior of 8-anilino-1-naphthalenesulphonate (ANS) in vesicles of dipalmitoylphosphatidylcholine (DPPC), a pulmonary surfactant, has been carried out in a detailed manner. ANS shows notable variations in fluorescence intensity, lifetime, and anisotropy parameters as it gets into the vesicle. It was found that ANS partitions well into the DPPC bilayer membrane with an estimated partition coefficient of ~2.0 × 10(5). Among the various fluorescence parameters of ANS, fluorescence anisotropy was found to be most responsive to the temperature induced phase change of the bilayer membrane. These interesting fluorescence parameters of ANS were then used to study the hydration of lipid bilayer membrane by submicellar concentration of bile salts. From the steady-state fluorescence intensity and dynamic fluorescence lifetime analyses it is clear that ANS is able to probe the submicellar concentration (≤1 mM) of bile salt induced hydration of lipid bilayer membrane that accompanies expulsion of ANS from the bilayer to the aqueous bulk phase. Lower-temperature shift in the phase transition of DPPC bilayer indicates that fluorescence anisotropy of ANS is sensitive enough to the bile salt induced perturbation in the packed acyl chains of DPPC bilayer and modification in the membrane fluidity. In presence of sodium deoxycholate (NaDC) and sodium cholate (NaC) in DPPC vesicles, ANS experiences restriction in rotational mobility which is evident from the variation in steady-state fluorescence anisotropy and fluorescence anisotropy decay parameters.

The Influence of Lithium Cations on Dynamics and Structure of Room Temperature Ionic Liquids

The orientational relaxation dynamics of perylene in the room temperature ionic liquid (RTIL) 1-butyl-3-methylimidazolium bis(trifluoromethyl)sulfonyl imide were studied as a function of lithium ion concentration. Perylene is nonpolar and locates in the alkyl regions of the RTIL. The lithium cation was added as lithium bis(trifluoromethyl)sulfonyl imide, so the addition of Li(+) did not change the anion. The Li(+) concentration ranged from 0 to 0.4 mol fraction. The dynamics were measured by observing the fluorescence anisotropy decay using time correlated single photon counting. The anisotropy experiments and viscosity measurements were performed as a function of temperature for each Li(+) concentration sample. Because perylene has high symmetry, it was possible to independently determine the in-plane and out-of-plane diffusion constants and friction coefficients. With increasing concentration of lithium salt the viscosity increases and both the in-plane and out-of-plane orientational relaxations of perylene become slower. However, the corresponding molecular friction coefficients decreased, with the in-plane coefficient decreasing to a greater extent than the out-of-plane coefficient. The decrease in the friction coefficients demonstrates that lithium ions, which are located in the ionic regions of the RTILs, change the structure of the alkyl regions of the RTIL.