Time-resolved Fluorescence Measurements Research Articles

Interaction of a sphingolipid with human serum albumin in the native, thermally denatured and chemically denatured states: Emission wavelength-dependent photophysical revelation

The present study exhibits a spectroscopic deciphering of the effect of a phosphosphingolipid, sphingomyelin (SM), on native as well as thermally disrupted (at ~65 °C), and urea-induced partially (in the presence of ~4.5 M urea) and completely (in the presence of ~8 M urea) chemically denatured human serum albumin (HSA). The steady-state absorption studies reveal ground-state complex formation between SM and the serum albumin. The modulations in the steady-state emission spectral profile of the Trp residue in HSA along with the time-resolved fluorescence measurements depict SM-induced modification of the micro-environment around the sole Trp residue of HSA in all the studied cases, with no significant structural modification of the protein. However, monitoring the emission wavelength-sensitive time-resolved decay behaviour of Trp, keeping the excitation wavelength constant, in the absence and presence of SM in all the cases provides an interesting result depicting a progressive increase of average fluorescence lifetime of Trp upon shifting the monitoring emission wavelength gradually to the red end of the spectrum. A significantly greater extent of increase in the average lifetime of Trp with added SM compared to that in the absence of the lipid in case of completely chemically denatured HSA than those in the other studied cases provides a probable indication that SM modifies the solvation structure of the microenvironment around Trp in presence of 8 M urea most effectively and thus leading to a significant modulation of the solvation dynamics of the flourophore in its excited-state.

Identification of pyrene in complex sample matrix using time-resolved fluorescence measurement coupled with PARAFAC analysis

This article reports an approach to use pyrene time-resolved fluorescence decay coupled with parallel factor analysis (PARAFAC) as an indicator of the presence of pyrene in complex and interfering sample matrix. Pyrene is selectively excited using 340 nm LED and the fluorescence decay is collected at 390 nm with a cut-off filter at 350 nm; pyrene reports through its long excited-state fluorescence lifetime. Humic acid (HA), a component of fluorescent dissolved organic matter, and diesel samples, consisting of PAHs at unknown concentration levels are used as interfering analytes in this study. Compared to previous reported methods (which used time-delayed technique with the help of delay generators or low temperature fluorescence decay measurement), this method uses a proven multivariate data analysis tool (PARAFAC) to segregate fluorescence decay profile of background intense and short-lived chromophores; it is verified that PARAFAC model effectively resolves the interfering decay components. Data are analysed by two approaches: in the first method, decay data are directly subjected to PARAFAC to extract pyrene contribution. In the second method, the decay curves are first globally analysed using separate curve fitting software and then the resultant relative amplitudes or fractional intensities are subjected to PARAFAC analysis. The second method provides better decomposition in terms of relative fluorescence decay contributions of pyrene.

Phosphatidylinositol 4,5-bisphosphate drives Ca2+-independent membrane penetration by the tandem C2 domain proteins synaptotagmin-1 and Doc2β

Exocytosis mediates the release of neurotransmitters and hormones from neurons and neuroendocrine cells. Tandem C2 domain proteins in the synaptotagmin (syt) and double C2 domain (Doc2) families regulate exocytotic membrane fusion via direct interactions with Ca2+ and phospholipid bilayers. Syt1 is a fast-acting, low-affinity Ca2+ sensor that penetrates membranes upon binding Ca2+ to trigger synchronous vesicle fusion. The closely related Doc2β is a slow-acting, high-affinity Ca2+ sensor that triggers spontaneous and asynchronous vesicle fusion, but whether it also penetrates membranes is unknown. Both syt1 and Doc2β bind the dynamically regulated plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), but it is unclear whether PIP2 serves only as a membrane contact or enables specialized membrane-binding modes by these Ca2+ sensors. Furthermore, it has been shown that PIP2 uncaging can trigger rapid, syt1-dependent exocytosis in the absence of Ca2+ influx, suggesting that current models for the action of these Ca2+ sensors are incomplete. Here, using a series of steady-state and time-resolved fluorescence measurements, we show that Doc2β, like syt1, penetrates membranes in a Ca2+-dependent manner. Unexpectedly, we observed that PIP2 can drive membrane penetration by both syt1 and Doc2β in the absence of Ca2+, providing a plausible mechanism for Ca2+-independent, PIP2-dependent exocytosis. Quantitative measurements of penetration depth revealed that, in the presence of Ca2+, PIP2 drives Doc2β, but not syt1, substantially deeper into the membrane, defining a biophysical regulatory mechanism specific to this high-affinity Ca2+ sensor. Our results provide evidence of a novel role for PIP2 in regulating, and under some circumstances triggering, exocytosis.

Perceptive of block co polymers as potential delivering system of synthesized pyridine and pyrimidine derivatives: Spectroscopic and computational approach

In this work, a pyridine containing Schiff-base, 1-((anthracen-10-yl)methylene)-2-(pyridin-2-yl)hydrazine (AP) and a pyrimidine containing Schiff-base 2-(2-(Anthracen-9-ylmethylene) hydrazinyl)-4, 6-dimethyl pyrimidine (ANHP) have been synthesized and characterized by IR, NMR, HRMS and different spectroscopic methods. Two different tri block co polymers (P-123 and F-127) have been used as delivering system of this poor water soluble drug molecule AP and ANHP which are the main investigation of this work. To find out which one is the better delivering systems of AP and ANHP, spectroscopic studies have been carried out in P-123 and F-127 polymeric media. From the interactions of AP and ANHP with P-123 and F-127 polymers individually, binding constant values have been determined. The drug delivering efficiency of block co polymers has been investigated by using different spectroscopic techniques like absorbance, fluorescence, DLS and time resolved fluorescence measurements. All these experimental results indicate that P-123 is the better AP and ANHP delivering system than F-127 polymer. In addition, the structural properties like bond length, bond angles etc. of AP have been calculated by using density functional theory (DFT).

Photocatalytic Oxygenation Reactions with a Cobalt Porphyrin Complex Using Water as an Oxygen Source and Dioxygen as an Oxidant.

Photocatalytic oxygenation of hexamethylbenzene occurs under visible-light irradiation of an O2-saturated acetonitrile solution containing a cobalt porphyrin complex CoII(TPP) (TPP2- = tetraphenylporphyrin dianion), water, and triflic acid (HOTf) via a one-photon-two-electron process, affording pentamethylbenzyl alcohol and hydrogen peroxide as products with a turnover number of >6000; in this reaction, H2O and O2 were used as an oxygen source and a two-electron oxidant, respectively. The photocatalytic mechanism was clarified by means of electron paramagnetic resonance, time-resolved fluorescence, and transient absorption measurements as well as 18O-labeling experiments with H218O and 18O2. To the best of our knowledge, we report the first example of efficient photocatalytic oxygenation of an organic substrate by a metal complex using H2O as an oxygen source and O2 as a two-electron oxidant.

Are water-xylitol mixtures heterogeneous? An investigation employing composition and temperature dependent dielectric relaxation and time-resolved fluorescence measurements

Aqueous xylitol solutions at six different concentrations were studied employing dielectric relaxation (DR) and time-resolved fluorescence (TRF) measurements in the temperature range 295–323 K. The focus was to explore the solution heterogeneity aspect via monitoring the viscosity coupling of the average relaxation rates at various temperatures. TRF measurements were done using both hydrophobic and hydrophilic probes to explore the preferences, if any, for solute locations in these binary mixtures. Energy-selective population excitations and the corresponding fluorescence emissions did not suggest any significant spatial heterogeneity in solution structure within the lifetimes of these probes. DR measurements and TRF experiments indicated mild deviations from the hydrodynamic viscosity dependence of the measured relaxation rates. All these suggest mild spatiotemporal heterogeneity for these water-xylitol mixtures in the temperature range considered. In addition, DR timescales appear to originate from reorientational and H-bond relaxation dynamics, excluding the possibility of full molecular rotations. Heterogeneity in water-xylitol mixtures was investigated employing dielectric relaxation (DR) and Time-resolved fluorescence (TRF) spectroscopic techniques. Time-resolved anisotropy and DR response were found to depend both on xylitol concentration and temperature. Signature of mild heterogeneity was detected along with presence of water molecules that were slower than bulk-like water. Slower than bulk DR response vanishes with an increase of solution temperature.

Photophysical insights on a new supramolecular recognition element comprising PyC60 and a bisporphyrin studied in solution

The present work reports detailed photophysical insights on supramolecular interaction between C60 pyrrolidine tris-acid ethyl ester (PyC60) and a designed diporphyrin (1) in toluene. Both UV–vis and steady state fluorescence studies indicate effective complexation between PyC60 and 1 as revealed from the value of binding constant (K) of PyC60–1 complex, i.e., KPyC60–1 (UV–vis) = 7625 dm3·mol−1 and KPyC60–1 (fluorescence) = 9550 dm3·mol−1. Time-resolved fluorescence measurements evoke that the decay of photoexcited 1 in presence of PyC60 occurs only via static quenching mechanism and the magnitude of bimolecular quenching constant value for such system is enumerated to be 9.033 × 1011 dm3·mol−1·sec−1. In 1,2-dichlorobenzene (DCB), PyC60–1 system exhibits appreciable value of rate-constant of charge-separation (kcs) and quantum yield of charge-separation (ϕCS) than that observed in toluene. However, magnitude and order of both kcs (=5.35 × 107) and ϕCS (= 0.085) value for the PyC60–1 system estimated in DCB indicate that like toluene, there is no possibility of electron transfer in DCB. Both 1H and 13C NMR spectra of PyC60 molecule show appreciable shift of functional protons and carbon moiety of parent molecule in presence of 1, respectively. Quantum chemical calculations by ab initio method in vacuo establish the geometric structure of PyC60–1 complex and predict that dispersive forces associated with π-π interaction play significant role behind non-covalent interaction between PyC60 and 1.

Exploring Aqueous Solution Dynamics of an Amphiphilic Diblock Copolymer: Dielectric Relaxation and Time-Resolved Fluorescence Measurements.

We explore in this work, after synthesizing and appropriately characterizing an amphiphilic diblock copolymer, its interaction with water molecules and the subsequent aqueous solution dynamics by employing time-resolved fluorescence measurements (TRF) and megahertz-gigahertz dielectric relaxation (DR) experiments. The synthesized amphiphilic diblock copolymer is poly(2-(((tert-butoxycarbonyl)alanyl)oxy)ethyl methacrylate)-b-poly(polyethylene glycol monomethyl ether methacrylate) (P(Boc-l-Ala-HEMA)-b-PPEGMA). Dynamic light scattering measurements of aqueous solutions indicate formation of 14-20 nm particles from a balance between the chain lengths of the hydrophobic (P(Boc-l-Ala-HEMA) and hydrophilic (PPEGMA) segments. Field-emission scanning electron microscopy, on the other hand, suggests a spherical shape for the dried micelles. The critical micelle concentration of the P(Boc-l-Ala-HEMA)-b-PPEGMA block copolymer at different block lengths in aqueous media, determined via steady-state fluorescence measurements, is very low (∼4-8 mg/L), and the resultant micellar size has been found to be insensitive to the polymer concentration. Interfacial and bulk aqueous dynamics have been investigated by tracking the solution frictional resistance on rotational motion of dissolved hydrophobic and hydrophilic dipolar solute probes of comparable sizes. TRF anisotropy measurements reflect the biphasic temporal profile for the frictional resistance. Interestingly, the hydrophobic probe, because of its preferential location at the micellar interface, experiences greater frictional resistance than the hydrophilic counterpart, although the latter reports stronger polymer concentration dependence of the frictional retardation than the former. DR measurements at the highest of the polymer concentrations considered suggest presence of aqueous dynamics slower than that for neat bulk water, although evidence for such "slow" dynamics at lower concentrations has not been detected in the present DR measurements.

Conformational plasticity in the KcsA potassium channel pore helix revealed by homo-FRET studies

Potassium channels selectivity filter (SF) conformation is modulated by several factors, including ion-protein and protein-protein interactions. Here, we investigate the SF dynamics of a single Trp mutant of the potassium channel KcsA (W67) using polarized time-resolved fluorescence measurements. For the first time, an analytical framework is reported to analyze the homo-Förster resonance energy transfer (homo-FRET) within a symmetric tetrameric protein with a square geometry. We found that in the closed state (pH 7), the W67-W67 intersubunit distances become shorter as the average ion occupancy of the SF increases according to cation type and concentration. The hypothesis that the inactivated SF at pH 4 is structurally similar to its collapsed state, detected at low K+, pH 7, was ruled out, emphasizing the critical role played by the S2 binding site in the inactivation process of KcsA. This homo-FRET approach provides complementary information to X-ray crystallography in which the protein conformational dynamics is usually compromised.

Large Crystalline Domains and an Enhanced Exciton Diffusion Length Enable Efficient Organic Solar Cells

We studied crystallinity and exciton harvesting in bulk heterojunctions of the semiconducting polymer PffBT4T-2OD and electron acceptor PC71BM that are used to make highly efficient organic solar cells. Grazing incidence wide-angle X-ray scattering shows that the size of crystalline domains of PffBT4T-2OD increases to ∼18 nm in photovoltaic blends upon thermal annealing at 100 °C for 5 min. These domains are larger than the typical exciton diffusion lengths in conjugated polymers. Time-resolved fluorescence measurements show that the exciton diffusion length in PffBT4T-2OD increases from ∼14 to ∼24 nm upon thermal annealing, which enables efficient charge generation in blends with large domains. Solar cells prepared using thermally annealed blends show higher photocurrents, open circuit voltages, and fill factors compared to unannealed blends, which indicates reduced recombination losses. Our results demonstrate the advantages of large crystalline domains in organic photovoltaics, providing exciton diffusion is sufficient.

FRET-Based Semiconducting Polymer Dots for pH Sensing.

Förster resonance energy transfer (FRET)-based polymer dots (Pdots), fabricated by semiconducting polymers and exhibiting excellent properties, have attracted much interest in the last decade, however, full polymer-dot-based pH sensors are seldom systematically exploited by researchers. In this work, we constructed a kind of blend polymer dot, utilizing poly[(9,9-dihexyl-9H-fluorene-2,7-vinylene)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-phenylenevinylene)] (PFV) as the donor, poly[2,5-bis(3′,7′-dimethyloctyloxy)-1,4-phenylenevinylene] (BDMO-PPV) as the acceptor, and polysytrene graft EO functionalized with carboxy (PS-PEG-COOH) to generate surface carboxyl groups. This type of Pdot, based on the FRET process, was quite sensitive to pH value changes, especially low pH environments. When the pH value decreases down to 2 or 1, the fluorescence spectrum of Pdots-20% exhibit spectral and intensity changes at the same time, and fluorescence lifetime changes as well, which enables pH sensing applications. The sharpening of the emission peak at ~524 nm, along with the weakening and blue shifts of the emission band at ~573 nm, imply that the efficiency of the energy transfer between PFV and BDMO-PPV inside the Pdots-20% decreased due to polymer chain conformational changes. The time-resolved fluorescence measurements supported this suggestion. Pdots constructed by this strategy have great potential in many applications, such as industrial wastewater detection, in vitro and intracellular pH measurement, and DNA amplification and detection.

Solvent-Modulated Charge-Transfer Resonance Enhancement in the Excimer State of a Bay-Substituted Perylene Bisimide Cyclophane.

Excimer, a configurational mixing between Frenkel exciton and charge-transfer resonance states, is typically regarded as a trap state that hinders desired energy or charge-transfer processes in artificial molecular assemblies. However, in recent days, the excimer has received much attention as a functional intermediate in the excited-state dynamics such as singlet fission or charge-separation processes. In this work, we show that the relative contribution to charge-transfer resonance of the excimer state in a bay-substituted perylene bisimide dimer cyclophane can be modulated by dielectric properties of the solvents employed. Solvent-dependent time-resolved fluorescence and absorption measurements reveal that an enhancement of charge-transfer resonance in the excimer state is reflected by incomplete symmetry-breaking charge-separation processes from the structurally relaxed excimer state by means of dipolar solvation processes in the high dielectric environment.

Probing visible light induced photochemical stabilization of collagen in green solvent medium.

Crosslinking of proteins such as collagen for enhanced stability and mechanical properties is an intriguing area in the context of both biomedical and industrial applications. Herein, we have shown the crosslinking of collagen fibers using visible light in a green solvent, ethanol, in the presence of photosensitizers such as methylene blue (Mb) and erythrosine B (Eb). The visible light induced crosslinking increases the shrinkage temperature of collagen fibers from 67 to 100 °C in a concentration dependent manner (1.0 mM Mb/Eb) as revealed by differential scanning calorimetry. Such manifestation was possible only in the presence of ammonium persulphate, a photo-initiator, which has been corroborated through enzymatic degradation, fluorescence and Raman spectroscopic studies. We have also demonstrated that the photoexcited tyrosine moiety of collagen fibers form di-tyrosine during the crosslinking between the peptide chains through steady-state and time-resolved fluorescence measurements. The results suggest a new and efficient photocrosslinking technique to stabilize proteins for diverse applications.

Interaction of repaglinide with bovine serum albumin: Spectroscopic and molecular docking approaches

Repaglinide (RPG) regulates the amount of glucose by stimulating the pancreas to release insulin in the blood. In view of its biological importance, we have examined the interaction between RPG and a model protein, bovine serum albumin (BSA) employing various spectroscopic, electrochemical and molecular docking methods. Fluorescence spectra of BSA were recorded in the presence and absence of RPG in phosphate buffer of pH 7.4. Fluorescence intensity of BSA was decreased upon the addition of increased concentrations of RPG, indicating the interaction between RPG and BSA. Stern-Volmer quenching analysis results revealed that RPG quenched the intensity of BSA through dynamic quenching mechanism. This was further confirmed from the time-resolved fluorescence measurements. The binding constant as calculated from the spectroscopic and voltammetric results was observed to be in the order of 104 M−1 at 298 K, suggesting the moderate binding affinity between RPG and BSA. Competitive experimental results revealed that the primary binding site for RPG on BSA was site II. Absorption and circular dichroism studies indicated the changes in the secondary structure of BSA upon its interaction with RPG. Molecular simulation studies pointed out that RPG was bound to BSA in the hydrophobic pocket of site II.

Ultrafast Intramolecular and Solvation Dynamics in 4,7-Bis (4,5-dibutylbenzo[1,2-b:4,3-b′]bisthiophene[1,2-b:4,3-b′]bisthiophen-2-yl)-2,1,3-benzothiadiazole

We report a combined approach of stationary and time-resolved fluorescence measurements and ultraviolet–visible (UV–vis) transient absorption spectroscopy (TAS) along with ab initio calculations, which provide an overall picture of the dynamics occurring after excitation in a push–pull molecule, namely, 4,7-bis (4,5-dibutylbenzo[1,2-b:4,3-b′]bisthiophene[1,2-b:4,3-b′]bisthiophen-2-yl)-2,1,3-benzothiadiazole. The analysis of the emission spectra in solvents of different polarities reveals the presence of three conformers whose structures differ in the orientation of the 4,5-dibutylbenzo-bisthiophene groups and in their planarity with respect to the benzothiadiazole acceptor group. The Kawski method allows us to estimate the ground- and first-excited state dipole moments (μg and μe) for the three conformers. We find values of μe similar for the three conformers and higher than the relative μg values as can be expected from a push–pull molecule undergoing a light-induced charge-transfer (CT) transition. UV–vis TAS in different solvents highlights the instantaneous (within our instrumental resolution) formation of a locally excited S1 state (accompanied by a big change in the dipole moment with respect to S0), which undergoes a rapid intramolecular CT (ICT) assisted by molecule planarization [planar ICT (PICT)]. The strong dipole–dipole interactions with the polarized solvent molecules stabilize the S1 CT state that decays principally through fluorescence emission. Both PICT and solvation dynamics are responsible for the big Stokes’ shift characterizing the molecule, particularly in polar solvents. The fluorescence lifetimes are substantially longer in polar solvents, and also fluorescence quantum yields are higher in polar solvents. We conclude that the radiative relaxation time increases when molecular planarization of the S1 emissive state takes place, and this condition is favored in polar solvents where local dipole–dipole interactions support the structural stabilization of the CT emissive state. In the poly(methyl methacrylate) matrix, the structural and solvation dynamics are strongly inhibited, leading to reduction of nonradiative processes and to shortening of the fluorescence relaxation time.

Unraveling the Role of Monoolein in Fluidity and Dynamical Response of a Mixed Cationic Lipid Bilayer.

The maintenance of cell membrane fluidity is of critical importance for various cellular functions. At lower temperatures when membrane fluidity decreases, plants and cyanobacteria react by introducing unsaturation in the lipids, so that the membranes return to a more fluidic state. To probe how introduction of unsaturation leads to reduced membrane fluidity, a model cationic lipid dioctadecyldimethylammonium bromide (DODAB) has been chosen, and the effects of an unsaturated lipid monoolein (MO) on the structural dynamics and phase behavior of DODAB have been monitored by quasielastic neutron scattering and time-resolved fluorescence measurements. In the coagel phase, fluidity of the lipid bilayer increases significantly in the presence of MO relative to pure DODAB vesicles and becomes manifest in significantly enhanced dynamics of the constituent lipids along with faster hydration and orientational relaxation dynamics of a fluorophore. On the contrary, MO restricts both lateral and internal motions of the lipid molecules in the fluid phase (>330 K), which is consistent with relatively slow hydration and orientational relaxation dynamics of the fluorophore embedded in the mixed lipid bilayer. The present study illustrates how incorporation of an unsaturated lipid at lower temperatures (below the phase transition) assists the model lipid (DODAB) in regulating fluidity via enhancement of dynamics of the constituent lipids.

Macromolecular crowding effects on energy transfer efficiency and donor-acceptor distance of hetero-FRET sensors using time-resolved fluorescence

Living cells are crowded with macromolecules and organelles, which affect a myriad of biochemical processes. As a result, there is a need for sensitive molecular sensors for quantitative, site-specific assessment of macromolecular crowding. Here, we investigated the excited-state dynamics of recently developed hetero-FRET sensors (mCerulean3–linker–mCitrine) in homogeneous and heterogeneous environments using time-resolved fluorescence measurements, which are compatible with fluorescence lifetime imaging microscopy (FLIM). The linker in these FRET constructs, which tether the mCerulean3 (the donor) and mCitrine (the acceptor), vary in both length and flexibility. Glycerol and Ficoll-70 solutions were used for homogeneous and heterogeneous environments, respectively, at variable concentrations. The wavelength-dependent studies suggest that the 425-nm excitation and the 475-nm emission of the donor are best suited for quantitative assessment of the energy transfer efficiency and the donor-acceptor distance of these FRET probes. Under the same experimental conditions, the enzymatically cleaved counterpart of these probes was used as a control as well as a means to account for the changes in the environmental refractive indices. Our results indicate that the energy transfer efficiency of these FRET probes increases as the linker becomes shorter and more flexible in pure buffer at room temperature. In addition, the FRET probes favor a compact structure with enhanced energy transfer efficiency and a shorter donor-acceptor distance in the heterogeneous, polymer-crowded environment due to steric hindrance. In contrast, the stretched conformation of these FRET probes is more favorable in the viscous, homogeneous environment with a reduced energy transfer efficiency and relatively larger donor-acceptor distance as compared with those in pure buffer, which was attributed to a reduced structural fluctuation of the mCerulean3–mCitrine FRET pair in the viscous, more restrictive glycerol-enriched buffer. Our findings will help to advance the potential of these hetero-FRET probes using FLIM for spatio-temporal assessment of the compartmentalized crowding in living cells.

Efficiency and temporal response of p-terphenyl based wavelength shifting films on H12700 multi anode photomultipliers

Abstract Wavelength-shifting (WLS) films of p-terphenyl have been applied by means of dip-coating on the entrance window of the H12700 multi anode photomultiplier (MAPMT) in order to enhance the UV sensitivity. Using coated and uncoated MAPMTs in a CBM RICH testbox in a proton testbeam at the COSY accelerator, an enhancement of the number of hits per ring of 15%–20% depending on cuts has been observed. Due to new fast readout electronics of the RICH detector for the CBM/ HADES experiments, the time response of p-terphenyl has been measured to show a decay constant of 2.35 ns. This compares well with time-resolved fluorescence measurements of the films.

Interactions between a Bioflavonoid and G-Quadruplex DNA at the Ensemble and Single-Molecule Level

G-Quadruplex (GQ) DNA has garnered the attention of the scientific community due to its role in important biological processes like telomerase activity inhibition, regulation of gene expression and genomic stability. This has triggered studies on understanding the interactions of GQ with small molecules of therapeutic significance. We have studied the interactions of bioflavonoid kaempferol (3,4',5,7-tetrahydroxyflavone, KF), known for its anti-cancer properties, with a c-myc promoter-based GQ at the ensemble and single-molecule level. The binding constant (Kb) is estimated using steady state and time-resolved fluorescence measurements. Upon binding no structural perturbation of GQ is observable from the circular dichroism (CD) data. However, an increase in melting temperature of GQ upon binding suggests stabilization of the DNA structure by the ligand, an observation substantiated by the ensemble FRET experiments. Fluorescence correlation spectroscopy (FCS) measurements, which probe the interactions at the single-molecule level, reveal a progressive increase in the diffusion time of KF with increase in the amount of GQ confirming the binding event. The rate constants of the association (k+) and dissociation (k-) processes have been estimated from the FCS time profiles. The nature of the binding interactions and possible binding site of KF in GQ have been determined from the molecular docking studies. The above findings are expected to enhance our understanding of flavonoid-GQ DNA interactions for development of therapies involving quadruplex DNA.

Single Molecule FRET - A Multi-Environment Ruler for Determining Structure and Dynamics

Studies of multi-domain proteins and multi-protein machines necessitate a deeper understanding of dynamic structure and transient conformations. Therefore, conventional structure determination methods need to be integrated into dynamic detection approaches. We developed a hybrid method that integrates x-ray structure information into self-consistent distance networks based on single-molecule Förster resonance energy transfer (FRET) [1,2]. By analyzing time-correlated distance distributions globally, we can separate real protein dynamics and fluctuations from modelling uncertainties, and ultimately generate time-correlated structural ensembles. On top of that, we reveal correlated small- and large-scale intra-molecular fluctuations from time-resolved, color- and polarization-sensitive fluorescence measurements. We applied our approach to the heat shock protein Hsp90. The chaperone activates large sets of signal transduction proteins often assisted by co-chaperones [3]. These conformation- and nucleotide-dependent processes are not yet comprehensively understood. Our approach resembled the x-ray structure of Hsp90's closed conformation with an RMSD of 2.8 Å. Beyond that, we resolved the previously unknown dynamic open structure of this multidomain protein. The large-scale fluctuations on the lower millisecond timescale might be the basis of a general regulation mechanism. Finally, I want to show how FRET can be orthogonally integrated into cross-disciplinary platforms such as a microfluidics [4] and others, enabling the simultaneous detection of dynamic structure and low-affinity interactions. [1] Hellenkamp B, Wortmann P, Kandzia F, Zacharias M, Hugel T. Multidomain structure and correlated dynamics determined by self-consistent FRET networks. Nature Methods 14(2), 174-180 (2017) [2] Hellenkamp B, Schmid S et al. Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study Nature Methods 15, 669-676 (2018) [3] Taipale, M et al. Quantitative Analysis of Hsp90-Client Interactions Reveals Principles of Substrate Recognition. Cell 140(5), 987-1001 (2012) [4] Hellenkamp B, Thurn J, Stadlmeier M, Hugel T., submitted