Time-resolved Anisotropy Measurements Research Articles

Photophysical properties of lumichrome in aqueous solution tri-block copolymers

We have reported the photophysics of lumichrome (LUM)) in tri-block copolymers micelles of F-127 and P-123, having differences in length of hydrophilic unit. Due to the amphiphilic nature of polymeric micelles, and hydrophilic corona region, we observed the existence of different forms of LUM. We observed an extra emission peak of LUM at ∼ 430 nm and less percentage of existence of the A10 anionic form of LUM in case F-127. From time resolved anisotropy measurement, we have found that the LUM in F-127 micelle faced a more restricted environment than P-123 micelle.

RNA-based liposomes for oral cancer: From biophysical characterization to biological evaluation

Oral cancer incidence and mortality are increasing over time. The most common therapies for oral cancers are surgery and radiotherapy, either used alone or combined, and immunotherapy can be also an option. Although there are several therapeutic options, none of them are completely effective, and in addition, there are numerous associated side effects. To overcome these limitations, researchers have been trying to reduce these drawbacks by using drug delivery systems that carry drugs for specific delivery to cancer cells. For that purpose, RNA-coated liposomes to selectively deliver the ligands C8 (acridine orange derivative) and dexamethasone to oral cancer cells were produced, characterized, and biologically evaluated.Firstly, the RNA structure and binding interaction with ligands (C8 and dexamethasone) were evaluated by circular dichroism (CD), thermal difference spectroscopy (TDS), nuclear magnetic resonance (NMR) and fluorescence titrations. The biophysical assays evidenced the formation of an RNA hairpin and duplex structure. Moreover, steady-state and time-resolved fluorescence intensity and anisotropy experiments show that C8 forms a complex with RNA and adopts an open conformation upon RNA binding.Then, RNA-coated liposomes were characterized by dynamic light scattering, and diameters near 160 nm were observed. Time-resolved anisotropy measurements of C8 loaded in RNA-functionalized liposomes indicate the co-existence of free C8 in solution (inside the liposome) and C8 bound to RNA at the external liposome surface. The RNA-functionalized liposomes loaded with C8 or dexamethasone mediated a significant reduction in the cell viability of malignant UPCI-SCC-154 cells while maintaining viable non-malignant NHDF cells. Additionally, the liposomes were able to internalize the cells, with higher uptake by the malignant cell line.Overall, the results obtained in this work can contribute to the development of new drug delivery systems based on RNA-coated liposomes.

Bioconjugation of a Near-Infrared DNA-Stabilized Silver Nanocluster to Peptides and Human Insulin by Copper-Free Click Chemistry.

DNA-stabilized silver nanoclusters (DNA-AgNCs) are biocompatible emitters with intriguing properties. However, they have not been extensively used for bioimaging applications due to the lack of structural information and hence predictable conjugation strategies. Here, a copper-free click chemistry method for linking a well-characterized DNA-AgNC to molecules of interest is presented. Three different peptides and a small protein, human insulin, were tested as labeling targets. The conjugation to the target compounds was verified by MS, HPLC, and time-resolved anisotropy measurements. Moreover, the spectroscopic properties of DNA-AgNCs were found to be unaffected by the linking reactions. For DNA-AgNC-conjugated human insulin, fluorescence imaging studies were performed on Chinese hamster ovary (CHO) cells overexpressing human insulin receptor B (hIR-B). The specific staining of the CHO cell membranes demonstrates that DNA-AgNCs are great candidates for bioimaging applications, and the proposed linking strategy is easy to implement when the DNA-AgNC structure is known.

Aliphatic and aromatic amine based nitrogen-doped carbon dots: a comparative photophysical study

The quantum confinement effect in nanoscale material is critical not just in the viewpoint of dimensions but also influences their photophysics such as optical, excitonic, and electrical properties compared to their bulk counterpart. This effect makes the semiconducting quantum dots fluorescent with size and band tunability, which is an asset for their applications in sensors, LEDs, solar cells, etc. The current study highlights the synthesis and photo-induced properties of N-doped carbon dots (CDs) from two precursors, one with ethylenediamine (ECDs) and the other with 2-phenethylamine (PCDs). With the use of both microscopic and spectroscopic approaches, we observe distinct changes in the dimensionality and optical properties of the phenylethylamine-induced CD compared to ethylenediamine-based CDs. The aftermath of doping with Zeta potential measurement indicates positively and negatively charged surfaces for ECDs and PCDs respectively. Through different time-resolved PL lifetime and anisotropy measurements, we report that the incorporation of the phenyl ring in PCDs significantly reduces their water solubility, rotational diffusivity, and PL quantum yield compared to that of the ECDs.

Role of Conformational Change and Glucose Binding Sites in the Enhanced Glucose Tolerance of Agrobacterium tumefaciens 5A GH1 β-Glucosidase Mutants.

β-Glucosidases are often inhibited by their reaction product glucose and a barrier to the efficient lignocellulosic biomass hydrolysis to glucose. We had previously reported the mutants, C174V, and H229S, with a nearly 2-fold increased glucose tolerance over the wild type (WT), H0HC94, encoded in Agrobacterium tumefaciens 5A (apparent Ki,Glc = 686 mM). We report our steady-state and time-resolved intrinsic fluorescence spectroscopy, circular dichroism, and isothermal titration calorimetry (ITC) studies to further understand increased glucose tolerance. Changes in the mutants' emission intensity and the differential change in quenching rate in the absence and presence of glucose reflect changes in protein conformation by glucose. Time-resolved lifetime and anisotropy measurements further indicated the microenvironment differences across solvent-exposed tryptophan residues and a higher hydrodynamic radius due to glucose binding, respectively. ITC measurements confirmed the increase of glucose binding sites in the mutants. The experiment results were supported by molecular dynamics simulations, which revealed significant variations in the glucose-protein hydrogen-bonding profiles. Protein structure network analysis of the simulated structures further indicates the mutants' conformation change than the WT. Computational studies also indicated additional glucose binding sites in mutants. Our results indicate the role of glucose binding in modulating the enzyme response to glucose.

A Comparative Study on DMSO-Induced Modulation of the Structural and Dynamical Properties of Model Bilayer Membranes

Modulating the structures and properties of biomembranes via permeation of small amphiphilic molecules is immensely important, having diverse applications in cell biology, biotechnology, and pharmaceuticals, because their physiochemical and biological interactions lead to new pathways for transdermal drug delivery and administration. In this work, we have elucidated the role of dimethyl sulfoxide (DMSO), broadly used as a penetration-enhancing agent and cryoprotective agent on model lipid membranes, using a combination of fluorescence microscopy and time-resolved fluorescence spectroscopy. Spatially resolved fluorescence lifetime imaging microscopy (FLIM) has been employed to unravel how the fluidity of the DMSO-induced bilayer regulates the structural alteration of the vesicles. Moreover, we have also shown that the dehydration effect of DMSO leads to weakening of the hydrogen bond between lipid headgroups and water molecules and results in faster solvation dynamics as demonstrated by femtosecond time-resolved fluorescence spectroscopy. It has been gleaned that the water dynamics becomes faster because bilayer rigidity decreases in the presence of DMSO, which is also supported by time-resolved rotational anisotropy measurements. The enhanced diffusivity and increased membrane fluidity in the presence of DMSO are further ratified at the single-molecule level through fluorescence correlation spectroscopy (FCS) measurements. Our results indicate that while the presence of DMSO significantly affects the 1,2-dimyristoyl-rac-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-rac-glycero-3-phosphatidylcholine (DPPC) bilayers, it has a weak effect on 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (DMPG) vesicles, which might explain the preferential interaction of DMSO with the positively charged choline group present in DMPC and DPPC vesicles. The experimental findings have also been further verified with molecular dynamics simulation studies. Moreover, it has been observed that DMSO is likely to have a differential effect on heterogeneous bilayer membranes depending on the structure and composition of their headgroups. Our results illuminate the importance of probing the lipid structure and composition of cellular membranes in determining the effects of cryoprotective agents.

Effects of the Flanking polyQ Regions and Membrane Physical Properties on Huntingtin Binding to Lipid Vesicles

The pathological expansion of the polyglutamine (polyQ) repeat within the first exon of huntingtin (HTTex1) protein is a hallmark of Huntington’s disease (HD). Multiple evidences support that the membrane interaction of huntingtin is critical for its misfolding and aggregation in HD. Here, we focus on obtaining a detailed understanding of the initial steps of HTTex1-lipid interaction. This characterization has been exceptionally challenging using traditional ensemble methods due to its heterogeneous nature and the high propensity of HTTex1 to aggregate. To overcome these limitations, we used single-molecule approaches based mainly on fluorescence correlation spectroscopy (FCS) to monitor the binding of HTTex1 to synthetic lipid vesicles. The dependence on membrane curvature and lipid composition was studied, considering as models anionic lipids, raft-mimicking mixtures, and total brain lipid extract. The FCS results show a preferential binding of HTTex1 towards POPS (anionic lipid)-containing vesicles of small curvature. Moreover, time-resolved anisotropy measurements reveal distinct conformational dynamics of the adjacent polyQ regions in the HTTex1 membrane-bound state. Notably, the proline-rich domain remains highly flexible and solvent exposed even upon membrane binding. instead, the N17 segment converts into a less dynamic state. Our findings provide unique insight into how membrane curvature/composition and flanking polyQ sequences modulate the early stages of HTTex1 aggregation at the membrane surface. Work supported by FCT-Portugal (PTDC/BIA-BFS/30959/2017 grant and CEECIND/00884/2017 contract to AMM, and UIDB/04565/2020 funding to iBB-IST).

Deeper insight into the multifaceted photodynamics of a potential organic functional material emphasizing aggregation induced emission enhancement (AIEE) properties

The multifaceted photodynamics of a simple azine based organic functional molecule namely 2-((Z)-((E)-(pyren-1-ylmethylene)hydrazono)methyl)quinolin-8-ol (PHQ) emphasizing its brilliant fluorescence emission redemption properties in aggregation state (AIEE) have been primarily explored through absorption and steady state emission techniques. The governing role of photo-induced electron transfer (PET) rates and active intramolecular motions of non-interacting PHQ monomers at lower water fraction comprising mixed solvent systems have been identified to be the prime reasons for non-radiative annihilation of photoexcited states. The transition from weakly emissive to highly emissive state has been substantiated through elaborate study using time-resolved photoluminescence (TRPL), fluorescence quantum yield and variation of external control experiments. In the current study, the increased rotational relaxation time of aggregated hydrosol, responsible for AIEE, is investigated using time-resolved anisotropy measurement (TRAM) of different PHQ microenvironments, which is unprecedented to the best of our knowledge in AIEE research. Interestingly, the high-angle annular dark field-scanning transmission electron microscopy (HAADF-STEM) image shows one dimensionally grown molecular entity of a single nano-sheet like structure of PHQ, which is a unique observation. The average particle size of PHQ aggregates is also increased from 84.5 nm to 814 nm corresponding to 10% and 90% fw (water volume %) system respectively. The energy-dispersive X-ray spectroscopy (EDX) has also been employed, for the first time in AIEE research, which reveals a gradual increase in the amount of carbon within the aggregated microstructure with addition of water. The present molecular system PHQ, being a molecular rotor system, provides future prospect for probing local microenvironmental viscosities within biological systems.

Determining the Stoichiometry of Small Protein Oligomers Using Steady-State Fluorescence Anisotropy

A large fraction of soluble and membrane-bound proteins exists as non-covalent dimers, trimers, and higher-order oligomers. The experimental determination of the oligomeric state or stoichiometry of proteins remains a nontrivial challenge. In one approach, the protein of interest is genetically fused to green fluorescent protein (GFP). If a fusion protein assembles into a non-covalent oligomeric complex, exciting their GFP moiety with polarized fluorescent light elicits homotypic Förster resonance energy transfer (homo-FRET), in which the emitted radiation is partially depolarized. Fluorescence depolarization is associated with a decrease in fluorescence anisotropy that can be exploited to calculate the oligomeric state. In a classical approach, several parameters obtained through time-resolved and steady-state anisotropy measurements are required for determining the stoichiometry of the oligomers. Here, we examined novel approaches in which time-resolved measurements of reference proteins provide the parameters that can be used to interpret the less expensive steady-state anisotropy data of candidates. In one approach, we find that using average homo-FRET rates (kFRET), average fluorescence lifetimes (τ), and average anisotropies of those fluorophores that are indirectly excited by homo-FRET (rET) do not compromise the accuracy of calculated stoichiometries. In the other approach, fractional photobleaching of reference oligomers provides a novel parameter a whose dependence on stoichiometry allows one to quantitatively interpret the increase of fluorescence anisotropy seen after photobleaching the candidates. These methods can at least reliably distinguish monomers from dimers and trimers.

Excitation/detection energy controlled anisotropy dynamics in asymmetrically cyano substituted tri-podal molecules.

In the present work, the photophysical properties of two series of asymmetrical tri-podal molecules are studied, in order to determine the dependence of energy localization/delocalization phenomena on excitation and detection wavelength, by means of steady state, femtosecond time-resolved fluorescence and anisotropy spectroscopy. The molecules bear triphenylamine as an electron donating core and an acetylenic or olefinic π-conjugated bridge. At the periphery, they are substituted by no, one, two or three -CN groups used as electron acceptors. Thus, the compounds with only one or two -CN groups are asymmetrically substituted. As a comparison, the photophysics of their dipolar and quadrupolar analogues is also presented. The steady state absorption spectra of the asymmetrical tri-podal compounds exhibit a broadening and a low energy shoulder due to the splitting of the excited states. The fluorescence spectra are more red-shifted in the tri-podal molecules with a single -CN group, providing the first evidence of its mostly dipolar nature. Time-resolved anisotropy measurements by using different excitation and detection wavelengths provide clear evidence that the asymmetrical tri-podal molecules with one or two -CN groups behave like octupolar molecules upon high-energy excitation (the initial anisotropy is found 0.1-0.15), while upon low-energy excitation they reveal a behavior expected for linear dipolar or V-shaped quadrupolar molecules (the initial anisotropy is very close to 0.4 and 0.17, respectively). The symmetrical tri-podal compounds with no or three cyano groups, exhibit an anisotropy depolarization time of 2.5 ps attributed to energy hopping. The amplitude of this energy hopping component is wavelength dependent and increases as the excitation is shifted towards the long wavelength edge.

An Insulating Al2O3 Overlayer Prevents Lateral Hole Hopping Across Dye-Sensitized TiO2 Surfaces.

Three chromophores of the general form [Ru(bpy')2(4,4'-(PO3H2)2-2,2'-bipyridine)]2+, where bpy' is 4,4'-(C(CH3)3)2-2,2'-bipyridine (Ru(dtb)2P); 4,4'-(CH3O)2-2,2'-bipyridine (Ru(OMe)2P), and 2,2'-bipyridine (RuP) were anchored to mesoporous thin films of TiO2 nanocrystallites at saturation surface coverages to investigate lateral self-exchange RuIII/II intermolecular hole hopping in 0.1 M LiClO4/CH3CN electrolytes. Hole hopping was initiated by a potential step 500 mV positive of the E1/2 (RuIII/II) potential or by pulsed laser (532 nm, 8 ns fwhm) excitation and monitored by visible absorption chronoabsorptometry and time-resolved absorption anisotropy measurements, respectively. The hole hopping rate constant kR extracted from the potential step data revealed self-exchange rate constants that followed the trend: TiO2|Ru(OMe)2P (ket = 1.4 × 106 s-1) > TiO2|RuP (7.1 × 105 s-1) > TiO2|Ru(dtb)2P (6.5 × 104 s-1). Analysis of the anisotropy data with Monte Carlo simulations provided hole hopping rate constants for TiO2|RuP and TiO2|Ru(dtb)2P that were within experimental error the same as that measured with the potential step. The hole hopping rate constants were found to trend with the TiO2(e-)|RuIII → TiO2|RuII charge recombination rate constants. The atomic layer deposition of an ∼10 Å layer of Al2O3 on top of the dye-sensitized films was found to prevent hole hopping by both initiation methods even though the chromophore surface coverage exceeded the percolation threshold and excited-state injection was efficient. The dramatic hole hopping turnoff was attributed to a larger outer-sphere reorganization energy for self-exchange due to the restricted access of electrolyte to the redox active chromophores. The implications of these findings for solar energy conversion applications are discussed.

Solvent dependent relaxation dynamics in lithium ion battery electrolytes: Coupling to medium friction

Solution heterogeneity and dynamics of the industrially relevant electrolyte system, [9.795{xEC + (1 − x)PC} + 0.869LiClO4] with x representing mole fraction, were explored at different loadings of ethylene carbonate (EC) via temperature dependent measurements of time-resolved spectral response and physico-chemical properties. This system was chosen because polarity differed considerably on replacing propylene carbonate (PC) by EC but the solution viscosity remained nearly the same, allowing to investigate the polarity impact on solution inhomogeneity and solute dynamics. Coumarin 153 (C153) and trans‑2‑[4‑(dimethylamino)styryl]‑benzothiazole (trans-DMASBT) were used as fluorescent probes which differ widely in their excited state lifetimes. Driven by the potential for applications of these electrolyte systems in the energy sector, we have investigated them at an optimal composition with (PC + EC)/LiClO4 mole ratio of ~11.3, where direct current (DC) conductivity reaches at the maximum. Signatures of spatio-temporal heterogeneity were detected in steady state fluorescence and time-resolved anisotropy measurements with DMASBT (lifetime ~100–200 ps) but not with C153, suggesting a timescale over which the inhomogeneity density distributions persist in these electrolyte systems. Subsequent Stokes shift dynamics measurements using C153 captured a dynamic shift of ~700–900 cm−1 with an estimated missing portion of ~50%, and biphasic solvation response characterized by sub-100 ps and nanosecond components. Time-resolved fluorescence anisotropy measurements reflect partial viscosity decoupling for the short lifetime probe and bimodal frictional response in these electrolyte systems.

Influence of Trivalent Metal Ions on Lipid Vesicles: Gelation and Fusion Phenomena.

In this contribution, we report the interaction of 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC)lipid vesicles with a series of trivalent metal ions of the same group, namely, Al3+, Ga3+, and In3+, to get a distinct view of the effect of size, effective charge, and hydration free energy of these metal ions on lipid vesicles. We employed steady-state and time-resolved spectroscopic techniques including time-resolved anisotropy measurement, confocal imaging, and dynamic light scattering (DLS) measurement to probe the interaction. Our study reveals that all of the three trivalent metal ions induce gelation in lipid vesicles by removing water molecules from the interfacial region. The extent of gelation induced by the metal ions follows the order of In3+ > Ga3+ ≥ Al3+. We explain this observation in light of different free-energy terms. Notably, the degree of interaction for trivalent metal ions is higher as compared to that for divalent metal ions at physiological pH (pH ∼ 7.0). Most importantly, we observe that unlike divalent metal ions, trivalent metal ions dehydrate the lipid vesicles even at lower pH. The DLS measurement and confocal imaging indicate that In3+ causes significant aggregation or fusion of the PC vesicles, while Al3+ and Ga3+ did not induce any aggregation at the experimental concentration. We employ Derjaguin-Landau-Vervey-Overbeek (DLVO)theory to explain the aggregation phenomena induced by In3+.

Sensing of Different Human Telomeric G-Quadruplex DNA Topologies by Natural Alkaloid Allocryptopine Using Spectroscopic Techniques.

This article describes how a natural alkaloid allocryptopine (ALL) is able to differentiate two forms of biologically relevant human telomeric (htel22) G-quadruplex DNAs (GQ-DNA) depending on the presence of K+ and Na+ ions by steady-state and time-resolved spectroscopic techniques. For both interactions, predominant involvements of static-type quenching mechanism with the negligible influence of dynamic collision are established by UV-vis absorption and fluorescence emission study, which is further supported by fluorescence lifetime measurements. ALL exhibits appreciable affinity toward both GQ-DNAs. Both the mixed-hybrid (3 + 1) quadruplex structures in K+ ions and the basket-type antiparallel quadruplex structure under Na+ condition are converted to parallel types in the presence of ALL. Fluorescence intercalator displacement assay experiment revealed modest selectivity of ALL to both quadruplexes over duplex DNA along with higher selectivity for antiparallel types among the two quadruplexes via groove and/or loop binding, which is distinct from the conventional π-stacking of the ligands on external G-quartets. ALL stabilized both GQ-DNA topologies moderately. The differences in the dynamics of ALL within both DNA environments have been demonstrated vividly by time-resolved anisotropy measurements using the wobbling-in-cone model. These results suggest groove binding with antiparallel G-quartet with high affinity and moderate loop binding with mixed-hybrid G-quartet accompanied by the partial end stacking additionally in both of the cases. Our conclusions are further supported by steady-state anisotropy measurements and molecular docking. The present investigation can be used in the development of a biocompatible antitumour/anticancer agent targeting particular GQ-DNA conformation.

Enhanced Luminescent Properties of Photo-Stable Copper Nanoclusters through Formation of "Protein-Corona"-Like Assemblies.

In this study, interactions of synthesized copper nanoclusters (CuNCs) with a model transport protein, human serum albumin (HSA), have been systematically investigated by using various spectroscopic approaches. The interactions give rise to the formation of "protein-corona" like assemblies and the luminescence properties (both steady-state and time-resolved) are enhanced due to gradual adsorption of the protein on the surface of the NCs. The associated thermodynamics and binding parameters have been estimated resorting to luminescent experimental techniques as well as isothermal titration calorimetry (ITC) studies, indicating that every NC is surrounded by (4±1) protein molecules. The adsorption of HSA on the surface of the NCs has been characterized by dynamic light scattering (DLS) and time-resolved anisotropy measurements. Finally, fluorescence correlation spectroscopy (FCS) data substantiate the emergence of new "protein-corona" like assemblies resulting in slower translational diffusion motions and concomitant rise of the hydrodynamic diameters.

Förster resonance energy transfer and charge transfer dynamics in ternary organic nanoparticles

Abstract Binary and ternary organic nanoparticles (NPs) comprising 7,7-(4,4-bis(2-Ethylhexyl)-4H-silolo [3,2-b:4,5-b′]dithiophene-2,6-diyl)bis (6-fluoro-4-(5′-hexyl-[2,2′-bithiophen]-5-yl)benzo-[c] [1,2,5]thiadiazole) (p-DTS(FBTTH2)2), poly (3-hexylthiophene) (P3HT) or/and [6,6]-phenyl-C71-butyric acid methyl ester (PC71BM) were prepared by precipitation method. The formation of the organic NPs is confirmed by the atomic force microscope (AFM). The absorption spectra show that the blended organic NPs display increased interchain interactions compared to the polymer blends dissolved in the solution. The processes of exciton separation and charge transfer in the organic NPs have been investigated using time-resolved photoluminescence (TRPL) spectroscopy and anisotropy measurements. The results demonstrate that the charge transfer efficiency is improved in the NPs in comparison with that in solution, due to the enhanced interchain interactions and intimate contact between the donor and acceptor materials in NPs. Due to Forster resonance energy transfer (FRET) between P3HT and p-DTS(FBTTH2)2, the photodetectors with ternary NPs in the active layers exhibit improved on/off ratios and enhanced detectivity compared with the binary system, demonstrating the promising applications of organic NPs in the ecofriendly fabrication of organic optoelectronic devices.

Influence of Eugenol on the Organization and Dynamics of Lipid Membranes: A Phase-Dependent Study.

Eugenol is known for its antimicrobial effects against microorganisms responsible for infectious diseases in humans, food-borne pathogens, and oral pathogens. In spite of several reports on the antimicrobial function of eugenol by modulating the structural properties of cell membranes, there is limited information on the influence of eugenol in the lipid membrane. In this work, we explored the effect of eugenol on the organization and dynamics of large unilamellar vesicles (LUVs) of DMPC using the intrinsic fluorescence of eugenol and an extrinsic hydrophobic probe, DPH, in varying phases. The organization and dynamics of the bilayers of DMPC vesicles were monitored by utilizing varieties of steady-state and time-resolved fluorescence measurements. Our results show that eugenol stabilizes the gel phase and elevates the phase-transition temperature of DMPC in a concentration-dependent fashion. Fluorescence lifetime measurements demonstrate that higher eugenol-induced water penetration was observed in fluid-phase membranes. Time-resolved anisotropy measurements demonstrate that eugenol reduces the semiangle of DPH wobbling-in-a-cone in gel-phase membranes, whereas the semiangle remains unaffected in fluid-phase membrane. This implies that the eugenol further orders the gel-phase membrane, and this could be a plausible reason for the eugenol-dependent elevation of the phase-transition temperature of DMPC. We envisage that these results will contribute important information to understand the interaction of eugenol with biological membranes.

Fluorescence Dynamics of a FRET Probe Designed for Crowding Studies.

Living cells are crowded with macromolecules and organelles. As a result, there is an urgent need for molecular sensors for quantitative, site-specific assessment of the macromolecular crowding effects on a myriad of biochemical processes toward quantitative cell biology and biophysics. Here we investigate the excited-state dynamics and translational diffusion of a novel FRET sensor (mCerulean-linker-mCitrine) in a buffer (PBS, pH 7.4) at room temperature. Complementary experiments were carried out on free CFP, YFP, and the cleaved FRET probe as controls. The wavelength-dependent fluorescence lifetime measurements of the donor and acceptor in the FRET probe, using the time-correlated single-photon counting technique, indicate an energy transfer efficiency of 6.8 ± 0.9% in PBS, with distinct excited-state dynamics from the recombinant CFP and YFP. The estimated mCerulean-mCitrine distance in this FRET probe is 7.7 ± 0.2 nm. The energy transfer efficiency increases (11.5 ± 0.9%) as the concentration of Ficoll-70 increases over the range of 0-300 g/L with an estimated mCerulean-mCitrine distance of 6.1 ± 0.2 nm. Complementary time-resolved anisotropy measurements suggest that the rotational diffusion of hetero-FRET in PBS is sensitive to the energy transfer from the donor to the acceptor. The results also suggest that the linker, -(GSG)6A(EAAAK)6A(GSG)6A(EAAAK)6A(GSG)6-, is rather flexible, and the observed rotational dynamics is likely to be due to a segmental mobility of the FRET pairs rather than an overall tumbling motion of a rigid probe. Comparative studies on a new construct of a FRET probe with a shorter, more flexible linker, mCerulean-(GSG)18-mCitrine, reveal enhanced energy transfer efficiency. On the millisecond time scale, fluorescence fluctuation analyses of the acceptor (excited at 488 nm) provide a means to examine the translational diffusion coefficient of the FRET probe. The results also suggest that the linker is flexible in this FRET probe, and the observed diffusion coefficient is faster than predicted as compared to the cleaved FRET probe. Our results serve as a point of reference for this FRET probe in a buffer toward its full potential as a sensor for macromolecular crowding in living cells and tissues.

Au21S(SAdm)15: An Anisotropic Gold Nanomolecule. Optical and Photoluminescence Spectroscopy and First-Principles Theoretical Analysis.

We introduce a class of gold nanomolecules exhibiting anisotropy as a major feature by reporting steady-state and time-resolved photoluminescence and anisotropy measurements and in-depth theoretical analysis of energetics and optical response of a recently synthesized Au21S(SAdm)15 nanomolecule (SAdm = adamantanethiol). Starting from single-crystal X-ray data showing that Au21S(SAdm)15 exhibits a symmetry-broken structure, we unambiguously demonstrate how this translates into a striking anisotropy of its properties, for example, of its (chiro)optical absorption spectrum of great promise for sensing, optoelectronic, and electrochemical applications, and argue about the abundance and general significance of this class of compounds.

Excited state dynamics and conformations of a Cu(ii)-phthalocyanine-perylenebisimide dyad.

We investigate the excited state dynamics and the conformations of a new molecular donor-bridge-acceptor system, a Cu(ii)-phthalocyanine (CuPc) covalently linked via a flexible aliphatic spacer to a perylenebisimide (PBI). We performed time-resolved polarization anisotropy and pump-probe measurements in combination with molecular dynamics simulations. Our data suggest the existence of three conformations of the dyad: two more extended, metastable conformations with centre-of-mass distances >1 nm between the PBI and CuPc units of the dyad, and a highly stable folded structure, in which the PBI and CuPc units are stacked on top of each other with a centre-of-mass distance of 0.4 nm. In the extended conformations the dyad shows emission predominantly from the PBI unit with a very weak contribution from the CuPc unit. In contrast, for the folded conformation the PBI emission of the dyad is strongly quenched due to fast energy transfer from the PBI to the CuPc unit (3 ps) and subsequent intersystem-crossing (300 fs) from the first excited singlet state of CuPc unit into its triplet state. Finally, the CuPc triplet state is deactivated non-radiatively with a time constant of 25 ns.