Fluorophore-quencher Pair Research Articles

Peptidic "Molecular Beacon" for Collagen.

Collagen-mimetic peptides (CMP) have been invaluable tools for understanding the structure and function of collagen, which is the most abundant protein in animals. CMPs have also been developed as probes that detect damaged collagen because of the specificity required to form a collagen triple helix. These probes are not, however, ratiometric. Here, we used EPR spectroscopy to determine the end-to-end distances of CMPs that do not form stable homotrimeric helices. We found that those distances are shorter than the distances in the context of a collagen triple helix, suggesting their potential utility as a "molecular beacon" and guiding the choice and location of a pendant fluorophore-quencher pair. We then showed that a molecular beacon based on a glycine-(2S,4S)-4-fluoroproline-(2S,4R)-4-hydroxyproline tripeptide repeat and EDANS-DABCYL pair enabled the ratiometric detection of its binding to both other CMPs and natural mammalian collagen. These results provide guidance for the development of a new modality for detecting damaged collagen in physiological settings.

N-Perfluorooctane versus n-Octane: Pyrene Fluorescence to Compare and Contrast Solute Solvation.

Fluorous solvents may offer distinctly different solvation environments to a solute compared to their hydrocarbon analogues due to the inherently high electronegativity associated with fluorine. Solute solvation within n-perfluorooctane (PFO) is compared with that in n-octane using the well-established polycyclic aromatic hydrocarbon (PAH) fluorescence probe pyrene in the temperature range of 288 to 318 K. Both density (ρ) and dynamic viscosity (η) of PFO are considerably higher than those of n-octane. UV-vis molecular absorbance, fluorescence emission/excitation, and excited-state emission intensity decay reveal the cybotactic region of pyrene to be more nonpolar in PFO than that in n-octane. Bimolecular quenching rate constants (kq) for the pyrene-nitrobenzene fluorophore-quencher pair adhere to the Stokes-Einstein formulation; however, they are considerably higher than the estimated rate constants for the diffusion-controlled process (kdiff). This is due to the high electron affinity of nitrobenzene leading to aromatic π-π interactions between pyrene and nitrobenzene. For a nonaromatic low electron affinity quencher, such as nitromethane, while kq < kdiff in n-octane, kq > kdiff in PFO. This is due to the fact that highly electronegative fluorines on PFO stabilize the partial positive charge (δ+) that develops on excited pyrene during electron/charge transfer to the quencher nitromethane, facilitating quenching in the process. Exciplex formation between pyrene and triethylamine (TEA) is more favored in PFO as opposed to n-octane although ηPFO > ηn-octane. The developing charge on the exciplex is stabilized by the electronegative fluorines of the PFO. The pyrene-TEA exciplex appears to form exclusively in the excited state of pyrene, and the kinetics of exciplex formation is in the subnanosecond regime. On the contrary, the efficiency of exciplex formation between pyrene and N,N-dimethylaniline (DMA) is comparable in PFO and n-octane, and the kinetics is slower in comparison to that of the pyrene-TEA exciplex. Certain ground-state heterogeneity is detected for the pyrene-DMA system in PFO due to the low solubilizing ability of the fluorous solvent. Highly electronegative fluorines on perfluorohydrocarbon solvents are found to offer unusual and unique solvation characteristics.

Fluorescent probe for imaging intercellular tension: molecular force approach.

Cellular mechanical force plays a crucial role in numerous biological processes, including wound healing, cell development, and metastasis. To enable imaging of intercellular tension, molecular tension probes were designed, which offer a simple and efficient method for preparing Au-DNA intercellular tension probes with universal applicability. The proposed approach utilizes gold nanoparticles linked to DNA hairpins, enabling sensitive visualization of cellular force in vitro. Specifically, the designed Au-DNA intercellular tension probe includes a molecular spring flanked by a fluorophore-quencher pair, which is anchored between cells. As intercellular forces open the hairpin, the fluorophore is de-quenched, allowing for visualization of cellular force. The effectiveness of this approach was demonstrated by imaging the cellular force in living cells using the designed Au-DNA intercellular tension probe.

Fluorescence-Based Multimodal DNA Logic Gates.

The use of DNA structures in creating multimodal logic gates bears high potential for building molecular devices and computation systems. However, due to the complex designs or complicated working principles, the implementation of DNA logic gates within molecular devices and circuits is still quite limited. Here, we designed simple four-way DNA logic gates that can serve as multimodal platforms for simple to complex operations. Using the proximity quenching of the fluorophore-quencher pair in combination with the toehold-mediated strand displacement (TMSD) strategy, we have successfully demonstrated that the fluorescence output, which is a result of gate opening, solely relies on the oligonucleotide(s) input. We further demonstrated that this strategy can be used to create multimodal (tunable displacement initiation sites on the four-way platform) logic gates including YES, AND, OR, and the combinations thereof. The four-way DNA logic gates developed here bear high promise for building biological computers and next-generation smart molecular circuits with biosensing capabilities.

3D microscaffolds with triple-marker sensitive nanoprobes for studying fatty liver disease in vitro.

Non-alcoholic fatty liver disease (NAFLD) is a heterogeneous condition that encompasses a wide range of liver diseases that progresses from simple hepatic steatosis to the life-threatening state of cirrhosis. However, due to the heterogeneity of this disease, comprehensive analysis of several physicochemical and biological factors that drive its progression is necessary. Therefore, an in vitro platform is required that would enable real-time monitoring of these changes to better understand the progression of these diseases. The earliest stage of NAFLD, i.e. hepatic steatosis, is characterised by triglyceride accumulation in the form of lipid vacuoles in the cytosol of hepatocytes. This fatty acid accumulation is usually accompanied by hepatic inflammation, leading to tissue acidification and dysregulated expression of certain proteases such as matrix metalloproteinases (MMPs). Taking cues from the biological parameters of the disease, we report here a 3D in vitro GelMA/alginate microscaffold platform encapsulating a triple-marker (pH, MMP-3 and MMP-9) sensitive fluorescent nanoprobe for monitoring, and hence, distinguishing the fatty liver disease (hepatic steatosis) from healthy livers on the basis of pH change and MMP expression. The nanoprobe consists of a carbon nanoparticle (CNP) core, which exhibits intrinsic pH-dependent fluorescence properties, decorated either with an MMP-3 (NpMMP3) or MMP-9 (NpMMP9) sensitive peptide substrate. These peptide substrates are flanked with a fluorophore-quencher pair that separates on enzymatic cleavage, resulting in fluorescence emission. The cocktail of these nanoprobes generated multiple fluorescence signals corresponding to slightly acidic pH (blue) and overexpression of MMP-3 (green) and MMP-9 (red) enzymes in a 3D in vitro fatty liver model, whereas no/negligible fluorescence signals were observed in a healthy liver model. Moreover, this platform enabled us to mimic fatty liver disease in a more realistic manner. Therefore, this 3D in vitro platform encapsulating triple-marker sensitive fluorescent nanoprobes would facilitate the monitoring of the changes in pH and MMP expression, thereby enabling us to distinguish a healthy liver from a diseased liver and to study liver disease stages on the basis of these markers.

CRISPR/Cas-Assisted Nanoneedle Sensor for Adenosine Triphosphate Detection in Living Cells.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) (CRISPR/Cas) systems have recently emerged as powerful molecular biosensing tools based on their collateral cleavage activity due to their simplicity, sensitivity, specificity, and broad applicability. However, the direct application of the collateral cleavage activity for in situ intracellular detection is still challenging. Here, we debut a CRISPR/Cas-assisted nanoneedle sensor (nanoCRISPR) for intracellular adenosine triphosphate (ATP), which avoids the challenges associated with intracellular collateral cleavage by introducing a two-step process of intracellular target recognition, followed by extracellular transduction and detection. ATP recognition occurs by first presenting in the cell cytosol an aptamer-locked Cas12a activator conjugated to nanoneedles; the recognition event unlocks the activator immobilized on the nanoneedles. The nanoneedles are then removed from the cells and exposed to the Cas12a/crRNA complex, where the activator triggers the cleavage of an ssDNA fluorophore-quencher pair, generating a detectable fluorescence signal. NanoCRISPR has an ATP detection limit of 246 nM and a dynamic range from 1.56 to 50 μM. Importantly, nanoCRISPR can detect intracellular ATP in 30 min in live cells without impacting cell viability. We anticipate that the nanoCRISPR approach will contribute to broadening the biomedical applications of CRISPR/Cas sensors for the detection of diverse intracellular molecules in living systems.

Correlation of solute diffusion with dynamic viscosity in lithium salt-added (choline chloride + glycerol) deep eutectic solvents.

Due to their favorable physicochemical properties, deep eutectic solvents (DESs) are finding increased use in chemistry. Metal salt-added DESs are currently being investigated for their potential applications in electrochemistry as a replacement for organic electrolytes. Insights into solute diffusion in salt-added DESs, in this context, are of the utmost importance. Solute diffusion in a LiCl-added DES composed of the H-bond acceptor choline chloride and the H-bond donor glycerol in a 1 : 2 mole ratio, named glyceline, is assessed as a function of temperature and LiCl concentration. For relative translational diffusion, the fluorophore-quencher pair of pyrene-nitromethane is used, whereas for rotational diffusion a fluorescent anisotropic rotor, perylene, is selected. The fluorescence quenching of pyrene by nitromethane was found to be purely dynamic in nature. The estimated bimolecular quenching rate constant (kq) exhibits excellent adherence to the Stokes-Einstein relation, suggesting relative translational diffusion of the solute to be controlled by the dynamic viscosity of the LiCl-added glyceline solution. The rotational reorientation time (θ) of the rotor perylene is also found to scale with dynamic viscosity and obey the Stokes-Einstein relation satisfactorily. Linear correlation between θ and dynamic viscosity (η) improves for glyceline solutions with fixed LiCl concentrations hinting at the possible change in the hydrodynamic volume with LiCl concentration within the DES. Control of rotational diffusion of the solute by the dynamic viscosity is established nonetheless. The effect of earlier reported micro- and/or nano-heterogeneities within salt-added DES systems on solute diffusion dynamics is found to be minimal. The work highlights DESs in offering a solubilizing medium for solutes where the diffusion dynamics are simply controlled by the dynamic viscosity.

Development of a highly sensitive lateral flow strip device for nucleic acid detection using molecular beacons

Extensive research is focused on the development of highly sensitive, rapid on-site diagnostic devices. The lateral flow strip (LFS) is a paper-based point-of-care diagnostic device, which is highly promising because of its ease of use and low cost. Despite these advantages, LFS device is still less popular than other methods such as enzyme-linked immunosorbent assay (ELISA) or real-time polymerase chain reaction (qPCR) due to its low sensitivity. Here, we have developed a fluorescence-based lateral flow strip (f-LFS) device for DNA detection using a molecular beacon (MB), a short hairpin-forming DNA strand tagged with a fluorophore-quencher pair. Each paper and membrane component of f-LFS device was carefully selected based on their physicochemical properties including porosity, surface functionality, and autofluorescence. The limit of detection (LOD) of this device was substantially improved to 2.1 fg/mL by adding MgCl2 to the reaction buffer and narrowing the test membrane dimension. Also, a portable fluorescence detection system for f-LFS was developed using a multi-pixel photon counter (MPPC), a sensitive detector detecting the signal on site. We anticipate that this highly sensitive paper-based diagnostic device can be utilized for on-site diagnosis of various diseases.

Digital E. coli Counter: A Microfluidics and Computer Vision-Based DNAzyme Method for the Isolation and Specific Detection of E. coli from Water Samples.

Biological water contamination detection-based assays are essential to test water quality; however, these assays are prone to false-positive results and inaccuracies, are time-consuming, and use complicated procedures to test large water samples. Herein, we show a simple detection and counting method for E. coli in the water samples involving a combination of DNAzyme sensor, microfluidics, and computer vision strategies. We first isolated E. coli into individual droplets containing a DNAzyme mixture using droplet microfluidics. Upon bacterial cell lysis by heating, the DNAzyme mixture reacted with a particular substrate present in the crude intracellular material (CIM) of E. coli. This event triggers the dissociation of the fluorophore-quencher pair present in the DNAzyme mixture leading to a fluorescence signal, indicating the presence of E. coli in the droplets. We developed an algorithm using computer vision to analyze the fluorescent droplets containing E. coli in the presence of non-fluorescent droplets. The algorithm can detect and count fluorescent droplets representing the number of E. coli present in the sample. Finally, we show that the developed method is highly specific to detect and count E. coli in the presence of other bacteria present in the water sample.

One-Component DNA Mechanoprobes for Facile Mechanosensing in Photopolymerized Hydrogels and Elastomers

DNA mechanosensors offer unique properties for mechano-adaptive and self-reporting materials, such as programmable bond strength and geometrical strain response, tunable fluorescent strain sensing, interfacing to biological systems, and the ability to store mechanical information. However, the facile incorporation of advanced DNA motifs into polymer networks and achieving robustness in application settings remain difficult. Herein, we introduce one-component DNA mechanoprobes that can be easily polymerized into polymer hydrogels and even elastomers to allow strain-induced fluorescence sensing. The all-in-one mechanoprobe contains a DNA hairpin for programmable force sensing, an internal fluorophore-quencher pair as a reporter, and methacrylamide groups on both ends for rapid and facile photopolymerization into networks based on the nontoxic water-soluble monomer methoxy triethylene glycol acrylate (mTEGA). In addition to mechanosensing hydrogels, we utilize the low Tg of p(mTEGA) to develop the first bulk elastomer materials with DNA force sensors, which show high elasticity and stronger mechanofluorescence. The system makes decisive steps forward for DNA-based mechanoprobes by overcoming the classical multicomponent design of such probes, allowing photopolymerization useful for the design of complex objects or even 3D printing and demonstrating that such motifs may even be useful in dry bulk materials.

Electric field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2

The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In one basic form of this assay, the CRISPR-Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex becomes activated only when it specifically binds to target DNA and cleaves it. The activated complex thereafter nonspecifically cleaves single-stranded DNA reporter probes labeled with a fluorophore-quencher pair. We discovered that electric field gradients can be used to control and accelerate this CRISPR assay by cofocusing Cas12-gRNA, reporters, and target within a microfluidic chip. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab samples. We here combine this ITP purification with loop-mediated isothermal amplification and the ITP-enhanced CRISPR assay to achieve detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA (from raw sample to result) in about 35 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables an alternate modality for a suite of microfluidic CRISPR-based diagnostic assays.

Fluorophore photostability and saturation in the hotspot of DNA origami nanoantennas

Fluorescent dyes used for single-molecule spectroscopy can undergo millions of excitation-emission cycles before photobleaching. Due to the upconcentration of light in a plasmonic hotspot, the conditions for fluorescent dyes are even more demanding in DNA origami nanoantennas. Here, we briefly review the current state of fluorophore stabilization for single-molecule imaging and reveal additional factors relevant in the context of plasmonic fluorescence enhancement. We show that despite the improved photostability of single-molecule fluorophores by DNA origami nanoantennas, their performance in the intense electric fields in plasmonic hotspots is still limited by the underlying photophysical processes, such as formation of dim states and photoisomerization. These photophysical processes limit the photon count rates, increase heterogeneity and aggravate quantification of fluorescence enhancement factors. These factors also reduce the time resolution that can be achieved in biophysical single-molecule experiments. Finally, we show how the photophysics of a DNA hairpin assay with a fluorophore-quencher pair can be influenced by plasmonic DNA origami nanoantennas leading to implications for their use in fluorescence-based diagnostic assays. Especially, we show that such assays can produce false positive results by premature photobleaching of the dark quencher.

An aptamer based fluorometric microcystin-LR assay using DNA strand-based competitive displacement.

The high-affinity region of a truncated aptamer was applied to the development of a sensitive method for the determination of microcystin-LR (MC-LR) using competitive displacement and molecular beacons. In this assay, the fluorophore and quencher labelled complementary sequences of the aptamer are hybridized with the truncated aptamer to form a fluorophore-quencher pair. In the presence of MC-LR, the aptamer duplex dissociates, and the fluorophore-quencher pair is separated. This turn leads to an increase in the yellow fluorescence which is best measured at excitation/emission wavelengths of 555/580nm. One of the truncated aptamers showed a 50-fold increase in the affinity (0.93nM) compared to the wild type aptamer (50nM). The truncated sequence shows considerable cross-reactivity with L congeners but none with other congeners. The assay works in 0.5 to 200nM MC-LR concentration range. It was applied to spiked tap water samples and gave recoveries around 95 ± 5%. Graphical abstract Schematic representation of a method for determination of microcystin-LR via fluorescence that is induced by competitive displacement of complementary DNA strands in a truncated dsDNA aptamer.

A tunable assay for modulators of genome-destabilizing DNA structures.

Regions of genomic instability are not random and often co-localize with DNA sequences that can adopt alternative DNA structures (i.e. non-B DNA, such as H-DNA). Non-B DNA-forming sequences are highly enriched at translocation breakpoints in human cancer genomes, representing an endogenous source of genetic instability. However, a further understanding of the mechanisms involved in non-B DNA-induced genetic instability is needed. Small molecules that can modulate the formation/stability of non-B DNA structures, and therefore the subsequent mutagenic outcome, represent valuable tools to study DNA structure-induced genetic instability. To this end, we have developed a tunable Förster resonance energy transfer (FRET)-based assay to detect triplex/H-DNA-destabilizing and -stabilizing ligands. The assay was designed by incorporating a fluorophore-quencher pair in a naturally-occurring H-DNA-forming sequence from a chromosomal breakpoint hotspot in the human c-MYC oncogene. By tuning triplex stability via buffer composition, the assay functions as a dual-reporter that can identify stabilizers and destabilizers, simultaneously. The assay principle was demonstrated using known triplex stabilizers, BePI and coralyne, and a complementary oligonucleotide to mimic a destabilizer, MCRa2. The potential of the assay was validated in a 384-well plate with 320 custom-assembled compounds. The discovery of novel triplex stabilizers/destabilizers may allow the regulation of genetic instability in human genomes.

Highly sensitive multiplex detection of microRNA by competitive DNA strand displacement fluorescence assay

MicroRNA (miRNA) is a small non-coding RNA with the size of 18–22 nucleotide. MiRNAs play a major role in the gene expression and regulation. They influence more than 50% of protein coding genes in mammalian genome which regulate many cellular functions. Their dysregulation can lead to cancer and other diseases. MiRNAs have been shown to be associated with breast cancers. Previously q-PCR based assays have been used successfully for detection and quantification of miRNAs, however, these assays are expensive and cumbersome. Here, we report the detection of few breast cancer microRNA sequences using fluorescence displacement assay. The assay employed a fluorophore-quencher pair by hybridization of fluorescently labelled cDNA of miRNA and a quencher labelled short DNA. Upon hybridization, the fluorophore and the quencher become in close vicinity to each other leading to significant fluorescence quenching. In the presence of target miRNA, the fluorophore and quencher are separated due to the duplex dissociation and form stable cDNA-miRNA double strand which leads to increase in the fluorescence intensity. The assay has dynamic range from 0.2 to 250 nM miRNA concentration. We could detect miRNA as low as 1 pM level. The method was applied to detect the miRNA spiked to the total RNA extracted from whole blood. The fluorescence based method has been validated using standard Q-PCR method. This could prove a promising method for the detection of miRNAs.

Magnetic Field-Sensitive Radical Pair Dynamics in Polymethylene Ether-Bridged Donor-Acceptor Systems.

Donor–acceptor systems forming exciplexes are versatile models for the study of magnetic field effects (MFEs) on charge recombination reactions. The MFEs originate from singlet–triplet interconversion within transient radical ion pairs (RIPs), which exist in a dynamic equilibrium with the exciplexes. Here, we describe the synthesis and MFEs of the chain-linked N,N-dimethylaniline (DMA)/9-methylanthracene (MAnt) donor–acceptor system MAnt–(CH2)n–O–CH2–CH2–DMA for n = 6, 8, 10, and 16. The MFEs are found to increase with increasing chain length. Effects as large as 37.5% have been observed for the long-chain compound with n = 16. The solvent dependence of the MFEs at magnetic field intensity 75 mT is reported. For the range of solvent static dielectric constants εs = 6.0–36.0, the MFEs go through a maximum for intermediate polarities, for which the direct formation of RIPs prevails and their dissociation and reencounter are balanced. Field-resolved measurements (MARY spectra) are reported for solutions in butyronitrile. The MARY spectra reveal that for n = 8, 10, 16, the average exchange interaction is negligible during the coherent lifetime of the radical pair. However, singlet–triplet dephasing broadens the lineshape; the shorter the linker, the more pronounced this effect is. For n = 6, a dip in the fluorescence intensity reveals a nonzero average exchange coupling of the order of ±5 mT. We discuss the field-dependence in the framework of the semiclassical theory taking spin-selective recombination, singlet–triplet dephasing, and exchange coupling into account. Singlet recombination rates of the order of 0.1 ns–1 and various degrees of singlet–triplet dephasing govern the spin dynamics. In addition, because of a small free energy gap between the exciplex and the locally excited fluorophore quencher pair, a fully reversible interconversion between the RIP, exciplex, and locally excited fluorophore is revealed by spectrally resolved MFE measurements for the long-chain systems (n = 10, 16).

Label-free detection of exonuclease III activity and its inhibition based on DNA hairpin probe

In this paper, we have developed a label-free and rapid fluorescence assay for the detection of exonuclease III (exo III) activity via thioflavin T (ThT) as the G-quadruplex inducer. In this assay, a hairpin probe (HP) with a 5′-guanine-rich (G-rich) sequence is employed as the substrate for exo III. In the presence of exo III, HP can be digested at 3′-OH termini releasing 5′-G-rich sequence. Then, the 5′-G-rich sequence folds into a G-quadruplex, which can be recognized quickly by the ThT dye resulting in an increase in fluorescence emission. This strategy can detect exo III activity as low as 0.5 U/mL. This assay is simple and of low cost without the requirement of labeling with a fluorophore-quencher pair.

Design and Synthesis of Quenched Activity-based Probes for Diacylglycerol Lipase and α,β-Hydrolase Domain Containing Protein 6.

Diacylglycerol lipases (DAGL) are responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol. The fluorescent activity-based probes DH379 and HT-01 have been previously shown to label DAGLs and to cross-react with the serine hydrolase ABHD6. Here, we report the synthesis and characterization of two new quenched activity-based probes 1 and 2, the design of which was based on the structures of DH379 and HT-01, respectively. Probe 1 contains a BODIPY-FL and a 2,4-dinitroaniline moiety as a fluorophore-quencher pair, whereas probe 2 employs a Cy5-fluorophore and a cAB40-quencher. The fluorescence of both probes was quenched with relative quantum yields of 0.34 and 0.0081, respectively. The probes showed target inhibition as characterized in activity-based protein profiling assays using human cell- and mouse brain lysates, but were unfortunately not active in living cells, presumably due to limited cell permeability.

Florescence Quenching within Lithium Salt-Added Ionic Liquid.

Salt-added ionic liquid media have emerged as a versatile alternative to the conventional electrolytes in several applications. A lithium bis(trifluoromethylsulfonyl)imide (LiTf2N)-added ionic liquid 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([emim][Tf2N]) system up to a LiTf2N mole fraction ( xLiTf2N) of 0.40 is investigated using a fluorophore-quencher pair of pyrene-nitromethane in the 298.15-358.15 K temperature range. Excited-state intensity decay of pyrene fits best to a single-exponential decay function irrespective of the concentration of nitromethane, xLiTf2N, and the temperature. Pyrene lifetimes decrease with increasing temperature at a given xLiTf2N with lifetime becoming more sensitive to temperature at higher LiTf2N concentration. The pyrene-nitromethane fluorophore-quencher pair follows a simplistic Stern-Volmer formulation, indicating the quenching to be purely dynamic in nature affording dynamic quenching constants ( KD) in the process. KD along with the estimated bimolecular quenching rate constant ( kq) within LiTf2N-added [emim][Tf2N] first increases with increasing LiTf2N until xLiTf2N ∼ 0.10, decreasing monotonically thereafter until xLiTf2N = 0.40. The decrease in KD and kq with increasing xLiTf2N is attributed to the exponentially increased dynamic viscosity with increasing xLiTf2N of the ([emim][Tf2N] + LiTf2N) system. The initial increase in KD and kq is controlled by the structural changes within the system as LiTf2N is added to [emim][Tf2N]. It is proposed that the presence of [Li(Tf2N)2]- anionic clusters stabilizes the partial positive charge that develops on excited pyrene during the electron/charge transfer to nitromethane during the quenching process. While the Stokes-Einstein formulation is not followed by the ([emim][Tf2N] + LiTf2N) system in general, it is found to be obeyed at fixed xLiTf2N. The role of structural changes within the system beyond viscosity increase on the quenching process is amply highlighted.

Kinetics of Membrane Bending by Protein Crowding

Dynamic shape changes in phospholipid membranes are considered critical to a number of important cellular processes. Such structural changes are thought to be driven by a number of possible mechanisms. Recently, the effects of protein crowding on shaping and remodeling membrane surfaces has attracted a lot of interest due to the ubiquity of crowded interfaces in biological systems. While a number of recent studies have confirmed the ability of proteins to drive curvature solely via a colligative, crowding-based mechanism- a number of key fundamental mechanistic details remain unsolved. Specifically, very little is known about the kinetics of this process. What are the timescales of membrane deformation process? How kinetically separated are protein binding and the membrane bending events? Is membrane deformation preceded by a previously unresolved intermediate? To address these questions, we provide a comprehensive study on the kinetics of these events through stopped-flow fluorescence using FRET as the spectroscopic handle. A fluorophore-quencher pair was used to study the protein binding kinetics to the bilayer, while a donor-acceptor pair was used as a reporter for membrane area expansion. We report a complex multi-phasic kinetic behavior for both the protein association and membrane expansion process. The association rates follow a surprising negative trend with respect to increasing protein concentration presumably due to saturation of membrane binding sites owing to protein crowding. We find that the membrane deformation happens at significantly slower timescales relative to the protein binding events. The complexity of the fits suggest that the membrane bending process might be preceded by an unresolved intermediate. Taken together with a predictive theoretical model that is currently underway, we believe that our observations will advance our knowledge about membrane bending driven by protein crowding.