Unusual similarity of DNA solvation dynamics in high-salinity crowding with divalent cations of varying concentrations.

Double-stranded DNA bears the highest linear negative charge density (2e- per base-pair) among all biopolymers, leading to strong interactions with cations and dipolar water, resulting in the formation of a dense 'condensation layer' around DNA. Interactions involving proteins and ligands binding to DNA are primarily governed by strong electrostatic forces. Increased salt concentrations impede such electrostatic interactions - a situation that prevails in oceanic species due to their cytoplasm being enriched with salts. Nevertheless, how these interactions' dynamics are affected in crowded hypersaline environments remains largely unexplored. Here, we employ steady-state and time-resolved fluorescence Stokes shifts (TRFSS) of a DNA-bound ligand (DAPI) to investigate the static and dynamic solvation properties of DNA in the presence of two divalent cations, magnesium (Mg2+), and calcium (Ca2+) at varying high to very-high concentrations of 0.15 M, 1 M and 2 M. We compare the results to those obtained in physiological concentrations (0.15 M) of monovalent Na+ ions. Combining data from fluorescence femtosecond optical gating (FOG) and time-correlated single photon counting (TCSPC) techniques, dynamic fluorescence Stokes shifts in DNA are analysed over a broad range of time-scales, from 100 fs to 10 ns. We find that while divalent cation crowding strongly influences the DNA stability and ligand binding affinity to DNA, the dynamics of DNA solvation remain remarkably similar across a broad range of five decades in time, even in a high-salinity crowded environment with divalent cations, as compared to the physiological concentration of the Na+ ion. Steady-state and time-resolved data of the DNA-groove-bound ligand are seemingly unaffected by ion-crowding in hypersaline solution, possibly due to ions being mostly displaced by the DNA-bound ligand. Furthermore, the dynamic coupling of cations with nearby water may possibly contribute to a net-neutral effect on the overall collective solvation dynamics in DNA, owing to the strong anti-correlation of their electrostatic interaction energy fluctuations. Such dynamic scenarios may persist within the cellular environment of marine life and other biological cells that experience hypersaline conditions.

G-Tetrad-Selective Ligand Binding Kinetics in G-Quadruplex DNA Probed with Fluorescence Correlation Spectroscopy.

Probing the kinetics of ligand binding to biomolecules is of paramount interest in biology and pharmacology. Measurements of such kinetic processes provide information on the rate-determining steps that control the binding affinity of ligands to biomolecules, thereby predicting the mechanism of the molecular interaction. In this context, ligand binding to G-quadruplex DNA (GqDNA) structures has attracted tremendous attention primarily because of their use in possible anticancer therapy. Although a large number of G-quadruplex-specific ligands have been proposed, probing the kinetics of G-tetrad-selective binding of (multiple) ligands within a G-quadruplex DNA (GqDNA) structure remains challenging. Most of the earlier studies focused on the thermodynamics of ligand binding; however, the kinetics of ligand association and dissociation with GqDNA, particularly binding of multiple ligands within a GqDNA structure, have not been explored. Here, we propose a simple fluorescence correlation spectroscopy-based method that measures the G-tetrad-selective association and dissociation rates of ligands within a GqDNA structure by correlating the fluorescence fluctuations of a site-specific (5' or 3' end-labeled) fluorophore (Cy3) in GqDNA due to quenching of Cy3 fluorescence, induced by the ligand binding to the G-tetrads. We show that well-known GqDNA ligands, BRACO19, TMPyP4, Hoechst 33258, and Hoechst 33342, have G-tetrad-selective association and dissociation rates, which suggest site-dependent variation of free energy barriers for binding/unbinding of the ligands with GqDNA. We also show that the measured kinetic rates depend not only on the G-tetrad site (5' vs 3' end) but also on the ligand and GqDNA structures.

DNA damage, cell cycle perturbation and cell death by naphthalene diimide derivative in gastric cancer cells.

Stomach cancer causes the third-highest cancer-related deaths worldwide. Limited availability of anticancer measures with higher efficiency and low unwanted toxicities necessitates the development of better cancer chemotherapeutics. Naphthalene diimide (NDI) derivatives have gained significant attention owing to their excellent anticancer potential. We evaluated the anticancer properties of NDI derivatives, 1a and 2a in cancer cell lines and found that 1a showed higher efficacy as compared to 2a exhibiting a remarkable difference in activity upon single atom substitution of C with N. Particularly, NDI 1a showed potent inhibitory activity against gastric cancer cell line AGS with IC50 of 2.0 µM. NDI 1a induced remarkable morphological changes and reduced clonogenicity as well as the migratory ability of AGS cells. The reduction in AGS cell migration was mediated through inhibition of Tyr397 p-FAK dephosphorylation at focal adhesion points leading to enhanced attachment of cells at contact points. NDI 1a caused extensive DNA double-strand-breaks (DSBs) leading to activation of p53 and its transcriptional target p21. Reduced nuclear BRCA1 but enhanced nuclear p53BP1 foci formation upon 1a treatment suggests that DNA DSB repair is mediated through error-prone NHEJ which led to the accumulation of extensive DNA damage. Combinatorial effects mediated by interactions of 1a with double-stranded DNA through minor groove binding as well as induction of intracellular ROS exacerbated the loss of genomic integrity induced by 1a. NDI 1a mediated DNA damage-induced S phase arrest; however, cells experiencing extensive and irreparable DNA damage underwent mitochondrial apoptosis through downregulation of anti-apoptotic protein p21. Furthermore, proliferation inhibitory activity of 1a is also attributed to inhibition of ß-catenin/c-Myc axis in AGS cells with constitutively active ß-catenin pathway. In vivo toxicity analysis of 1a revealed minimal systemic toxicity suggesting that compound 1a is a safe and potential candidate for the development of gastric cancer chemotherapeutics.

Molecular Picture of the Effect of Cosolvent Crowding on Ligand Binding and Dispersed Solvation Dynamics in G-Quadruplex DNA.

Understanding molecular interactions and dynamics of proteins and DNA in a cell-like crowded environment is crucial for predicting their functions within the cell. Noncanonical G-quadruplex DNA (GqDNA) structures adopt various topologies that were shown to be strongly affected by molecular crowding. However, it is unknown how such crowding affects the solvation dynamics in GqDNA. Here, we study the effect of cosolvent (acetonitrile) crowding on ligand (DAPI) solvation dynamics within human telomeric antiparallel GqDNA through direct comparison of time-resolved fluorescence Stokes shift (TRFSS) experiments and molecular dynamics (MD) simulations results. We show that ligand binding affinity to GqDNA is drastically affected by acetonitrile (ACN). Solvation dynamics probed by DAPI in GqDNA groove show dispersed dynamics from ∼100 fs to 10 ns in the absence and presence of 20% and 40% (v/v) ACN. The nature of dynamics remain similar in buffer and 20% ACN, although in 40% ACN, distinct dynamics is observed in <100 ps. MD simulations performed on GqDNA/DAPI complex reveal preferential solvation of ligand by ACN, particularly in 40% ACN. Simulated solvation time-correlation functions calculated from MD trajectories compare very well to the overall solvation dynamics of DAPI in GqDNA, observed in experiments. Linear response decomposition of simulated solvation correlation functions unfolds the origin of dispersed dynamics, showing that the slower dynamics is dominated by DNA-motion in the presence of ACN (and also by the ACN dynamics at higher concentration). However, water-DNA coupled motion controls the slow dynamics in the absence of ACN. Our data, thus, unravel a detailed molecular picture showing that though ACN crowding affect ligand binding affinity to GqDNA significantly, the overall dispersed solvation dynamics in GqDNA remain similar in the absence and the presence of 20% ACN, albeit with a small effect on the dynamics in the presence of 40% ACN due to preferential solvation of ligand by ACN.

Graphene Quantum Dot-Based Optical Sensing Platform for Aflatoxin B1 Detection via the Resonance Energy Transfer Phenomenon.

An optical sensing platform for the detection of an important mycotoxin, aflatoxin B1 (AFB1), in the absence of a bioactive environment is explored. In this work, a fluorescence-based sensing technique was designed by combining graphene quantum dots (GQDs) and AFB1 via fluorescence quenching, where AFB1 acts as the quencher of GQD fluorescence. GQDs were synthesized through a single-step hydrothermal reaction from the leaves of "curry tree" (Murraya Koenigii) at 200 °C. The fluorescent GQDs were quenched by AFB1 (quencher), which itself is detecting the analyte. Hence, this study reports the direct sensing of the mycotoxin AFB1 without the involvement of inhibitors or biological entities. The possible mode of quenching is the nonradiative resonance energy transfer between the GQDs and the AFB1 molecules. This innovative sensor could detect AFB1 in the range from 5 to 800 ng mL-1 with a detection limit of 0.158 ng mL-1. The interferent study was also carried out in the presence of different mycotoxins and carbohydrates (d-fructose, cellulose, and starch), which demonstrated the high selectivity and robustness of the sensor in the complex sample matrix. The recovery percentage of the spiked samples was also calculated to be up to 106.8%. Thus, this study reports the first GQD based optical sensor for AFB1.

Cdr1p highlights the role of the non-hydrolytic ATP-binding site in driving drug translocation in asymmetric ABC pumps.

ATP-binding cassette (ABC) transporters couple ATP binding and hydrolysis to the translocation of allocrites across membranes. Two shared nucleotide-binding sites (NBS) participate in this cycle. In asymmetric ABC pumps, only one of them hydrolyzes ATP, and the functional role of the other remains unclear. Using a drug-based selection strategy on the transport-deficient mutant L529A in the transmembrane domain of the Candida albicans pump Cdr1p; we identified a spontaneous secondary mutation restoring drug-translocation. The compensatory mutation Q1005H was mapped 60 Å away, precisely in the ABC signature sequence of the non-hydrolytic NBS. The same was observed in the homolog Cdr2p. Both the mutant and suppressor proteins remained ATPase active, but remarkably, the single Q1005H mutant displayed a two-fold reduced ATPase activity and a two-fold increased drug-resistance as compared to the wild-type protein, pointing at a direct control of the non-hydrolytic NBS in substrate-translocation through ATP binding in asymmetric ABC pumps.

Structure of an Unfolding Intermediate of an RRM Domain of ETR-3 Reveals Its Native-like Fold.

Prevalence of one or more partially folded intermediates during protein unfolding with different secondary and ternary conformations has been identified as an integral character of protein unfolding. These transition-state species need to be characterized structurally for elucidation of their folding pathways. We have determined the three-dimensional structure of an intermediate state with increased conformational space sampling under urea-denaturing condition. The protein unfolds completely at 10 M urea but retains residual secondary structural propensities with restricted motion. Here, we describe the native state, observable intermediate state, and unfolded state for ETR-3 RRM-3, which has canonical RRM fold. These observations can shed more light on unfolding events for RRM-containing proteins.

Author Correction: Ras hyperactivation versus overexpression: Lessons from Ras dynamics in Candida albicans.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Origin of Slow Solvation Dynamics in DNA: DAPI in Minor Groove of Dickerson-Drew DNA.

The measurement and understanding of collective solvation dynamics in DNA have vital biological implications, as protein and ligand binding to DNA can be directly controlled by complex electrostatic interactions of anionic DNA and surrounding dipolar water, and ions. Time-resolved fluorescence Stokes shift (TRFSS) experiments revealed anomalously slow solvation dynamics in DNA much beyond 100 ps that follow either power-law or slow multiexponential decay over several nanoseconds. The origin of such dispersed dynamics remains difficult to understand. Here we compare results of TRFSS experiments to molecular dynamics (MD) simulations of well-known 4',6-diamidino-2-phenylindole (DAPI)/Dickerson-Drew DNA complex over five decades of time from 100 fs to 10 ns to understand the origin of such dispersed dynamics. We show that the solvation time-correlation function (TCF) calculated from 200 ns simulation trajectory (total 800 ns) captures most features of slow dynamics as measured in TRFSS experiments. Decomposition of TCF into individual components unravels that slow dynamics originating from dynamically coupled DNA-water motion, although contribution from coupled water-Na+ motion is non-negligible. The analysis of residence time of water molecules around the probe (DAPI) reveals broad distribution from ∼6 ps to ∼3.5 ns: Several (49 nos.) water molecules show residences time greater than 500 ps, of which at least 14 water molecules show residence times of more than 1 ns in the first solvation shell of DAPI. Most of these slow water molecules are found to occupy two hydration sites in the minor groove near DAPI binding site. The residence time of Na+, however, is found to vary within ∼17-120 ps. Remarkably, we find that freezing the DNA fluctuations in simulation eliminates slower dynamics beyond ∼100 ps, where water and Na+ dynamics become faster, although strong anticorrelation exists between them. These results indicate that primary origin of slow dynamics lies within the slow fluctuations of DNA parts that couple with nearby slow water and ions to control the dispersed collective solvation dynamics in DNA minor groove.

Role of Ser65, His148 and Thr203 in the Organic Solvent-dependent Spectral Shift in Green Fluorescent Protein.

The photophysics of green fluorescent protein (GFP) is remarkable because of its exceptional property of excited state proton transfer (ESPT) and the presence of a functional proton wire. Another interesting property of wild-type GFP is that its absorption and fluorescence excitation spectra are sensitive to the presence of polar organic solvents even at very low concentrations. Here, we use a combination of methodologies including site-specific mutagenesis, absorption spectroscopy, steady-state and time-resolved fluorescence measurements and all-atom molecular dynamics simulations in explicit solvent, to uncover the mechanism behind the unique spectral sensitivity of GFP toward organic solvents. Based on the evidences provided herein, we suggest that organic solvent-induced changes in the proton wire prevent ground state movement of a proton through the wire and thus bring about the spectral changes observed. The present study can not only help to understand the mechanism of proton transfer by further dissecting the intricate steps in GFP photophysics but also encourages to develop GFP-based organic solvent biosensors.

Dynamics of water and ions around DNA: What is so special about them?

Water around biomolecules is special for behaving strangely - both in terms of structure and dynamics, while ions are found to control various interactions in biomolecules such as DNA, proteins and lipids. The questions that how water and ions around these biomolecules behave in terms of their structure and dynamics, and how they affect the biomolecular functions have triggered tremendous research activities worldwide. Such activities not only unfolded important static and dynamic properties of water and ions around these biomolecules, but also provoked heated debate regarding their explanation and role in biological functions. DNA, being negatively charged, interacts strongly with the surrounding dipolar water and positively charged counterions, leading to complex electrostatic coupling of water and ions with the DNA. Recent timeresolved fluorescence Stokes shift experiments and related computer simulation studies from our and other laboratories have unfolded some unique dynamic characteristics of water and ions near different structures of DNA. These results are discussed here to showcase the specialty of water and ion dynamics around DNA.

Ras hyperactivation versus overexpression: Lessons from Ras dynamics in Candida albicans.

Ras signaling in response to environmental cues is critical for cellular morphogenesis in eukaryotes. This signaling is tightly regulated and its activation involves multiple players. Sometimes Ras signaling may be hyperactivated. In C. albicans, a human pathogenic fungus, we demonstrate that dynamics of hyperactivated Ras1 (Ras1G13V or Ras1 in Hsp90 deficient strains) can be reliably differentiated from that of normal Ras1 at (near) single molecule level using fluorescence correlation spectroscopy (FCS). Ras1 hyperactivation results in significantly slower dynamics due to actin polymerization. Activating actin polymerization by jasplakinolide can produce hyperactivated Ras1 dynamics. In a sterol-deficient hyperfilamentous GPI mutant of C. albicans too, Ras1 hyperactivation results from Hsp90 downregulation and causes actin polymerization. Hyperactivated Ras1 co-localizes with G-actin at the plasma membrane rather than with F-actin. Depolymerizing actin with cytochalasin D results in faster Ras1 dynamics in these and other strains that show Ras1 hyperactivation. Further, ergosterol does not influence Ras1 dynamics.

Effect of T·T Mismatch on DNA Dynamics Probed by Minor Groove Binders: Comparison of Dynamic Stokes Shifts of Hoechst and DAPI.

Recognition of DNA base mismatches and their subsequent repair by enzymes is vital for genomic stability. However, it is difficult to comprehend such a process in which enzymes sense and repair different types of mismatches with different ability. It has been suggested that the differential structural changes of mismatched bases act as cues to the repair enzymes, although the effect of such DNA structural changes on surrounding water and ion dynamics is inevitable due to strong electrostatic coupling among them. Thus, collective dynamics of DNA, water, and ions near the mismatch site is believed to be important for mismatch recognition and repair mechanism. Here we show that introduction of a T·T mismatch in the minor groove of DNA induces dispersed (collective) power-law solvation dynamics (of exponent ∼0.24), measured by monitoring the time-resolved fluorescence Stokes shifts (TRFSS) of two popular minor groove binders (Hoechst 33258 and DAPI) over five decades of time from 100 fs to 10 ns. The same ligands however sense different dynamics (power-law of exponent ∼0.15 or power-law multiplied with biexponential relaxation) in the minor groove of normal-DNA. The similar fluorescence anisotropy decays of ligands measured in normal- and T·T-DNA suggest that Stokes shift dynamics and their changes in T·T-DNA purely originate from the solvation process, and not from any internal rotational motion of probe-ligands. The dispersed power-law solvation dynamics seen in T·T-DNA indicate that the ligands do not sense any particular (exponential) relaxation specific to T·T wobbling and/or other conformational changes. This could be the reason why T·T mismatch is recognized by enzymes with lower efficiency compared to purine-pyrimidine and purine-purine mismatches.

Probe-location dependent resonance energy transfer at lipid/water interfaces: comparison between the gel- and fluid-phase of lipid bilayer.

Despite significant interest in understanding the role of the local dielectric environment and lipid-bilayer fluidity/rigidity in resonance energy transfer between chromophores at lipid/water interfaces, a comprehensive approach to quantify such environmental dependence on energy transfer is missing - primarily because of the scarcity of suitable probes. Here we present the results on multi-chromophoric Förster resonance energy transfer (FRET) from a series of 4-aminophthalimide-based molecules (4AP-Cn; n = 2-10, 12) of different lipophilicity (donors), which reside at different depths across the lipid/water interfaces, to rhodamine-6G (Rh6G; acceptor) molecules that stay in a water-rich region near the lipid headgroups. We apply steady-state and time-resolved fluorescence spectroscopy, and find that multi-chromophoric FRET from the series of 4AP-Cn donors to the Rh6G acceptor occurs in a peculiar stepwise fashion at the lipid/water interface of a gel-phase (Lß') DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) bilayer at room temperature. However, the same donor-acceptor pairs show only subtle but continuous donor-depth-dependent FRET at the lipid/water interface of a fluid-phase (Lα) DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) bilayer. These features were found to correlate with the lipid-phase dependent local environmental polarity sensed by 4AP-Cn donors at the interfaces. Molecular dynamics (MD) simulations, combined with experimental results, show that relative depth (and angle) variation of the 4AP-Cn donors and Rh6G acceptor directly controls the FRET efficiencies through fine tuning of the emission and absorption spectra of the donors and acceptor, respectively. The results indicate that the 4AP-Cn probes are well-suited as donors for FRET studies, which allow the FRET parameters at lipid/water interfaces of gel- and fluid-phases of lipid-bilayers to be quantified and compared simultaneously.

Multidrug ABC transporter Cdr1 of Candida albicans harbors specific and overlapping binding sites for human steroid hormones transport.

The present study examines the kinetics of steroids efflux mediated by the Candida drug resistance protein 1 (Cdr1p) and evaluates their interaction with the protein. We exploited our in-house mutant library for targeting the 252 residues forming the twelve transmembrane helices (TMHs) of Cdr1p. The screening revealed 65 and 58 residues critical for ß-estradiol and corticosterone transport, respectively. Notably, up to 83% critical residues for corticosterone face the lipid interface compared to 54% for ß-estradiol. Molecular docking identified a possible peripheral corticosterone-binding site made of 8/14 critical/non-critical residues between TMHs 3, 4 and 6. ß-estradiol transport was severely hampered by alanine replacements of Cdr1p core residues involving TMHs 2, 5 and 8, in a binding site made of 10/14 critical residues mainly shared with rhodamine 6G with which it competes. By contrast, TMH11 was poorly impacted, although being part of the core domain. Finally, we observed the presence of several contiguous stretches of 3-5 critical residues in TMHs 2, 5 and 10 that points to a rotation motion of these helices during the substrate transport cycle. The selective structural arrangement of the steroid-binding pockets in the core region and at the lipid-TMD interface, which was never reported before, together with the possible rotation of some TMHs may be the structural basis of the drug-transport mechanism achieved by these type II ABC transporters.

Newly identified motifs in Candida albicans Cdr1 protein nucleotide binding domains are pleiotropic drug resistance subfamily-specific and functionally asymmetric.

An analysis of Candida albicans ABC transporters identified conserved related α-helical sequence motifs immediately C-terminal of each Walker A sequence. Despite the occurrence of these motifs in ABC subfamilies of other yeasts and higher eukaryotes, their roles in protein function remained unexplored. In this study we have examined the functional significance of these motifs in the C. albicans PDR transporter Cdr1p. The motifs present in NBD1 and NBD2 were subjected to alanine scanning mutagenesis, deletion, or replacement of an entire motif. Systematic replacement of individual motif residues with alanine did not affect the function of Cdr1p but deletion of the M1-motif in NBD1 (M1-Del) resulted in Cdr1p being trapped within the endoplasmic reticulum. In contrast, deletion of the M2-motif in NBD2 (M2-Del) yielded a non-functional protein with normal plasma membrane localization. Replacement of the motif in M1-Del with six alanines (M1-Ala) significantly improved localization of the protein and partially restored function. Conversely, replacement of the motif in M2-Del with six alanines (M2-Ala) did not reverse the phenotype and susceptibility to antifungal substrates of Cdr1p was unchanged. Together, the M1 and M2 motifs contribute to the functional asymmetry of NBDs and are important for maturation of Cdr1p and ATP catalysis, respectively.

New insight into probe-location dependent polarity and hydration at lipid/water interfaces: comparison between gel- and fluid-phases of lipid bilayers.

Environment polarity and hydration at lipid/water interfaces play important roles in membrane biology, which are investigated here using a new homologous series of 4-aminophthalimide-based fluorescent molecules (4AP-Cn; n = 2-10, 12) having different lipophilicities (octanol/water partition coefficient - log P). We show that 4AP-Cn molecules probe a peculiar stepwise polarity (E) profile at the lipid/water interface of the gel-phase (Lß') DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) bilayer at room temperature, which was not anticipated in earlier studies. However, the same molecules probe only a subtle but continuous polarity change at the interface of water and the fluid-phase (Lα) DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) bilayer at room temperature. Fluorescence quenching experiments indicate that solutes with different log P values adsorb at different depths across DPPC/water and DOPC/water interfaces, which correlate with the polarity profiles observed at the interfaces. Molecular dynamics simulations performed on eight probe-lipid systems (four in each of the DPPC and DOPC bilayers - a total run of 2.6 µs) support experimental results, providing further information on the relative position and angle distributions as well as hydration of probes at the interfaces. Simulation results indicate that besides positions, probe orientations also play an important role in defining the local dielectric environment by controlling the probes' exposure to water at the interfaces especially of the gel-phase DPPC bilayer. The results suggest that 4AP-Cn probes are well suited for studying solvation properties at lipid/water interfaces of gel- and fluid-phases simultaneously.

Measuring Size, Size Distribution, and Polydispersity of Water-in-Oil Microemulsion Droplets using Fluorescence Correlation Spectroscopy: Comparison to Dynamic Light Scattering.

Water-in-oil microemulsion droplets (MEDs) are thermodynamically stable supramolecular structures formed in a mixture of water and oil, stabilized by surfactant layer. Here we use fluorescence correlation spectroscopy (FCS) to measure the diffusion, and the size, size distribution, and polydispersity of MEDs prepared in ternary mixtures of water/oil/sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in heptane, isooctane, and nonane at (near) single droplet level. We compare FCS data directly to dynamic light scattering (DLS) data, which shows that the optical matching point (OMP) conditions of MEDs in different oils (where excess optical polarizability of droplets vanish) severely influence DLS data, while FCS extracts the accurate size, size distribution, and polydispersity of AOT-MEDs in all three oils. This suggests that extreme precaution must be taken in acquiring and explaining DLS data of MEDs in solution. FCS data show nearly identical W0-dependent (peak) size variations of AOT-MEDs in all three oils, though a subtle increase in (average) polydispersity of droplets is observed with increase in carbon chain length of oils. Establishing the accuracy of FCS data for AOT-MEDs, we further apply FCS to measure the size parameters of MEDs prepared in a quaternary mixture of water/oil/cetyltrimethylammonium bromide (CTAB)/1-butanol in hexane, heptane, and isooctane. Unlike AOT-MEDs, FCS data show substantial effect of added cosurfactant (1-butanol) and external oil on size, size distribution and polydispersity of quaternary CTAB-MEDs. Analysis of size distributions reveals large variation of polydispersity which possibly indicates the existence of larger shape heterogeneity, together with size heterogeneity, of CTAB-MEDs compared to AOT-MEDs in solution.