Sobhan Sen

Dynamics of Water and Ions Near DNA: Comparison of Simulation to Time-Resolved Stokes-Shift Experiments

Time-resolved Stokes-shift experiments measure the dynamics of biomolecules and of the perturbed solvent near them on subnanosecond time scales, but molecular dynamics simulations are needed to provide a clear interpretation of the results. Here we show that simulations using standard methods quantitatively reproduce the main features of TRSS experiments in DNA and provide a molecular assignment for the dynamics. The simulations reproduce the magnitude and unusual power-law dynamics of the Stokes shift seen in recent experiments [ Andreatta, D., et al. J. Am. Chem. Soc. 2005, 127, 7270 ]. A polarization model is introduced to eliminate cross-correlations between the different components contributing to the signal. Using this model, well-defined contributions of the DNA, water, and counterion to the experimental signal are extracted. Water is found to have the largest contribution and to be responsible for the power-law dynamics. The counterions have a smaller, but non-negligible, contribution with a time constant of 220 ps. The contribution to the signal of the DNA itself is minor and fits a 30 ps stretched exponential. Both time-averaged and dynamic distributions are calculated. They show a small subset of ions with a different coupling but no other evidence of substates or rate heterogeneity.

Fluorescence Correlation Spectroscopy: An Efficient Tool for Measuring Size, Size-Distribution and Polydispersity of Microemulsion Droplets in Solution

Fluorescence correlation spectroscopy (FCS) is an ideal tool for measuring molecular diffusion and size under extremely dilute conditions. However, the power of FCS has not been utilized to its best to measure diffusion and size parameters of complex chemical systems. Here, we apply FCS to measure the size, and, most importantly, the size distribution and polydispersity of a supramolecular nanostructure (i.e., microemulsion droplets, MEDs) in dilute solution. It is shown how the refractive index mismatch of a solution can be corrected in FCS to obtain accurate size parameters of particles, bypassing the optical matching problem of light scattering techniques that are used often for particle-size measurements. We studied the MEDs of 13 different W(0) values from 2 to 50 prepared in a ternary mixture of water, sodium bis(2-ethylhexyl) sulfosuccinate (AOT), and isooctane, with sulforhodamine-B as a fluorescent marker. We find that, near the optical matching point of MEDs, the dynamic light scattering (DLS) measurements underestimate the droplet sizes while FCS estimates the accurate ones. A Gaussian distribution model (GDM) and a maximum-entropy-based FCS data fitting model (MEMFCS) are used to analyze the fluorescence correlation curves that unfold Gaussian-type size distributions of MEDs in solution. We find the droplet size varies linearly with W(0) up to ~20, but beyond this W(0) value, the size variation deviates from this linearity. To explain nonlinear variation of droplet size for W(0) values beyond ~20, we invoke a model (the coated-droplet model) that incorporates the size polydispersity of the droplets.

Solvation dynamics of DCM in micelles

The solvation dynamics of 4-(dicyanomethylene)-2-methyl-6(p-dimethylaminostyryl) 4H-pyran (DCM) have been studied in neutral (Triton X-100, TX), cationic (cetyl trimethyl ammonium bromide, CTAB) and anionic (sodium dodecyl sulfate, SDS) micelles using picosecond time-resolved Stokes shift. Above the critical micellar concentration for all three micelles, DCM exhibits wavelength-dependent fluorescence decays. At short wavelengths, a fast decay is observed while, at long wavelengths, a distinct growth precedes the decay. The time-dependent Stokes shift indicates that the water molecules in the Stern layer of the micelles relax on a timescale which is markedly slower than the sub-picosecond relaxation dynamics in pure water.

Femtosecond study of solvation dynamics of DCM in micelles

Solvation dynamics of 4-(dicyanomethylene)-2-methyl-6-(p-dimethylamino-styryl) 4H-pyran (DCM) has been studied in neutral (triton X-100, TX) and cationic (cetyl trimethyl ammonium bromide, CTAB) micelles using femtosecond upconversion. Since DCM is insoluble in bulk water the observed solvation dynamics reports the relaxation dynamics exclusively at the micellar interface. It is observed that the solvation dynamics in TX is slower than that in CTAB. The solvation dynamics is described by components of 2.1, 165 and 2050 ps for TX and 0.23, 6.5 (average 1.75 ps) and 350 ps for CTAB.

Solvation dynamics in a protein-surfactant complex

Solvation dynamics in the denatured state of a protein, lysozyme (denatured by sodium dodecyl sulfate, SDS) is markedly slower than that in the native state. For coumarin 153 bound to lysozyme, the average solvation time, hssi is 330 ps. In the lysozyme–SDS complex, the solvation dynamics is markedly slower with hss i¼ 7250 ps. On addition of dithiothreitol (DTT) to the lysozyme–SDS complex, when the di-sulfide bonds are destroyed, hssi is found to be 1140 ps. The slow dynamics in the denatured protein is attributed to the polymer chain dynamics and the exchange of bound and free water molecules. 2003 Elsevier B.V. All rights reserved.

Insight into Pleiotropic Drug Resistance ATP-binding Cassette Pump Drug Transport through Mutagenesis of Cdr1p Transmembrane Domains

Background: The Candida albicans efflux pump Cdr1p causes clinically significant antifungal resistance. Results: Biochemical mapping of Cdr1p transmembrane domain mutants reveals residues affecting drug transport. Conclusion: Functional characterization and homology modeling provide insight into the drug binding cavity and substrate promiscuity of Cdr1p. Significance: A platform is provided for systematic Cdr1p structure/function analysis and the rational design of transport modulators/inhibitors. The fungal ATP-binding cassette (ABC) transporter Cdr1 protein (Cdr1p), responsible for clinically significant drug resistance, is composed of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). We have probed the nature of the drug binding pocket by performing systematic mutagenesis of the primary sequences of the 12 transmembrane segments (TMSs) found in the TMDs. All mutated proteins were expressed equally well and localized properly at the plasma membrane in the heterologous host Saccharomyces cerevisiae, but some variants differed significantly in efflux activity, substrate specificity, and coupled ATPase activity. Replacement of the majority of the amino acid residues with alanine or glycine yielded neutral mutations, but about 42% of the variants lost resistance to drug efflux substrates completely or selectively. A predicted three-dimensional homology model shows that all the TMSs, apart from TMS4 and TMS10, interact directly with the drug-binding cavity in both the open and closed Cdr1p conformations. However, TMS4 and TMS10 mutations can also induce total or selective drug susceptibility. Functional data and homology modeling assisted identification of critical amino acids within a drug-binding cavity that, upon mutation, abolished resistance to all drugs tested singly or in combinations. The open and closed Cdr1p models enabled the identification of amino acid residues that bordered a drug-binding cavity dominated by hydrophobic residues. The disposition of TMD residues with differential effects on drug binding and transport are consistent with a large polyspecific drug binding pocket in this yeast multidrug transporter.

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.

Understanding Growth Kinetics of Nanorods in Microemulsion: A Combined Fluorescence Correlation Spectroscopy, Dynamic Light Scattering, and Electron Microscopy Study

Even though nanostructures of various shapes and sizes can be controlled by microemulsions, there is substantial difficulty in understanding their growth mechanism. The evolution of nanostructures from the time of mixing of reactants to their final stage is a heterogeneous process involving a variety of intermediates. To obtain a deeper insight into these kinetic steps, we studied the slow growth kinetics (extending over eight days) of iron oxalate nanorods inside the polar core of water-in-oil microemulsion droplets made of cetyltrimethylammonium bromide/1-butanol/isooctane. Fluorescence correlation spectroscopy (FCS), dynamic light scattering (DLS), and transmission electron microscopy (TEM) have been employed to monitor the nanostructure growth at (near) the single-droplet level and in an ensemble. Analyzing FCS data with suitable kinetic model we obtain transient dimer lifetime (28 μs) and the droplet fusion rates (and fusion tendency) on each day as the reaction proceeds. The droplet fusion rate is found to directly control the nanorod growth in microemulsion solution and attains its maximum value (3.55 × 10(4) s(-1)) on day 6, when long nanorods are found in TEM data, implying that more and more reactants are fed into the growing system at this stage. Combining FCS, DLS, and TEM results, we find three distinct periods in the entire growth process: a long nucleation-dominant nanoparticle growth period which forms nanoparticles of critical (average) size of ∼53 nm, followed by a short period where isotropic nanoparticles switch to anisotropic growth to form nanorods, and finally elongation of nanorods and growth (and shrinking) of nanoparticles.

Understanding ligand interaction with different structures of G-quadruplex DNA: evidence of kinetically controlled ligand binding and binding-mode assisted quadruplex structure alteration.

The study of ligand interaction with G-quadruplex DNA is an active research area, because many ligands are shown to bind G-quadruplex structures, showing anticancer effects. Here, we show, for the first time, how fluorescence correlation spectroscopy (FCS) can be used to study binding kinetics of ligands with G-quadruplex DNA at the single molecule level. As an example, we study interaction of a benzo-phenoxazine ligand (Cresyl Violet, CV) with antiparallel and (3 + 1) hybrid G-quadruplex structures formed by human telomeric sequence. By using simple modifications in FCS setup, we describe how one can extract the reaction kinetics from diffusion-coupled correlation curves. It is found that the ligand (CV) binds stronger, by an order of magnitude, to a (3 + 1) hybrid structure, compared to an antiparallel one. Ensemble-averaged time-resolved fluorescence experiments are also carried out to obtain the binding equilibrium constants (K) of ligand-quadruplex interactions in bulk solution for the first time, which are found to match very well with FCS results. Global analysis of FCS data provides association (k(+)) and dissociation (k(-)) rates of the ligand in the two structures. Results indicate that stronger ligand binding to the (3 + 1) hybrid structure is controlled by the dissociation rate, rather than the association rate of ligand in the quadruplexes. Circular dichroism (CD) and induced-CD spectra show that the ligand not only binds at different conformations in the quadruplexes, but also induces antiparallel structure to form a mixed-type hybrid structure in Na(+) solution. However, in K(+) solution, the ligand stabilizes the (3 + 1) hybrid structure. Molecular docking studies predict the possible differences in binding sites of the ligand inside two quadruplexes, which strongly support the experimental observations. Results suggest that different binding modes of the ligand to the quadruplex structures actually assist the alteration of structures differently.

Solvation dynamics of TNS in polymer (PEG)–surfactant (SDS) aggregate

Abstract Solvation dynamics of 2,6- p -toluidinonaphthalene sulfonate (TNS) is studied using picosecond time resolved emission spectroscopy in a polymer–surfactant aggregate consisting of polyethylene glycol (PEG) and sodium dodecyl sulfate (SDS). The critical association concentration (CAC) of SDS for PEG, is found to be 4.5±0.5 mM. Solvation dynamics of TNS in PEG–SDS aggregate is described by two components, 90±10 ps (31%) and 570±50 ps (69%). Solvation dynamics in PEG–SDS aggregate is slow compared to that in bulk water or in PEG alone or in SDS alone indicating restricted movement of the water molecules squeezed in between the polymer chains and the micellar (SDS) surface.