Alan Van Orden

Characterization of DNA-protein complexes by capillary electrophoresis-single molecule fluorescence correlation spectroscopy.

A high-speed capillary electrophoresis mobility shift assay (CEMSA) for determining the binding ratios of DNA-protein complexes in solution is demonstrated. Single molecule fluorescence correlation spectroscopy (FCS) was used to resolve the bound and unbound fluorescently labeled DNA molecules as they flowed continuously through a fused silica capillary under the influence of an applied electric field. Resolution of the bound and unbound complexes was based on the difference in their electrophoretic mobilities, and was accomplished without the need to perform a chemical separation. Data sufficient to perform the analysis was acquired in less than 10 s, compared to the minutes that are normally needed to carry out such measurement via CE separation. The binding ratios were determined with 5 to 10% precision and agreed with the results obtained by CE separation within experimental error. The resolution of the CEMSA based FCS analysis (CEMSA-FCS) was significantly higher than for the analysis performed by conventional diffusional FCS, due to the higher mass sensitivity of the electrophoretic mobility compared to the translational diffusion coefficient. Fluorescently labeled 39-mer single stranded DNA (ssDNA) and the single stranded binding protein (SSB) from Escherichia coli was used as the model system. The dissociation constant of the ssDNA-SSB complex was estimated to be approximately 2 nM based on the CEMSA-FCS analysis.

Insulin Receptors and Downstream Substrates Associate with Membrane Microdomains after Treatment with Insulin or Chromium(III) Picolinate

We have examined the association of insulin receptors (IR) and downstream signaling molecules with membrane microdomains in rat basophilic leukemia (RBL-2H3) cells following treatment with insulin or tris(2-pyridinecarbxylato)chromium(III) (Cr(pic)3). Single-particle tracking demonstrated that individual IR on these cells exhibited reduced lateral diffusion and increased confinement within 100xa0nm-scale membrane compartments after treatment with either 200xa0nM insulin or 10xa0μM Cr(pic)3. These treatments also increased the association of native IR, phosphorylated insulin receptor substrate 1 and phosphorylated AKT with detergent-resistant membrane microdomains of characteristically high buoyancy. Confocal fluorescence microscopic imaging of Di-4-ANEPPDHQ labeled RBL-2H3 cells also showed that plasma membrane lipid order decreased following treatment with Cr(pic)3 but was not altered by insulin treatment. Fluorescence correlation spectroscopy demonstrated that Cr(pic)3 did not affect IR cell-surface density or compete with insulin for available binding sites. Finally, Fourier transform infrared spectroscopy indicated that Cr(pic)3 likely associates with the lipid interface in reverse-micelle model membranes. Taken together, these results suggest that activation of IR signaling in a cellular model system by both insulin and Cr(pic)3 involves retention of IR in specialized nanometer-scale membrane microdomains but that the insulin-like effects of Cr(pic)3 are due to changes in membrane lipid order rather than to direct interactions with IR.

Actin-dependent clustering of insulin receptors in membrane microdomains

Recent evidence suggests that, after binding insulin, insulin receptors (IR) interact with specialized, cholesterol-containing, membrane microdomains and components of the actin cytoskeleton. Using single particle tracking techniques, we examined how binding of insulin, depletion of membrane cholesterol and disruption of actin filaments affect the lateral diffusion of individual quantum dot-labeled native IR on live rat basophilic leukemia 2H3 cells. We also examined the effects of similar treatments on IR clustering and multivalent insulin binding on these cells using both photon counting histogram analysis and polarization-based fluorescence resonance energy homo-transfer imaging. Our analyses indicate that binding of insulin to IR on these cells is multivalent, involving at least two insulin molecules per IR as labeling concentrations approach 1μM. Insulin binding also reduces lateral diffusion of IR and the size of membrane compartments accessed by IR. For IR that have not bound insulin, lateral diffusion of IR and the size of membrane compartments accessed by IR increase after disrupting actin filaments or depleting membrane cholesterol. However, clustering of insulin-occupied IR is reduced only by disrupting actin filaments or by fixing cells with paraformaldehyde prior to exposure to insulin, but not by depleting membrane cholesterol. Thus, it appears that, although restriction of IR lateral diffusion on these cells is sensitive to both actin filament dynamics and membrane cholesterol content, clustering of insulin-occupied IR primarily involves an actin-dependent mechanism.

Cy3 in AOT reverse micelles II. Probing intermicellar interactions using fluorescence correlation spectroscopy.

Cyanine-3 (Cy3) fluorescent dye molecules confined in sodium di-2-ethylhexyl sulfosuccinate (AOT) reverse micelles were examined using dynamic light scattering and fluorescence correlation spectroscopy to probe the kinetics of Cy3 dye and reverse micelle aggregation. This study explored a range of reverse micelle sizes, defined as w(0) = [H(2)O]/[AOT], in which the occupation number ranged from one Cy3 molecule per ∼10(5) to ∼10(6) reverse micelles. These measurements reveal that in the smallest reverse micelle, w(0) = 1, the Cy3 molecules aggregate to form H-aggregate dimers, and the Cy3 dimerization is accompanied by the formation of a transient dimer between reverse micelles. Transient reverse micelle dimer particles are only observed in the small fraction of Cy3-labeled reverse micelles probed by fluorescence correlation spectroscopy and are not observed in the bulk solution probed by dynamic light scattering. Furthermore, fluorescence correlation spectroscopy makes it possible to probe the size and shape of these dimers, revealing prolate ellipsoid-shaped particles with twice the volume and surface area of a single reverse micelle.

Fluorescence correlation spectroscopy for ultrasensitive DNA analysis in continuous flow capillary electrophoresis

Continuous flow capillary electrophoresis (CFCE) is non-separations based analytical technique based on the free solution electrophoretic mobility of biological molecules such as DNA, RNA, peptides, and proteins. The electrophoretic mobilities and translational diffusion constants of the analyte molecules are determined using single molecule detection methods, including fluorescence correlation spectroscopy (FCS). CFCE is used to resolve multiple components in a mixture of analytes, measure electrophoretic mobility shifts due to binding interactions, and study the hydrodynamic and electrostatic properties of biological molecules in solution. Often this information is obtained with greater speed and sensitivity than conventational separations-based capillary-zone electrophoresis. This paper will focus on the application of two-beam fluorescence cross-correlation spectroscopy as a versatile detection method for CFCE and explore several applications to the study of the solution properties of single-stranded DNA.

Fluorescence correlation spectroscopic examination of insulin and insulin-like growth factor 1 binding to live cells

We used fluorescence correlation spectroscopy to examine the binding of insulin, insulin-like growth factor 1 (IGF1) and anti-receptor antibodies to insulin receptors (IR) and IGF1 receptors (IGF1R) on individual 2H3 rat basophilic leukemia cells. Experiments revealed two distinct classes of insulin binding sites with K(D) of 0.11 nM and 75 nM, respectively. IGF1 competes with insulin for a portion of the low-affinity insulin binding sites with K(D) of 0.14 nM and for the high-affinity insulin binding sites with K(D) of 10 nM. Dissociation rate constants of insulin and IGF1 were determined to be 0.015 min(-1) and 0.013 min(-1), respectively, allowing estimation of ligand association rate constants. Combined, our results suggest that, in addition to IR and IGF1R homodimers, substantial numbers of hybrid IR-IGF1R heterodimers are present on the surface of these cells.

Effect of loop composition on the stability and folding kinetics of RNA hairpins with large loops.

RNA hairpins are ubiquitous structural elements in biological RNAs, where they have the potential to regulate RNA folding and interactions with other molecules. There are established methods for predicting the thermodynamic stability of an RNA hairpin, but there are still relatively few detailed examinations of the kinetics of folding. Nonetheless, several recent studies indicate that hairpin folding does not proceed via a simple two-state model. Here, we monitor fluorescence from hairpins constructed as molecular beacons in ensemble, fluorescence correlation spectroscopy, and stopped-flow experiments to describe the folding of RNA hairpins with long (15 nucleotide) loops. Our results show that folding of these hairpins occurs through more than two states and that the mechanism of folding includes a fast intermediate phase observed on the tens of microseconds time scale and a slow phase, attributed to formation of the native folded hairpin loop and stem, observed on the milliseconds time scale. The composition of the RNA loop determines the time scale of intermediate and native folded states. Hairpins with a polyuracil loop sequence exhibit slower relaxation of the intermediate state and faster relaxation of the native folded state when compared to that of hairpins with cytosine or adenine in the loop. We hypothesize this composition dependence could be attributed to nucleobase stacking in cytosine and adenine containing regions of the loop, which would be absent in hairpins containing polyuracil loops. Such base stacking could destabilize the intermediate folds, thereby speeding the relaxation of the intermediate relative to similar sized hairpins with no base stacking in the loop. Likewise, the lower intermediate stability could prolong the relaxation of the native folded state.

Correlating structure and fluorescence dynamics of quantum dot clusters using super-resolution imaging

Clusters of quantum dots exhibit fluorescent behavior that differs from that of individual particles. Bulk measurements involving a large number of particles obscure these dynamics. Synthesizing clusters with 5–10 particles enables the study of collective behavior where single-molecule fluorescence techniques can be applied. Super-resolution microscopy of these clusters correlated with SEM imaging reveals the influence of geometry and structure on emission dynamics. Signatures of energy transfer can be seen in the form of enhanced blinking. Motion of the emission center of the cluster is tracked, made possible by the independent blinking events of the individual particles. Discrete steps in the localization are observed as random switching between various on/off configurations moves the location of the emission center.