Changes In Fluorescence Anisotropy Research Articles

Increase of Fluorescence Anisotropy Upon Self‐Assembly in Headgroup‐Labeled Surfactants

The change in fluorescence anisotropy upon micellization in headgroup-labeled surfactants is investigated. After eliminating the likelihood of depolarizing RET, anisotropy is shown to increase upon self-assembly due to increased rotational correlation times of the fluorophore. This is shown using two surfactant-fluorophore systems. Anisotropy in NBD-labeled phospholipids is studied both in chloroform (unaggregated) and in water (unilamellar vesicles), while in tryptophan-containing peptide-amphiphiles, the variation of anisotropy with concentration leads to a reasonable measurement of CAC. Anisotropy increase is shown to be largely the product of increased rotational correlation times for the fluorophore, relative to its tau. These results serve as a basis for future work that measures the amount of depolarizing energy transfer, characterizing distances between similar fluorescent headgroups on mixed micelles.

Interactions of PAMAM dendrimers with model lipid membranes

Dendrimers and other cationic macromolecules in general are considered potential vehicles for intracellular drug delivery. We used fluorescence spectroscopy to study the interaction of cationic PAMAM (polyamidoamine) dendrimers with model lipid bilayers. When the dendrimers interacted with small unilamellar vesicles (SUV) loaded with the self-quenching fluorophore calcein they did not induce release of calcein neither with neutral SUV (made of egg phosphatidylcholine) nor negatively charged SUV (with 25% egg phosphatidylglycerol). The G1 (generation 1, Mr = 1430) dendrimer was labeled with fluorescein and the G4 dendrimer (Mr = 14215) with a similar fluorophore Oregon green, and their membrane interactions were followed by changes in fluorescence anisotropy during titrations with lipid. The anisotropy changed linearly with the density of negative charge in the membrane. The addition of high salt caused redissociation of the dendrimers from SUV. These two observations prove the electrostatic nature of the interaction. Analysis of the binding data yielded values of the dissociation constants Kd. For G1-dendrimer, Kd = 30 +/- 16 uM with neutral SUV and Kd = 11 +/- 3 M with negatively charged SUV. For G4, which carries much higher charge on its surface, exhibited lower dissociation constants, Kd = 16 +/- 7 uM with neutral SUV and 5 +/- 1 uM with negatively charged SUV. We conclude that the interaction of PAMAM dendrimers with lipid membrane is electrostatic and the dendrimer binding does not significantly disrupt the bilayer integrity.

Physical Responses of Bacterial Chemoreceptors

Chemoreceptors of the bacterium Escherichia coli are thought to form trimers of homodimers that undergo conformational changes upon ligand binding and thereby signal a cytoplasmic kinase. We monitored the physical responses of trimers in living cells lacking other chemotaxis proteins by fluorescently tagging receptors and measuring changes in fluorescence anisotropy. These changes were traced to changes in energy transfer between fluorophores on different dimers of a trimer: attractants move these fluorophores farther apart, and repellents move them closer together. These measurements allowed us to define the responses of bare receptor oligomers to ligand binding and compare them to the corresponding response in kinase activity. Receptor responses could be fit by a simple “two-state” model in which receptor dimers are in either active or inactive conformations, from which energy bias and dissociation constants could be estimated. Comparison with responses in kinase-activity indicated that higher-order interactions are dominant in receptor clusters.

Extended Upstream A-T Sequence Increases T7 Promoter Strength

Bacteriophage T7 promoters contain a consensus sequence from -17 to +6 relative to the transcription start site, +1. In addition, the strong class III promoters are characterized by an extended AT-rich region upstream of -17, which is often interrupted by one or more GC base pairs in the weaker class II promoters. Herein we studied the role of the AT-rich region upstream of -17 in transcription regulation of T7 RNA polymerase. Equilibrium DNA binding studies with promoter fragments of consensus sequence truncated at various positions between -17 and -27 showed that the polymerase-promoter complex is significantly stabilized as the upstream AT-rich sequence is extended to and beyond -22. Similarly, promoters in which the AT-rich region from -17 to -22 is interrupted by several GC base pairs showed weak binding. Kinetic studies indicated that the presence of extended AT-rich sequence slows the dissociation rate constant of the polymerase-promoter complex and slightly stimulates the association rate constant, thereby increasing the stability of the complex. Measurement of the transcription activity revealed that the extended AT-rich region does not affect the kinetics of abortive synthesis up to the formation of 8-nucleotide RNA but causes accumulation of longer abortive products between 9 and 13 nucleotides. The observed effects of the upstream DNA region were AT sequence-specific, and the results suggested a larger role for the extended AT-rich sequence that has been unappreciated previously. We propose that the AT-rich DNA sequence upstream of -17 plays a role in modulating the efficiency of transcription initiation by affecting both the affinity of T7 RNA polymerase for the promoter and the efficiency of promoter clearance.

Spontaneously Formed Vesicles of SodiumN-(11-Acrylamidoundecanoyl)-glycinate andl-Alaninate in Water

Two N-acyl amino acid surfactants, sodium N-(11-acrylamidoundecanoyl)-glycinate (SAUG) and L-alaninate (SAUA), were synthesized and characterized in aqueous solution. A number of techniques, such as surface tension, fluorescence probe, light scattering, and transmission electron microscopy were employed for characterization of the amphiphiles in water. The surface and interfacial properties were measured. The amphiphiles have two critical aggregation concentrations. The results of surface tension and fluorescence probe studies suggested formation of bilayer self-assemblies in dilute aqueous solutions of the amphiphiles. The magnitudes of free energy change of aggregation have indicated that bilayer formation is more favorable in the case of SAUG. Steady-state fluorescence measurements of pyrene and 1,6-diphenyl-1,3,5-hexatriene (DPH) were used to study the microenvironment of the molecular self-assemblies. Temperature-dependent fluorescence anisotropy change of DPH probe revealed phase transition temperature of the bilayer self-assemblies. The effects of pH on the structure of the self-assemblies of SAUG and SAUA have been studied. The role of intermolecular hydrogen bonding between amide groups upon aggregation toward microstructure formation in solution has been discussed. Circular dichroism spectra suggested the presence of chiral aggregates in an aqueous solution of SAUA. The transmission electron micrographs revealed the presence of closed spherical vesicles in aqueous solutions of the amphiphiles. Dynamic light scattering measurements were performed to obtain average size of the aggregates.

Discrimination between two steps in the mitochondrial permeability transition process

It is well known that a lag phase generally elapses between the addition of inducers of the mitochondrial permeability transition and the opening of the pore. To advance our present understanding as regards the significance of this phenomenon, we used experimental approaches which are sensitive to different aspects of the permeability transition process. The pore conformation was sensed by the fluorescence anisotropy changes of hematoporphyrin-labelled mitochondria. Membrane permeabilization was ascertained by following the matrix swelling consequent to external solute equilibration. We show that the anisotropy changes of mitochondria-bound hematoporphyrin precede both membrane depolarization (proton permeation) and matrix swelling (solute permeation), thus sensing a step of the permeability transition process that involves the pore in its closed state. We suggest that the opening of the pore is preceded by a structural remodelling of mitochondrial domains containing hematoporphyrin-near, pore-regulating histidines. Such a perturbation is strongly inhibited at acidic matrix pH and completely blocked by cyclosporin A. In sucrose-based media the opening of the pore can be strongly delayed, as compared to salt-based media, a fact which probably reflects perturbation of mitochondrial membranes by sugar. We conclude that the mitochondrial permeability transition could be described as an at least two-step process which is mainly regulated by conformational changes of the pore components.

The Molecular Chaperone, ClpA, Has a Single High Affinity Peptide Binding Site per Hexamer

Substrate recognition by Clp chaperones is dependent on interactions with motifs composed of specific peptide sequences. We studied the binding of short motif-bearing peptides to ClpA, the chaperone component of the ATP-dependent ClpAP protease of Escherichia coli in the presence of ATPgammaS and Mg2+ at pH 7.5. Binding was measured by isothermal titration calorimetry (ITC) using the peptide, AANDENYALAA, which corresponds to the SsrA degradation motif found at the C terminus of abnormal nascent polypeptides in vivo. One SsrA peptide was bound per hexamer of ClpA with an association constant (K(A)) of 5 x 10(6) m(-1). Binding was also assayed by changes in fluorescence of an N-terminal dansylated SsrA peptide, which bound with the same stoichiometry of one per ClpA hexamer (K(A) approximately 1 x 10(7) m(-1)). Similar results were obtained when ATP was substituted for ATPgammaS at 6 degrees C. Two additional peptides, derived from the phage P1 RepA protein and the E. coli HemA protein, which bear different substrate motifs, were competitive inhibitors of SsrA binding and bound to ClpA hexamers with K(A)' > 3 x 10(7) m(-1). DNS-SsrA bound with only slightly reduced affinity to deletion mutants of ClpA missing either the N-terminal domain or the C-terminal nucleotide-binding domain, indicating that the binding site for SsrA lies within the N-terminal nucleotide-binding domain. Because only one protein at a time can be unfolded and translocated by ClpA hexamers, restricting the number of peptides initially bound should avoid nonproductive binding of substrates and aggregation of partially processed proteins.

Monitoring cholesterol organization in membranes at low concentrations utilizing the wavelength-selective fluorescence approach

We previously showed using a fluorescent analogue of cholesterol (NBD-cholesterol, or 25-[ N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol), that cholesterol may exhibit local organization at low concentrations in membranes by the formation of transbilayer tail-to-tail dimers of cholesterol (Rukmini, R., Rawat, S.S., Biswas, S.C., Chattopadhyay, A., 2001. Biophys. J. 81, 2122–2134). In this report, we have monitored the microenvironmental features of cholesterol monomers and dimers utilizing wavelength-selective fluorescence spectroscopy. Our results utilizing red edge excitation shift (REES) and wavelength-dependent change in fluorescence anisotropy show that the microenvironment around the NBD moieties in the dimer form is more rigid possibly due to steric constraints imposed by the dimer conformation. These results provide new information and are relevant in understanding the organization of cholesterol in membranes at low concentrations.

Incorporation of fluorescent probes into PAMAM dendrimers

Interactions of two fluorescent probes 1-(trimethylammoniumphenyl)-6-phenyl-1,3,5 hexatriene p-toluenesulfonate (TMA-DPH) and 12-(9-anthroyloxy) stearic acid (12-AS) with polyamidoamine (PAMAM) dendrimers were studied. Changes in fluorescence intensity and steady-state fluorescence anisotropy of TMA-DPH and 12-AS were monitored. It was found that 12-AS molecules incorporated into dendrimer cavities whereas TMA-DPH molecules aggregated on the surface of polymer. Dendrimer size had not significant impact on its host properties.

Binding of the Factor IX γ-Carboxyglutamic Acid Domain to the Vitamin K-dependent γ-Glutamyl Carboxylase Active Site Induces an Allosteric Effect That May Ensure Processive Carboxylation and Regulate the Release of Carboxylated Product

Propeptides of the vitamin K-dependent proteins bind to an exosite on gamma-glutamyl carboxylase; while they are bound, multiple glutamic acids in the gamma-carboxyglutamic acid (Gla) domain are carboxylated. The role of the propeptides has been studied extensively; however, the role of the Gla domain in substrate binding is less well understood. We used kinetic and fluorescence techniques to investigate the interactions of the carboxylase with a substrate containing the propeptide and Gla domain of factor IX (FIXproGla41). In addition, we characterized the effect of the Gla domain and carboxylation on propeptide and substrate binding. For the propeptide of factor IX (proFIX18), FIXproGla41, and carboxylated FIXproGla41, the Kd values were 50, 2.5, and 19.7 nM and the koff values were 273 x 10(-5), 9 x 10(-5), and 37 x 10(-5) s(-1), respectively. The koff of proFIX18 is reduced 3-fold by FLEEL and 9-fold by the Gla domain (residues 1-46) of FIX. The pre-steady state rate constants for carboxylation of FIXproGla41 was 0.02 s(-1) in enzyme excess and 0.016 s(-1) in substrate excess. The steady state rate in substrate excess is 4.5 x 10(-4) s(-1). These results demonstrate the following. 1) The pre-steady state carboxylation rate constant of FIXproGla41 is significantly slower than that of FLEEL. 2) The Gla domain plays an allosteric role in substrate-enzyme interactions. 3) Carboxylation reduces the allosteric effect. 4) The similarity between the steady state carboxylation rate constant and product dissociation rate constant suggests that product release is rate-limiting. 5) The increased dissociation rate after carboxylation contributes to the release of product.

Effects of Subzero Temperatures on Fluorescent Probes Sequestered within Aerosol-OT Reverse Micelles

We report on the effects of temperature (+30 to −100 °C) and water loading on the steady-state fluorescence from four fluorescent probe molecules when they are sequestered within the interior of sodium bis(2-ethyl-1-hexyl) sulfosuccinate (Aerosol OT, AOT) reverse micelles. Each probe locates itself to different average microenvironments within the reverse micelle, and these individual microenvironments behave differently to changes in the molar ratio of water-to-AOT (R) and temperature. Changes in probe steady-state fluorescence emission spectra or fluorescence anisotropy indicate that “freezing” occurs within the water pool at temperatures between −10 and −60 °C. The lowest freezing points are generally observed at the lowest R values.

Controlling the DNA Binding Specificity of bHLH Proteins through Intramolecular Interactions

Reversible control of the conformation of proteins was employed to probe the relationship between flexibility and specificity of the basic helix-loop-helix protein MyoD. A fusion protein (apaMyoD) was designed where the basic DNA binding helix of MyoD was stablized by an amino-terminal extension with a sequence derived from the bee venom peptide apamin. The disulfide-stabilized helix from apamin served as a nucleus for a helix that extended for a further ten residues, thereby holding apaMyoD's DNA recognition helix in a predominantly alpha-helical conformation. The thermal stability of the DNA complexes of apaMyoD was increased by 13 degrees C relative to MyoD-bHLH. Measurements of the fluorescence anisotropy change on DNA binding indicated that apaMyoD bound to E-box-containing DNA sequences with enhanced affinity relative to MyoD-bHLH. Consequently, the DNA binding specificity of apaMyoD was increased 10-fold.

HDL derived from the different phases of conjugated diene formation reduces membrane fluidity and contributes to a decrease in free cholesterol efflux from human THP-1 macrophages

Oxidized HDL (ox-HDL) has been reported to reduce free cholesterol efflux from cells. In this study we investigate the effect of different stages of ox-HDL on macrophage membrane fluidity and its effect on free cholesterol efflux from macrophages as a cell function influenced by ox-HDL. HDL was oxidized by means of conjugated diene production using copper as a prooxidant. Fluidity of HDL and human THP-1 macrophage membranes was evaluated by changes in fluorescence anisotropy ( r) by DPH probe where lower ( r) values give higher fluidity. We found that ox-HDL derived from the propagation phase (PP-HDL) and the decomposition phase (DP-HDL) became less fluid (( r): 0.263±0.001, 0.279±0.002, respectively) than HDL from the lag phase (LP-HDL) and native HDL (nat-HDL) (( r): 0.206±0.001) ( P<0.05). Macrophages incubated with PP-HDL and DP-HDL had less fluid membranes (( r): 0.231±0.001, 0.243±0.002, respectively) than those incubated with LP-HDL and nat-HDL (( r): 0.223±0.001) ( P<0.05). Consequently, fluidity was reduced not only in ox-HDL but also in the cell membranes exposed to ox-HDL. A significant negative correlation was observed between macrophage membrane fluorescence anisotropy ( r) and free cholesterol efflux from these cells (−0.876; P<0.05). Thus, lower membrane fluidity was associated with lower free cholesterol efflux from cells. In conclusion, the increase in the HDL oxidation process leads to a lost of macrophage membrane fluidity that could contribute to an explanation of the reduction of free cholesterol efflux from cells by ox-HDL.

Structural modifications of the permeability transition pore complex in resealed mitochondria induced by matrix-entrapped disaccharides

Mitochondrial resealing after the opening of the permeability transition (PT) pore was studied in saline- and sugar-based media by following the fluorescence anisotropy changes of mitochondria-bound hematoporphyrin (HP), a probe sensitive to conformational variations of the pore complex [Biochemistry 38 (1999) 9300]. The HP anisotropy changes correlated well with complete mitochondrial resealing in saline media and suggested that the pore complex regained the native structure after closure. Rebuilding of the pore complex structure was also achieved in monosaccharide-based media, thus ruling out a major influence of the swollen state of mitochondria on the reconstitution properties of the pore components. On the contrary, when sucrose or other disaccharides were used as osmotic support, restoration of the native mitochondrial structure, as monitored by HP anisotropy, was not achieved, though the proton barrier of the inner membrane and respiration functions were reestablished. Infrared spectroscopy experiments indicated the occurrence of strong perturbations of the mitochondrial membrane structure after disaccharide entrapment in the matrix space. These data suggest that mitochondria are able to reseal and regain functional activity after opening of the PT pore irrespective of the incubation medium but in sucrose (and other disaccharides) the pore complex adopts a conformation different from that existing before permeabilization. In general, our data indicate that the pore complex can exist in different conformations which are modulated by the nature of the interactions with the medium cosolvents.

Interaction of tubulin with a new fluorescent analogue of vinblastine.

Vinblastine is an antimitotic agent that has been used extensively in cancer chemotherapy. The biological effects of the drug are believed to be the result of its interaction with tubulin, the major component of cellular microtubules. Fluorescence spectroscopy is a powerful and versatile technique for studying drug-tubulin interactions, but it rarely has been applied to studies involving vinca alkaloids. We have prepared a new fluorescent derivative of vinblastine designed to retain high affinity for tubulin while possessing a fluorophore that absorbs and emits visible light. A coumarin derivative of vinblastine, 17-deacetyl-O-(3-carbonylamino-7-diethylaminocoumarin) vinblastine (F-VLB), was prepared by reaction of 17-deacetylvinblastine with 7-diethylaminocoumarin-3-carbonyl azide. F-VLB was a potent inhibitor of in vitro microtubule assembly (IC(50) = 0.5 microM). F-VLB binding to tubulin was inhibited by vinblastine. Tubulin binding induced an increase in the F-VLB emission intensity and shifted the emission maximum to higher energy (from 500 to 480 nm). The Stokes shift of tubulin-bound F-VLB was about the same as the Stokes shift of the molecule in ethanol, indicating that the tubulin-bound fluorophore is probably on the exterior of the vinblastine binding site. Unlike vinblastine, F-VLB failed to induce self-assembly of tubulin that could be detected by light scattering or electron microscopy, although some self-association could be detected by analytical ultracentrifugation. Equilibrium binding parameters were quantitatively determined by monitoring the change in fluorescence anisotropy of F-VLB upon tubulin binding. The apparent equilibrium constant for F-VLB binding to tubulin [K(a)(app) = (7.7 +/- 0.5) x 10(4) M(-1) at 25 degrees C] was identical to the equilibrium constant for vinblastine binding to 2 microM tubulin (K(1)) measured under similar buffer and temperature conditions using ultracentrifugation [Vulevic, B., Lobert, S., and Correia, J. J. (1997) Biochemistry 36, 12828-12835]. Binding allocolchicine to tubulin did not significantly affect F-VLB's affinity for the protein [K(a)(app) = (9.1 +/- 0.4) x 10(4) M(-1) at 25 degrees C]. Analysis of the steady-state emission spectra yielded a distance between the colchicine and vinca binding sites on tubulin of approximately 40 A. F-VLB bound to paclitaxel- and glutaraldehyde-stabilized microtubules, with approximately equal affinity. We conclude that F-VLB can be used to obtain information about the vinblastine binding site on tubulin under equilibrium conditions.

Binding affinity of Escherichia coli RNA polymerase*sigma54 holoenzyme for the glnAp2, nifH and nifL promoters.

Escherichia coli RNA polymerase associated with the sigma54 factor (RNAP*sigma54) is a holoenzyme form that transcribes a special class of promoters not recognized by the standard RNA polymerase*sigma70 com plex. Promoters for RNAP*sigma54 vary in their overall 'strength' and show differences in their response to the presence of DNA curvature between enhancer and promoter. In order to examine whether these effects are related to the promoter affinity, we have determined the equilibrium dissociation constant K(d) for the binding of RNAP*sigma54 to the three promoters glnAp2, nifH and nifL. Binding studies were conducted by monitoring the changes in fluorescence anisotropy upon titrating RNAP*sigma54 to carboxyrhodamine-labeled DNA duplexes. For the glnAp2 and nifH promoters similar values of K(d) = 0.94 +/- 0.55 nM and K(d) = 0.85 +/- 0.30 nM were determined at physiological ionic strength, while the nifL promoter displayed a significantly weaker affinity with K(d) = 8.5 +/- 1.9 nM. The logarithmic dependence of K(d) on the ionic strength I was -Deltalog(K(d))/Deltalog(I) = 6.1 +/- 0.5 for the glnAp2, 5.2 +/- 1.2 for the nifH and 2.1 +/- 0.1 for the nifL promoter. This suggests that the polymerase can form fewer ion pairs with the nifL promoter, which would account for its weaker binding affinity.

Biochemical Defects in Retina-specific Human ATP Binding Cassette Transporter Nucleotide Binding Domain 1 Mutants Associated with Macular Degeneration

The retina-specific human ABC transporter (ABCR) functions in the retinal transport system and has been implicated in several inherited visual diseases, including Stargardt disease, fundus flavimaculatus, cone-rod dystrophy, and age-related macular degeneration. We have previously described a general ribonucleotidase activity of the first nucleotide binding domain (NBD1) of human ABCR (Biswas, E. E. (2001) Biochemistry 40, 8181-8187). In this communication, we present a quantitative study analyzing the effects of certain disease-associated mutations, Gly-863 --> Ala, Pro-940 --> Arg, and Arg-943 --> Gln on the nucleotide binding, and general ribonucleotidase activities of this domain. NBD1 proteins, harboring these mutations, were created through in vitro site-specific mutagenesis and expressed in Escherichia coli. Results of the enzyme-kinetic studies indicated that these mutations altered the ATPase and CTPase activities of NBD1. The G863A and P940R mutations were found to have significant attenuation of the rates of nucleotide hydrolysis and binding affinities. On the other hand, the R943Q mutation had small, but detectable reduction in its nucleotidase activity and nucleotide binding affinity. We have measured the nucleotide binding affinities of NBD1 protein and its mutants quantitatively by fluorescence anisotropy changes during protein binding to ethenoadenosine ATP (epsilonATP), a fluorescent ATP analogue. We have correlated the dissociation constant (K(D)) and the rates of nucleotide hydrolysis (V(max)) of NBD1 and its mutants with the available genetic data for these mutations.

A STOPPED-FLOW FLUORESCENCE ANISOTROPY METHOD FOR MEASURING HORMONE BINDING AND DISSOCIATION KINETICS WITH CELL-SURFACE RECEPTORS IN LIVING CELLS

ABSTRACTWe have developed a system for extending stopped-flow analysis to the kinetics of ligand capture and release by cell surface receptors in living cells. While most mammalian cell lines cannot survive the shear forces associated with turbulent, stopped-flow mixing, we determined that 32D cells, murine hematopoietic precursor cells, can survive rapid mixing, even at the high flow rates necessary to achieve dwell times as short as 10 msec. In addition, 32D cells do not express any member of the ErbB family of receptors, providing a null background for studying this receptor family. We have established a series of stable, monoclonal 32D-derived cell lines that express the epidermal growth factor (EGF) receptor, ErbB2, or a combination of both at different ratios. Using these cell lines and a homogeneous fluorescent derivative of H22Y-mEGF modified with fluorescein at the amino terminus (F-EGF), we have measured association and dissociation of F-EGF with its receptor. Association was measured by following the time-dependent changes in fluorescence anisotropy after rapidly mixing cells at various cell densities with F-EGF at 1–15 nM. Dissociation was measured both by chase experiments in which unlabeled EGF was mixed with cells pre-equilibrated with F-EGF or by dilution of cells equilibrated with F-EGF. Comparison of these dissociation experiments demonstrated that little or no ligand-induced dissociation occurs in the chase dissociation experiments. For each cell line, data from a series of association experiments and dilution dissociation experiments were subjected to global analysis using a two independent receptor-class model. Our analysis is consistent with the presence of two distinct receptor populations, even in cells bearing only the EGF receptor. Increasing the relative expression of ErbB2 leads to an increase in the fraction of high affinity class receptors observed, without altering the total number of EGF binding sites.

Effect of ErbB2 coexpression on the kinetic interactions of epidermal growth factor with its receptor in intact cells.

We have extended the use of stopped-flow mixing and fluorescence anisotropy detection to investigate in real-time the effects of ErbB2 coexpression on the kinetic interactions of epidermal growth factor (EGF) with the EGF receptor. Using stable 32D-derived cell lines expressing both the EGF receptor and ErbB2, and fluorescein-labeled H22Y murine EGF (F-EGF), a series of association and dissociation experiments were performed in which the kinetic interaction of F-EGF with cells was monitored by observing time-dependent changes in fluorescence anisotropy following rapid mixing. Data were collected at various concentrations of F-EGF and multiple cell densities, using cells that express similar levels of the EGF receptor but different levels of ErbB2, and then analyzed by fitting to a two independent receptor-class model using global analysis techniques. The recovered kinetic parameters indicated that the coexpression of ErbB2 had relatively modest effects on recovered rate constants and calculated K(d) values, but a significant effect on the fraction of receptors associated with the high-affinity receptor class. This effect on the fraction of high-affinity receptors depended on the relative expression of ErbB2, as higher ErbB2 expression levels correlated with a larger fraction of high-affinity receptors. Further, the increase in the fraction of high-affinity receptors due to the presence of ErbB2 occurred without any change in the total number of EGF binding sites per cell. Thus, we have identified modulation of the relative populations of high- and low-affinity classes of EGF receptors as a consequence of coexpression of ErbB2 with the EGF receptor.

Molecular aptamer for real-time oncoprotein platelet-derived growth factor monitoring by fluorescence anisotropy.

Monitoring proteins in real time and in homogeneous solution has always been a difficult task. We have applied a fluorophore-labeled molecular probe based on a high-affinity platelet-derived growth factor (PDGF) aptamer for the ultrasensitive detection of PDGF in homogeneous solutions. The aptamer is labeled with fluorescein to specifically bind with the PDGF protein. Fluorescence anisotropy is used for the real-time monitoring of the binding between the aptamer and the protein. When the labeled aptamer is bound with its target protein, the rotational motion of the fluorophore attached to the complex becomes much slower because of an increased molecular weight after binding, resulting in a significant fluorescence anisotropy change. Using the anisotropy change, we are able to detect the binding events between the aptamer and the protein in real time and in homogeneous solutions (detection without separation). This assay is highly selective and ultrasensitive. It can detect PDGF in the subnanomolar range. The new method for protein detection is simple and inherits all of the advantages of molecular aptamers. Efficient oncoprotein detection using aptamer-based fluorescence anisotropy measurement will find wide applications in protein monitoring, in cancer diagnosis as well as other studies in which protein analysis is important.