Catherine A. Royer

Equilibrium Binding of Estrogen Receptor with DNA Using Fluorescence Anisotropy

Interaction of estrogen receptor (ER) with DNA sequences known as estrogen response elements (ERE) is required for estrogen regulation of the expression of target genes. To characterize the affinity and specificity of ER interaction with ERE sequencesin vitro under equilibrium conditions, fluorescence anisotropy assays were performed using recombinant, purified ER and a fluorescein-labeled 35-base pair oligonucleotide bearing an idealized palindromic ERE. In buffer containing 100 mm KCl, the baculovirus-expressed, purified human ER bound with similar affinity to the consensus ERE and a mutant ERE with a single base pair change per half-site. Above 225 mm KCl, ER exhibited discrimination between the consensus and mutated ERE targets. Between 225 and 275 mm KCl, binding to the consensus ERE was independent of salt concentration and occurred with an equilibrium dissociation constant (K d ) of 1.8 ± 0.6 nm, whereas binding to the mutant ERE was not detected at ER concentrations below 100 nm under the same conditions. At 300 mm KCl, the K d for the consensus ERE increased approximately 25-fold, suggesting complex salt concentration dependence. Both estrogen-occupied and unoccupied ER bound to the consensus ERE sequence with similar affinity, indicating that estrogen affects ER activity at a step other than DNA binding. Unlike the full-length ER, the recombinant DNA binding domain of ER did not discriminate between the consensus and mutated ERE sequences even at buffer salt concentrations greater than 200 mm NaCl, suggesting that ER sequences outside the DNA binding domain may be important in promoting specific binding.

ANALYSIS OF BINDING IN MACROMOLECULAR COMPLEXES : A GENERALIZED NUMERICAL APPROACH

In this work, we introduce a generalized, global numerical methodology for analysis of binding phenomena in complex macromolecular assemblies. On the basis of a numerical algorithm (EQS) to solve systems of simultaneous free energy equations, binding profiles of simple to highly complex interacting systems can be analyzed over any concentration region without any need to generate an analytical form to describe the data. The output of the numerical algorithm is the concentration of each individual species in solution, allowing the generation of all possible binding profiles of the system (e.g., protein saturation by ligand). We present here the application of this approach to the DNA-protein subunit-ligand interactions of the trp repressor system as a typical example. From a practical point of view, the analysis program is capable of the rapid and simultaneous analysis of multiple binding profiles in terms of internally consistent sets of free energies. Given both the enormous complexity, as well as the underlying subtlety, involved in the regulation of biological function, the present generalized approach to analyzing macromolecular binding should find wide applications.

FLUORESCENCE SPECTROSCOPY AS A TOOL TO INVESTIGATE PROTEIN INTERACTIONS

Recent advances in the use of fluorescence spectroscopy to study protein interactions have primarily involved combinations of classic fluorescence techniques, novel probe and coupling chemistries, and advances in laser excitation and detection capabilities. For example, new coupling strategies for fluorescent probes have allowed the first determination of the DeltaG° describing the insertion of a protein into a membrane. Fluorescently labeled oligonucleotides with specific protein-binding sequences have been used to study both protein-DNA associations and oligonucleotide hybridization using anisotropy changes. The first kinetic data describing a DNA-protein binding event was collected with stopped-flow fluorescence instrumentation. Combining scanning fluctuation correlation spectroscopy with a two-photon excitation source improved this technique so that it may now be used to study protein self-associations.

Solution studies of the interactions between the histone core proteins and DNA using fluorescence spectroscope

The equilibrium interactions between histone H2A-H2B and H3/H4 subunits with 200 base pair chicken erythrocyte DNA have been studied by monitoring the fluorescence polarization of a long-lived fluorescence probe covalently bound to the histone subunits. These studies have brought to light the formation of highly asymmetric complexes exhibiting very high histone/DNA stoichiometries as well as very high apparent affinities. The stoichiometries observed for these non-nucleosome complexes depended both upon the concentration of the histones and the concentration of the DNA 200mer. The observed stoichiometries varied approximately between 4 and 16 histone octamers/DNA 200mer and the affinities were in the nanomolar range. These results are discussed in terms of their in vitro as well as their possible in vivo significance.

The dissociation rate of estrogen receptor alpha from the consensus estrogen response element.

Abstract The rate of dissociation of recombinant, purified human estrogen receptor α (ERα) from a fluorescein-labeled DNA containing the consensus vitellogenin ERE sequence (F-vitERE) was determined in real time using fluorescence anisotropy. The complex of estradiol-occupied ERα with F-vitERE had an apparent dissociation rate of 1.48±0.06×10 −2 s −1 and a half-life of 46.6 s at room temperature. The dissociation rate was characterized by a single exponential decay, suggesting that ER dissociates from the DNA as a preformed dimer, rather than as two individual monomers. The association rate of estradiol-occupied ERα for the F-vitERE was calculated as 7×10 6 M −1 s −1 based on the dissociation rate measured and previous determinations of the equilibrium dissociation constant ( K d ) in similar assay conditions ( Ozers et al., 1997 ). In buffer containing various concentrations of salt, the rate of dissociation of estradiol-occupied ERα from F-vitERE was accelerated by increasing salt concentrations. Compared to estradiol-occupied ERα, the rate of dissociation of unoccupied ERα from the F-vitERE was very similar, indicating that estradiol occupancy does not affect the dissociation rate of ERα from the ERE.

Probing the relation between protein structure and intrinsic tryptophan fluorescence using superrepressor mutants of thetrp repressor

The wavelength dependence of the intrinsic tryptophan fluorescence lifetime of a series of mutants of thetrp repressor protein was characterized in both the native and the denatured states. These mutants belong to a particular class, called superrepressors, as their phenotype, when expressedin vivo, is to repress transcription at lower concentrations of the corepressor, tryptophan. It has been demonstrated previously that these mutations result in distinct and profound modifications of the structural and dynamic properties of the protein [Reedstrom and Royer (1995)J. Mol. Biol.253, 266; Reedstromet al. (1996)J. Mol. Biol.264, 32; Smithet al. (1995)Biochemistry34, 13183]. The present observations reveal that in the native state, these structural and dynamic modifications result in subtle, yet significant alterations in the intrinsic tryptophan fluorescence decay characteristics. Surprisingly, significant differences in the fluorescence decays between the mutants and the wild-type protein were also observed for the guanidine hydrochloride unfolded states. These results are discussed in terms of the various models which have been proposed to explain the decay properties of tryptophan in proteins.

Fluorescence dynamics of biological systems using synchrotron radiation (invited)

A beamline for time‐resolved fluorescence spectroscopy of biological systems is under construction at the Synchrotron Radiation Center. The fluorometer, operating in the frequency domain, will take advantage of the time structure of the synchrotron radiation light pulses to determine fluorescence lifetimes. Using frequency‐domain techniques, the instrument can achieve an ultimate time resolution on the order of picoseconds. Preliminary experiments have shown that reducing the intensity of one of the fifteen electron bunches in the storage ring allows measurement of harmonic frequencies equivalent to the single‐bunch mode. This mode of operation of the synchrotron significantly extends the range of lifetimes that can be measured. The wavelength range (encompassing the visible and ultraviolet), the range of measurable lifetimes, and the stability and reproducibility of the storage ring pulses should make this beamline a versatile tool for the investigation of the complex fluorescence decay of biological sys...

fluorescence approaches to the study of protein-DNA interactions

The three-dimensional structures of a number of DNA-binding proteins alone and complexed with their cognate DNA sequences have recently been reported. One of these is the trp repressor from E. coli, for which the structure in the absence and the presence of the co- repressor, tryptophan, has been deduced from x-ray crystallographic data, as well as that of the ternary complex between protein, co-repressor, and operator DNA. Rather than provide a definitive answer for the mechanism of the activation of the repressor by tryptophan binding, these structures have raised even more questions concerning the basis for the affinity changes observed. DNA binding studies of this protein using a number of biochemical techniques have brought to light the existence of repressor multimer-DNA complexes at physiological concentrations, although these have not been well characterized. Recently, we have demonstrated, using fluorescence polarization techniques, that the repressor oligomerizes in absence of DNA and that co-repressor binding destabilizes the oligomers. Clearly, in this case, as in many others, there are subtle thermodynamic relationships between protein-protein, protein-ligand, and protein-DNA interactions. The role of these energetic couplings in the allosteric regulation of the repressor by tryptophan is, however, not understood. Because of the non-equilibrium nature of filter-binding, gel mobility shift, and nuclease protection assays it is not possible to fully explore these different binding phenomena over broad concentration ranges of each component and under different solution conditions. Fluorescence methodologies provide observables of the multiple binding equilibria in solution between nanomolar and micromolar concentrations. Variables such as the protein and ligand concentration dependence of repressor-DNA interactions, as well as different solution conditions (i.e., salt concentration), have been probed using these techniques. The intrinsic tryptophan fluorescence of the protein has been used to characterize dimer stability. The fluorescence polarization of a covalently bound DNS label has been used to characterize the higher order protein-protein interactions and the effect of tryptophan and salt concentration upon them. DNS polarization has also been used to probe the protein and tryptophan concentration dependence of operator DNA binding. Finally, fluorescence quenching of coumarin covalently coupled to the trp repressor protein upon binding the operator fragment has been used to study these protein- DNA complexes. The combination of appropriate fluorescence methodologies allows for a detailed characterization of binding affinities and stoichiometries in such systems.