Fluorescence anisotropy or polarisation provides a sensitive tool to measure the binding of ligands to proteins when a fluorophore is attached to the ligand. This method is particularly useful if no changes in other fluorescence properties are seen. Changes in the anisotropy are caused by changes of the mobility of the fluorophore. This is the case when a small ligand binds to a macromolecule (e.g. proteins) that moves much slower than the ligand free in solution. If a fluorescence-labelled ligand is used only as a probe in competition experiments, dissociation constants for label free compounds under investigation can be obtained. This assay can provide useful data even for low affinity ligands and can be automated with a titration device.
Drug discovery and mechanistic biological studies on proteins require quantitative ligand binding data. A multitude of methods are available to obtain these data and the choice is often directed by the amounts and quality of target protein available, the number and nature of ligands to test, the binding kinetics and affinities. Often it is desirable to apply several independent methods for cross validation. Fluorescence has the advantage that only small amounts of target protein and ligands are required, that it can easily be automated and that even low affinity binding can be characterised. Beside changes in the fluorescence intensity, changes in the emission peak position and steady-state anisotropy may also change with the binding of a ligand which can be used to obtain binding constants.
This application note describes as example the binding of a label-free ligand in a competition experiment shown for the regulatory domain of the potassium efflux system (Kef) from Shewanella denitrificans. Kef protects Gram-negative bacteria against toxic electrophilic compounds.
Polarised emission spectra were recorded in an FLS980 Fluorescence Spectrometer equipped with double excitation and emission monochromators. Calcite polarisers were used in the excitation and emission, while for detection a photomultiplier tube detector (PMT-900) with 0.2 s dwell was used.
Figure 1: FLS980 Fluorescence Spectrometer from Edinburgh Instruments
For fluorescence anisotropy measurements linearly polarised light is used for excitation with a polariser placed between light source and sample and the emission intensity is measured dependent on the polarisation plane by using a second polariser between sample and detector. The anisotropy is then obtained as shown in Equation 1 where the first subscript indicates the position of the excitation polariser, the second of the emission polariser and G(λem) is an instrumental correction factor G(λem)=IHH(λem)/IHV(λem). The polarisation either vertical (V) or horizontal (H):
The fluorescence anisotropy is sensitive to the mobility of the fluorophore as it may move between excitation and emission resulting in a changed polarisation plane of the emitted light. Thus anisotropy measurements are especially useful when the ligand is labelled because the ligand is immobilised upon binding.
A soluble construct of the ligand binding domain of Kef from S. denitrificans was purified for binding experiments. In theshown experiment a high concentration of 54 μM Kef was used which provided low noise levels, but requires corrections during the analysis for the depletion of the ligands.
In this example, a specific fluorescence probe was synthesised, but general probes are commercially available. Kef is activated by adducts of glutathione and electrophiles. These adducts are formed when electrophiles enter the cell. Therefore, a fluorescence probe was developed with the fluorophore dansyl attached to a glutathione backbone: S-{[5-(dimethylamino)naphthalen-1-yl]sulfonylaminopropyl} glutathione (DNGSH) (synthesised by Conway et al., Oxford). The dansyl group was chosen as it is a small fluorophore which reduces the chance of steric clashes during binding to Kef. It was established that this probe binds to Kef and a dissociation constant of Kd=6 μM was determined. Twice the Kef concentration (100 μM) of DNGSH was used in the competition experiment.
The binding of the ligand S-Octan-3-on-1-yl glutathione (OctSG; adduct of the electrophile 1-octen-3-one and glutathione) was used as an example (synthesised by Conway et al., Oxford). This ligand, as well as DNGSH, was dissolved in the measuring buffer.
A micro fluorescence cuvette (Hellma, 105.254-QS) with 3×3 mm light paths was used to minimise the required sample volume to 100 μl. The temperature was kept constant at 20°C.
It was a prerequisite to establish the Kd for the fluorescence probe DNGSH beforehand (see above). In addition, it is required to approximate properties of the bound DNGSH before the competition experiment can be started. Therefore, a reverse titration of a small amount DNGSH with increasing concentration of Kef was performed. It was established that the anisotropy of DNGSH bound to Kef is r=0.180 and that the fluorescence intensity is 4 times as high as for the free ligand (Q=4). The anisotropy of the free DNGSH was measured directly r=0.020.
DNGSH was added to the Kef sample and the anisotropy was recorded. Then the ligand OctSG was added stepwise, the sample was equilibrated for 5 min, and the anisotropy was recorded.
The raw data of the OctSG titration are shown in Figure 2. To simplify analysis, mean values for the fluorescence anisotropy were calculated over the recorded wavelength range for each titration step.
Figure 2: OctSG titration to Kef in the presence of DNGSH.
Ligand Binding Assays on the Basis of Fluorescence AnisotropyÂ
In this fluorescence anisotropy application note, polarised emission spectra were recorded in an FLS980 Fluorescence Spectrometer equipped with double excitation and emission monochromators. The FLS980 has now been discontinued and has now been superseded by our FLS1000 Photoluminescence Spectrometer. For more information on upgrades to an existing FLS980 system, or to enquire about the FLS1000, contact a member of our sales team at sales@edinst.com.
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