Fluorescence Anisotropy
Fluorescently labelled ligands are being used increasingly as an alternative to radiolabelled ligands allowing monitoring and visualisation of ligand binding to the receptor (and other interactions/events with participation of fluorescently labelled partners) in biological samples. Novel fluorescent probes stand out with high molecular brightness, low photon-bleaching and insensitivity to solvent polarity and pH changes. These qualities have contributed to the development of a set of alternative methods to radioligand binding assays that exploit different intrinsic fluorescence aspects/properties of the fluorophore (intensity, wavelength, lifetime, anisotropy).
The fluorescence anisotropy (FA) method is based on the phenomenon that the population of fluorescent probes emits light with a certain degree of polarisation when excited by plane polarised light. The polarisation extent of emitted light of the fluorophore activated by polarised light depends on fluorophore’s freedom of movement within its fluorescence lifetime i.e., the average time the molecules stays in its excited state before emitting a photon. The binding of fluorescent ligands to bigger and more massive receptor proteins constrains their freedom of movement resulting in a greater extent of polarisation of the emitted fluorescence. The changes of FA can be detected as a change in emitted fluorescence intensities parallel (I↑↑) and perpendicular (I↑→) to the plane of excitation, respectively, and used for the calculation of FA signal.
Changes in FA signal can be followed in real-time without of any separation step requirement. FA signal at time t after the initiation of the binding reaction can be defined as parameter r(t) and calculated as following
The fluorescence anisotropy (FA) method is based on the phenomenon that the population of fluorescent probes emits light with a certain degree of polarisation when excited by plane polarised light. The polarisation extent of emitted light of the fluorophore activated by polarised light depends on fluorophore’s freedom of movement within its fluorescence lifetime i.e., the average time the molecules stays in its excited state before emitting a photon. The binding of fluorescent ligands to bigger and more massive receptor proteins constrains their freedom of movement resulting in a greater extent of polarisation of the emitted fluorescence. The changes of FA can be detected as a change in emitted fluorescence intensities parallel (I↑↑) and perpendicular (I↑→) to the plane of excitation, respectively, and used for the calculation of FA signal.
Changes in FA signal can be followed in real-time without of any separation step requirement. FA signal at time t after the initiation of the binding reaction can be defined as parameter r(t) and calculated as following
Thus, a simple principle, homogenous system and possibility of continuous online monitoring of the receptor-ligand interaction dynamics, as well as moderate requirements for equipment makes the polarisation-based FA assay attractive for the assessment of GPCR-ligand binding properties.
We have successfully implemented the FA-based assay systems for charaterization of the association and dissociation kinetics of ligands to and from the receptor, respectively, in membrane preparations from baculovirus infected Sf9 cells as well as budded baculovirus particles (Veikšina et al. 2010, Veikšina et al. 2014, Veikšina et al. 2015, Link et al. 2017).
We have successfully implemented the FA-based assay systems for charaterization of the association and dissociation kinetics of ligands to and from the receptor, respectively, in membrane preparations from baculovirus infected Sf9 cells as well as budded baculovirus particles (Veikšina et al. 2010, Veikšina et al. 2014, Veikšina et al. 2015, Link et al. 2017).
Figure 1.Application of fluorescence anisotropy for the charaterization of ligand binding to a GPCR. Upon binding to the receptor, the rotational mobility of fluorescent ligand decreases. The binding event can be detected as a change in intensities of emitted polarized light.