Fluorescence Anisotropy - Applications

Applications

Fluorescence anisotropy can be used to measure the binding constants and kinetics of reactions that cause a change in the rotational time of the molecules. If the fluorophore is bound to a small molecule, the rate at which it tumbles can decrease significantly when it is bound tightly to a large protein. If the fluorophore is attached to the larger protein in a binding pair, the difference in polarization between bound and unbound states will be smaller (because the unbound protein will already be fairly stable and tumble slowly to begin with) and the measurement will be less accurate. The degree of binding is calculated by using the difference in anisotropy of the partially bound, free and fully bound( large excess of protein) states measured by titrating the two binding partners.

If the fluorophore is bound to a relatively large molecule like a protein or an RNA, the change in the mobility accompanying folding can be used to study the dynamics of folding. This provides a measure of the dynamics of how the protein achieves its final, stable 3D shape.

Fluorescence anisotropy is also applied to microscopy, with use of polarizers in the path of the illuminating light and also before the camera. This can be used to study the local viscosity of the cytosol or membranes, with the latter giving information about the membrane microstructure and the relative concentrations of various lipids. This technique has also been used to detect the binding of molecules to their partners in signaling cascades in response to an certain cues.

The phenomenon of emFRET and the associated decrease in anisotropy when close interactions occur between fluorophores has been used to study the aggregation of proteins in response to signaling.