Experimental techniques for the study of the binding of a ligand
to a membrane receptor
Equations 17.1 to 17.11 allow the calculation of quantitative parameters that charac-
terise the binding of a ligand to a receptor. Such parameters are fundamental to the
understanding of the mechanism of the binding and its relationship to the subsequent
cellular response. They also allow comparisons to be made of the comparative efficacy
and affinity of a series of ligands for a common receptor, a process that is essential in
the development of new drugs (Chapter 18). Numerous techniques are available for the
study of ligand binding but those that are amenable to automation, do not require the
bound and unbound fraction of ligand to be separated and do not require the use of
radiolabelled ligand are generally the preferred methods. Examples are as follows.Fluorescence spectroscopy
Fluorescence-based techniques are ideal for the study of ligand–receptor binding as
they are ultra sensitive, being capable of studying binding involving a few or even
individual ligand molecules and single receptors. The general principles of fluorescence
spectroscopy are discussed in Chapter 12. The methods are based on either changes in
the intrinsic fluorescence of the receptor protein tagged with a suitable fluorescent
marker (fluor) or the induction of fluorescence in either the ligand or the receptor
protein as a result of receptor–ligand binding. Commonly used fluors include fluores-
cein, rhodamine and the dye Fluo-3 (Table 4.3.) but a better alternative is the green
fluorescent protein (GFP) of the jellyfishAequorea victoriaor the red fluorescent protein
ofDiscosoma striataeither of which can be attached to receptor proteins by gene
cloning without altering the normal function of the protein. The main advantage of
using either of these two autofluorescent proteins is that no cofactors are required for
fluorescence to occur hence the study protocols are relatively simple.
The most common forms of fluorescence spectroscopy applied to the study of
receptor-ligand binding are:- Fluorescence resonance energy transfer(FRET): This relies on the presence of two
fluors in distinct locations within the receptor protein such that the emission spectrum
of one and the excitation spectrum of the other overlap. In such circumstances, the
emission light of one fluor may be absorbed by the second (hence energy transfer) and
be emitted as part of its emission. The extent to which this may occur is proportional
to 1/R^6 , whereRis the distance between the two fluors and which is changed as a
result of ligand binding. - Fluorescence anisotropy: Anisotropy is the directional variation in optical properties,
in this case fluorescence, along perpendicular and parallel axes. In this technique
fluorescence is induced by plane-polarised (blue) light. Molecules of the fluor
orientated parallel to this plane of polarisation will be excited preferentially. However,
if some of these molecules rotate after the absorption of the light but before the
fluorescence has time to occur some of the resulting fluorescence will be depolarised
(i.e. no longer in one plane). The extent to which this occurs can be used to deduce
information about the size, shape and flexibility of the protein carrying the fluor. It
can also be used to monitor the binding of a ligand to the protein. The fluorescence
683 17.3 Ligand-binding and cell-signalling studies