intensity is measured parallel (i.e. in the same plane) to the absorbed plane-polarised
light and at right angles to it. From the two measurements it is possible to calculate the
degree of fluorescence depolarisation and hence the fluorescence anisotropy, both of
which are expressed in terms of the difference between the fluorescence parallel to the
absorbed plane-polarised light and that perpendicular to it, the difference being
expressed as a function of the sum of the fluorescence in the two planes.- Fluorescence cross-correlation spectroscopy(FCCS): This technique differs from other
forms of fluorescence spectroscopy in that it is not primarily concerned with
fluorescence intensity, but rather with small spontaneous fluorescence fluctuations
induced by molecules diffusing into and out of a small focal volume in aqueous
solution. Such fluctuations are related to changes in the diffusion coefficient of each
probe and hence can be correlated with receptor–ligand binding. Both the receptor
and the ligand are labelled with spectrally distinct fluors and their interaction studied
by confocal microscopy (Section 4.3.1). Two lasers are aligned to the same confocal
point and used to excite the two fluors. Following binding, the receptor–ligand
complex emits in both fluorescent wavelengths that are monitored to give a cross-
correlation signal that is directly related to the concentration of the receptor–ligand
complex.
Surface plasmon resonance (SPR) spectroscopy
The principles and experimental details of this technique are discussed in Section 13.3.
The advantages of the technique are that it does not require the molecules to be
fluorescent or radiolabelled; it can be used to study molecules as small as 100 Da and
can be used with coloured or opaque solutions. Such studies with G-protein-coupled
receptors have shown that agonists, inverse agonists and antagonists can readily
be distinguished by the conformational changes they induce in the membrane in
the region of the receptor. Specifically, agonists and inverse agonists increase mem-
brane thickness (agonists more so than inverse agonists) by causing an elongation of
the receptor whereas antagonists cause no change.Plasmon-waveguide resonance
(PWR)spectroscopyis closely related to SPR spectroscopy. It is more sensitive than
SPR spectroscopy and can be used to study receptor conformational changes in lipid
bilayers. Thus a single lipid bilayer is deposited on the resonator surface and the
receptor protein inserted from a detergent-solubilised solution. A solution containing
the ligand is then passed over the layer allowing the binding process to be studied.Isothermal titration calorimetry
This method is based on the measurement of the heat change, positive or negative,
associated with the binding and of the relationship between the enthalpy change
(▵H), Gibbs’ free energy change (▵G), the entropy change (▵S), the number of
molecules of ligand bound to each receptor protein (stoichiometry)(n) and the
binding constant (Ka). The experimental details are discussed in Section 15.3.3.
Its practical advantages are that it can be applied to the study of any receptor–ligand
pair without the need for radiolabelling or the attachment of fluors, or the need to
separate bound and unbound fractions.684 Cell membrane receptors and cell signalling