In this review paper, the conceptual basis and experimental design of total internal reflection with fluorescence correlation spectroscopy (TIR-FCS) is described. The few applications to date of TIR-FCS to supported membranes are discussed, in addition to a variety of applications not directly involving supported membranes. Methods related, but not technically equivalent, to TIR-FCS are also summarized. Future directions for TIR-FCS are outlined.

The combination of total internal reflection illumination and fluorescence correlation spectroscopy (TIR-FCS) is an emerging method useful for, among a number of things, measuring the thermodynamic and kinetic parameters describing the reversible association of fluorescently labeled ligands in solution with immobilized, nonfluorescent surface binding sites. However, there are many parameters (both instrumental and intrinsic to the interaction of interest) that determine the nature of the acquired fluorescence fluctuation autocorrelation functions. In this work, we define criteria necessary for successful measurements, and then systematically explore the parameter space to define conditions that meet the criteria. The work is intended to serve as a guide for experimental design; in other words, to provide a methodology to identify experimental conditions that will yield reliable values of the thermodynamic and kinetic parameters for a given interaction.

No abstract

The pregnane X receptor (PXR), a member of the nuclear receptor superfamily, regulates the expression of drug-metabolizing enzymes in a ligand-dependent manner. The conventional view of nuclear receptor action is that ligand binding enhances the receptor’s affinity for coactivator proteins, while decreasing its affinity for corepressors. To date, however, no known rigorous biophysical studies have been conducted to investigate the interaction among PXR, its coregulators, and ligands. In this work, steady-state total internal reflection fluorescence microscopy (TIRFM) and total internal reflection with fluorescence recovery after photobleaching were used to measure the thermodynamics and kinetics of the interaction between the PXR ligand binding domain and a peptide fragment of the steroid receptor coactivator-1 (SRC-1) in the presence and absence of the established PXR agonist, rifampicin. Equilibrium dissociation and dissociation rate constants of ~5 μM and ~2 s−1, respectively, were obtained in the presence and absence of rifampicin, indicating that the ligand does not enhance the affinity of the PXR and SRC-1 fragments. Additionally, TIRFM was used to examine the interaction between PXR and a peptide fragment of the corepressor protein, the silencing mediator for retinoid and thyroid receptors (SMRT). An equilibrium dissociation constant of ~70 μM was obtained for SMRT in the presence and absence of rifampicin. These results strongly suggest that the mechanism of ligand-dependent activation in PXR differs significantly from that seen in many other nuclear receptors.

Edited by Henrik G. Dohlman Upon activation by the G q family of G␣ subunits, G␤␥ subunits, and some Rho family GTPases, phospholipase C-␤ (PLC-␤) isoforms hydrolyze phosphatidylinositol 4,5-bisphosphate to the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. PLC-␤ isoforms also function as GTPase-activating proteins, potentiating G q deactivation. To elucidate the mechanism of this mutual regulation, we measured the thermodynamics and kinetics of PLC-␤3 binding to G␣ q. FRET and fluorescence correlation spectroscopy, two physically distinct methods, both yielded K d values of about 200 nM for PLC-␤3-G␣ q binding. This K d is 50-100 times greater than the EC 50 for G␣ q-mediated PLC-␤3 activation and for the G␣ q GTPase-activating protein activity of PLC-␤. The measured K d was not altered either by the presence of phospholipid vesicles, phosphatidylinositol 4,5-bisphosphate and Ca 2؉ , or by the identity of the fluorescent labels. FRET-based kinetic measurements were also consistent with a K d of 200 nM. We determined that PLC-␤3 hysteresis, whereby PLC-␤3 remains active for some time following either G␣ q-PLC-␤3 dissociation or PLC-␤3potentiated G␣ q deactivation, is not sufficient to explain the observed discrepancy between EC 50 and K d. These results indicate that the mechanism by which G␣ q and PLC-␤3 mutually regulate each other is far more complex than a simple, two-state allosteric model and instead is probably kinetically determined.