Using Fluorescence Anisotropy for Ligand Binding Kinetics of Membrane Proteins

Abstract

Determining ligand binding kinetics provides an indirect route to probe the functional capabilities of the binding pocket of a membrane protein receptor. Presented in this unit are four ligand-binding protocols that provide data useful for characterizing membrane proteins, including equilibrium binding, thermostability, competitive ligand binding, and kinetic ligand binding. These techniques use fluorescence anisotropy, which is safer, less costly, and simpler to execute than radioactive ligand binding. Each protocol may be used on its own or in combination with others to quantify a number of ligand binding constants. © 2018 by John Wiley & Sons, Inc.

Characterization of binding kinetics of A2AR to Gαs protein by surface plasmon resonance

“…Activity of purified A 2A R-1d4 and A 2A D316R-1d4 was confirmed by detection of a fluorescent adenosine receptor agonist (FITC-APEC) binding to purified receptors, as established in prior studies (35)(36)(37)(38)(39). Previously published work from our lab successfully characterized purified A 2A R binding to FITC-APEC by observing a shift in fluorescence anisotropy and saw similar K D values to those previously reported for whole cell binding K D (36,40).…”

“…Before SPR experiments, activity of purified A 2A R and A 2A D316R was verified by fluorescence anisotropy, as previously described (35) and detailed in the Supporting materials and methods. In previous studies, this results in similar activity and affinity levels to radioligand binding for A 2A R (33,36).…”

Fast peptide exchange on major histocompatibility complex class I molecules by acidic stabilization of a peptide‐empty intermediate

“…First, we reasoned that an increased k off , at a rather stable k on , should be accompanied by an increase in the equilibrium dissociation constant ( K D ), since $$$$ {K}_{\mathrm{D}}=\frac{k_{\mathrm{off}}}{k_{\mathrm{on}}}. $$$$ We, therefore, measured the K D by peptide titration with steady‐state anisotropy 29 and found that the pH did not significantly affect the peptide binding affinity of the complexes (Figure 2c,d). Second, another readout of peptide binding affinity to MHC‐I is the thermal stability of the peptide‐MHC‐I complexes 20,30–35 ; to measure it, we used nanoscale differential scanning fluorimetry (nanoDSF), which quantifies protein unfolding by the change in the fluorescence emission of tryptophan indoles and generates a thermal denaturation curve, the midpoint of which corresponds to the melting temperature ( T m ) 17 .…”

High‐Throughput Screening of Protein‐Detergent Complexes Using Fluorescence Polarization Spectroscopy

“…Steady‐state FP spectroscopy enables the examination of changes in the rotational mobility of a fluorescently labeled protein (Kwok & Cheung, ; Rossi & Taylor, ; Stoddart, White, Nguyen, Hill, & Pfleger, ; Turman, Nathanson, Stockbridge, Street, & Miller, ). This approach can be conducted by exciting a chemically attached fluorophore with plane‐polarized light (Moerke, ; Swonger & Robinson, ). Let us assume that the labeled membrane protein binds to detergent monomers, leading to the formation of a protein‐detergent complex (PDC), also called a proteomicelle.…”