2
APPLICATION NOTE
©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
Biophysical results using samples of GPCRs, in
general, are diff icult to obtain because of the
receptors' fl exibility and their conformational
heterogeneity. For biophysical and structural
characterization studies, GPCRs need to be extracted
stability for biophysical assays by screening buff er
conditions.
Introduction
G protein-coupled receptors (GPCRs) are implicated
in numerous diseases and are the target of many
drug treatments. Obtaining suff icient quantities of
purifi ed receptors for biophysical and structural
characterization has been a challenge, and is
still proving to be diff icult. Until recently, of the
almost thousand proteins in this family, only a few
crystallographic structures have been solved
(Figure 1) [1].
Figure 1: Structure of the human A2AR bound with the potential Parkinson's
drug ZM241385 (blue). Several lipid molecules are seen in the X-ray structure
(red). Four native disulfi de bonds are also indicated (yellow). The seven
transmembrane helices are colored in brown; the extracellular and intracellular
loops are shown in pink and turquoise, respectively.
Figure 2: Use of nanoDSF in early lead discovery, screening assays and structural
biology. A. Biophysical assays used in the early lead discovery process. B. Typical
construct optimization cycle diagram of a GPCR target for structural biology. The
nanoDSF assay can be used for various points in in vitro screening of compounds
and construct selection and optimization for biochemical and biophysical assays
such as X-ray crystallography (examples of assay possibilities are marked as *).
from a lipid environment using surfactants and
purifi ed to homogeneity in the presence of stabilizing
surfactants and a suitable buff er environment.
Receptors can also be rapidly denatured upon
concentration in absence of a high aff inity ligand.
Typically, receptor variants, surfactants, buff er
compositions (pH, additives, lipids) and ligands are
screened to optimize receptor stability for biophysical
and structural analysis as well as lead discovery
(Figure 2). Here we use the label-free diff erential
scanning fl uorimetry (nanoDSF) of the Prometheus
NT.48 to determine the thermal stability of two GPCRs
in the presence of a variety of diff erent ligands and in
a broad range of buff er systems.
