APPLICATION NOTE
Thermal Unfolding of GPCRs
nanoDSF: Label-free Thermal Unfolding Assay of G Protein-Coupled Receptors for
Compound Screening and Buffer Composition Optimization
Matthias Haff ke
1
, Gabriele Rummel
1
, Jacques Boivineau
1
,
Anna Münch
2
and Veli-Pekka Jaakola
1
1
Center for Proteomic Chemistry, Novartis Institutes
for Biomedical Research, Basel, Switzerland
2
NanoTemper Technologies GmbH Munich, Germany
Abstract
A thermal unfolding based assay using low volume diff erential intrinsic tryptophan
scanning fl uorimetry (nanoDSF) was applied to study the stabilizing eff ects of
ligands on G protein-coupled receptors (GPCRs). GPCRs are the fourth largest
superfamily in the human genome and are the largest class of targets for drug
discovery. The system has been validated using human adenosine A2A receptor
(A2AR). A2AR binds natural (adenosine and caff eine) and synthetic ligands with
diff erent aff inities to mediate a variety of physiological and pharmacological
responses. Several well-characterized ligands were used for the unfolding
experiments. The ∆T
m
shi values obtained from nanoDSF analysis and traditional
ligand binding studies correlate well with each other. We further characterized a
second human GPCR target (test-GPCR) for which traditional cysteine-reactive DSF
has been problematic. nanoDSF demonstrated that small molecule ligands can
stabilize the detergent-solubilized receptor, thus showing the target GPCR is active
in a selected detergent and lipid-free environment. In addition, we report a buff er
composition screen to further stabilize the receptor in its detergent environment
for biophysical assays.
Based on our results, we show that the nanoDSF technology will allow the
development of an automated screening platform in a label-free environment to
evaluate a large number of compounds for lead discovery and to improve receptor