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APPLICATION NOTE
©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
The adenosine A2AR is a class A GPCR, and is
important for neurotransmission, coronary blood
fl ow and respiration. It is blocked by caff eine and is
the subject of much research a er epidemiological
evidence suggested that coff ee drinkers have a lower
risk of Parkinson's disease [2]. Selective compounds
are likely to be useful for the treatment of pain,
cancer, Parkinson's disease and Huntington's
disease [3].
We fi rst validated the nanoDSF assay using a well-
behaving adenosine A2AR variant as a model system.
The wild-type amino acid sequence of A2AR contains
long disordered regions and post-translational
modifi cations. These are important for receptor
functionality and signaling but detrimental for
structural biology due to inherent conformational
fl exibility. There are various modifi ed and stable
A2AR constructs described in the literature. We
used an engineered A2AR - apocytochrome b562RIL
(BRIL) fusion variant rather than agonist/antagonist-
trapped variants because of their preferential binding
bias towards agonized or antagonized states of the
receptor.
A er assay validation we used another GPCR target
as a test case (test-GPCR) and show that a number
of parameters such as ligands, detergents and buff er
additives can be assessed for their eff ectiveness on
enhancing receptor thermostability.
Results
Assay development and validation with
Adenosine A2A receptor
The thermal stability of A2A-BRIL protein in diff erent
detergents was measured using CPM-DSF according
to the method described previously (Figure 3) [4].
From the infl ection point in a sigmoidal curve,
an apparent Tm can be calculated, signifying the
temperature at which 50 % of the A2A-BRIL is
unfolded. CPM dye specifi cally binds to the cysteine
A
B
Figure 3: nanoDSF validation with A2AR. A. A2A-BRIL protein quality used in the
assay development analyzed by SDS-PAGE and SEC. B. Reference CPM-DSF assay
∆T
m
curves of A2A-BRIL in presence of ZM241385 using DDM (51 °C) and LMNG (58
°C) as detergents, beta-Lactoglobulin is shown as reference in DDM (78 °C).
