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APPLICATION NOTE
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The assay clearly picks up the high aff inity ligands
and the main rank order between ∆T
m
and literature
values is maintained.
Test Case: screening compound and buffer conditions for
stability with a class A GPCR (test-GPCR)
We used a second human class A GPCR BRIL
fusion protein as a test case to further verify the
application of nanoDSF in compound and buff er
stability screening. Previously, we were not able to
obtain reliable melting curves for human test-GPCR
constructs using a conventional CPM-DSF assay. The
CPM-DSF assay worked reasonably well with the rat
isoform of test-GPCR using a sample quality similar
to the human receptor. We speculated that the lack
of cysteine residues in the transmembrane network
amino acid which o en resides in the hydrophobic
folds of the protein. Unbound CPM dye is non-
fl uorescent but becomes fl uorescent upon binding
with reactive cysteines.
To validate the nanoDSF technology, we compared
binding of ZM241385, a high aff inity A2AR subtype
specifi c antagonist, to samples without ligand (apo).
As expected, addition of ZM241385 increased the
thermostability of A2A-BRIL by 8.3 °C when compared
to the apo samples (Figure 4 and Table 1). The
stability eff ect is in very good agreement with the
CPM-DSF assay and with published data under similar
conditions. We further tested various commercially
available additional ligands to A2AR, including
antagonists and agonists (Figure 4 and Table 1).
Figure 4: Typical nanoDSF unfolding curves using A2A-BRIL as a reference for class A GPCRs. Recordings of tryptophan fl uorescence at 330 nm are
shown in the top half of the graph and the corresponding fi rst derivative is plotted in the bottom half. Infl ection points (equivalent to the T
m
) are shown
as vertical lines.
