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APPLICATION NOTE
©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
The receptor:#17:LMNG complex preferred a neutral
and slightly alkaline pH buff er (∆T
m
+ 1.7 °C). The
transition was lost in low pH buff er < 5.5. Notably, the
receptor complex was most stable in the presence of
high sodium chloride concentration (∆T
m
+ 2.6 °C).
Conclusion
In this case study, we demonstrate the usage of the
NanoTemper Technologies instrument Prometheus
NT.48 in defi ning thermal unfolding properties of two
class A GPCRs for further ligand screening, biophysical
characterization and structural biology.
The system was validated with human adenosine
A2AR-BRIL fusion protein by detecting changes in
intrinsic tryptophan fl uorescence at two wavelengths.
T
m
values of the receptor: ligand complexes could be
determined for the majority of the selected ligands. A
small portion of the selected ligands showed strong
autofl uorescence or fl uorescence quenching and was
not suitable for nanoDSF. Overall, the determined T
m
values show a good agreement with published and
in-house data with commonly used CPM-DSF assays.
Sample consumption and assay time is comparable
to standard CPM-DSF assays. A major advantage of
the nanoDSF technology compared to CPM-DSF assay
is the label-free assay format and straightforward
assay development.
We successfully applied nanoDSF for a test case
where the standard CPM-DSF assay did not yield
reliable and accurate determinations of T
m
values.
Using nanoDSF, we were able to confi rm a rank
order of ligands verifi ed with in-house radioligand
competition and Ca
2+
-FLIPR functional assays.
We could further improve the receptor stability
by including a matrix buff er screen in our assay
development for the class A test-GPCR.
compounds show a pronounced increase in
thermostability of the receptor (compound #17, #19
and #22). Unexpectedly, compounds #10 and #16 did
not show a strong eff ect on the thermostability of the
test-GPCR despite their high aff inity in the Ca
2+
-FLIPR
assay.
We wanted to further optimize the buff er conditions
in the presence of compound #17 which showed the
highest ∆T
m
in our nanoDSF assay. We constructed
a limited matrix screen with diff erent pH values and
sodium chloride concentrations (Figure 8 and Table 3).
Figure 8: Buff er composition and osmolality screen for human test-GPCR.
Receptor was purifi ed in presence of 10 µM compound #17. Best buff er condition
∆T
m
2.6 °C. Receptor-detergent micelles apparently prefer a high osmotic buff er
and slightly alkaline buff er.
