nanoDSF: Label-free thermal unfolding assay of G Protein-coupled receptors for compound screening and buffer composition optimization

November 9, 2018

A thermal unfolding based assay using low volume differential intrinsic tryptophan scanning fluorimetry (nanoDSF) was applied to study the stabilizing effects 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 caffeine) and synthetic ligands with different affinities to mediate a variety of physiological and pharmacological responses. Several well characterized ligands were used for the unfolding experiments. The ΔTm shift 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 buffer 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 stability for biophysical assays by screening buffer conditions.

Previous Video
Did you say, “membrane proteins”? Challenge accepted!
Did you say, “membrane proteins”? Challenge accepted!

Working with difficult targets like GPCRs and other membrane proteins? Because they live in the cell memb...

Up next
Lipids activate SecA for high affinity binding to the SecYEG complex
Lipids activate SecA for high affinity binding to the SecYEG complex

Working with tricky membrane proteins?

NanoTemper tools can help

Find out how

Sign up to receive
the latest NanoTemper news, product updates, event announcements and more

First Name
Last Name
Company Name
Agree to Subscribe & Privacy Policy*
*I have fully read, understood and agree to the Privacy Policy. I accept the storing and processing of my personal data by NanoTemper as described in the Privacy Policy.

By completing this form, you provide us consent to contact you with educational content, news and information about our products, services and events. You may unsubscribe at any time.
Thank you!
Error - something went wrong!