Probing the Conformational Space of the Cannabinoid Receptor 2 and a Systematic Investigation of DNP-Enhanced MAS NMR Spectroscopy of Proteins in Detergent Micelles

Tremendous progress has been made in determining the structures of G-protein coupled receptors (GPCR) and their complexes in recent years. However, understanding activation and signaling in GPCRs is still challenging due to the role of protein dynamics in these processes. Here, we show how dynamic nuclear polarization (DNP)-enhanced magic angle spinning nuclear magnetic resonance in combination with a unique pair labeling approach can be used to study the conformational ensemble at specific sites of the cannabinoid receptor 2. To improve the signal-to-noise, we carefully optimized the DNP sample conditions and utilized the recently introduced AsymPol-POK as a polarizing agent. We could show qualitatively that the conformational space available to the protein backbone is different in different parts of the receptor and that a site in TM7 is sensitive to the nature of the ligand, whereas a site in ICL3 always showed large conformational freedom.

Automated summary

Tremendous progress has been made in determining the structures of G-protein coupled receptors (GPCRs) and their complexes, but understanding their activation and signaling remains challenging due to protein dynamics. In this study, dynamic nuclear polarization (DNP)-enhanced magic angle spinning nuclear magnetic resonance was used to study the conformational ensemble of cannabinoid receptor 2 (CB2) at specific sites. The results showed that the conformational space of the protein backbone differs in different parts of the receptor, and a site in TM7 is sensitive to the ligand nature, while a site in ICL3 always exhibits large conformational freedom.