NMR chemical shift changes can report on the functional dynamics of biomacromolecules in solution with sizes >1 MDa. However, their interpretation requires chemical shift assignments to individual nuclei, which for large molecules often can only be obtained by tedious point mutations that may interfere with function.
Recently we in silico predicted and in practice created a global positioning system (GPS) that relies on pseudocontact shifts (PCSs) induced by paramagnetic thulium tag attached at various sites to an antibody in order to precisely position magnetic nuclei at distances >60 Å within a protein of interest1. This information is subsequently used to obtain the NMR assignment for the nuclei. Using the GPS-PCS method, we successfully assigned all 1H-15N-valine and tyrosine resonances of a G protein-coupled β1-adrenergic receptor (β1AR) in various functional forms, including the apo state, antagonist-bound inactive state, agonist-bound preactive state, and the active state.
We have now carried an in-depth analysis of the completely assigned 1H-15N valine and tyrosine β1AR backbone resonances in these different functional forms. The resonance shifts reveal backbone perturbations within β1AR across multiple states at unprecedented detail. We will discuss pertinent results. In particular, we show that under full agonist binding the entire extracellular region and parts of the β1AR center undergo slow exchange between two conformations (preactive and active). Furthermore, activation of β1AR by the G protein mimicking Nb80 induces very strong 1H-15N shift perturbations in transmembrane helix 3 (TM3), which only experiences minor structural changes in crystal structures. Thus TM3 appears as a stabilizing hub absorbing the forces exerted by receptor activation in its electronic structure, an important mechanism that is likely conserved within all class A GPCRs.