Motion of carboxyl terminus of Gα is restricted upon G protein activation: A solution NMR study using semisynthetic Gα subunits

The carboxyl terminus of the G protein α subunit plays a key role in interactions with G protein-coupled receptors. Previous studies that have incorporated covalently attached probes have demonstrated that the carboxyl terminus undergoes conformational changes upon G protein activation. To examine the conformational changes that occur at the carboxyl terminus of Gα subunits upon G protein activation in a more native system, we generated a semisynthetic Gα subunit, site-specifically labeled in its carboxyl terminus with ¹³C amino acids. Using expressed protein ligation, 9-mer peptides were ligated to recombinant Gα_i1 subunits lacking the corresponding carboxyl-terminal residues. In a receptor-G protein reconstitution assay, the truncated Gα_i1 subunit could not be activated by receptor; whereas the semisynthetic protein demonstrated functionality that was comparable with recombinant Gα_i1. To study the conformation of the carboxyl terminus of the semisynthetic G protein, we applied high resolution solution NMR to Gα subunits containing ¹³C labels at the corresponding sites in Gα_i1: Leu-348 (uniform), Gly-352 (α carbon), and Phe-354 (ring). In the GDP-bound state, the spectra of the ligated carboxyl terminus appeared similar to the spectra obtained for ¹³C-labeled free peptide. Upon titration with increasing concentrations of AlF₄^-, the ¹³C resonances demonstrated a marked loss of signal intensity in the semisynthetic Gα subunit but not in free peptide subjected to the same conditions. Because AlF₄^- complexes with GDP to stabilize an activated state of the Gα subunit, these results suggest that the Gα carboxyl terminus is highly mobile in its GDP-bound state but adopts an ordered conformation upon activation by AlF₄^-.