Motion of carboxyl terminus of Gα is restricted upon G protein activation

The carboxyl terminus of the G protein alpha subunit plays a key role in interactions with G protein- coupled receptors. Previous studies that have incorporated co-valently 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 G alpha subunits upon G protein activation in a more native system, we generated a semisynthetic G alpha subunit, site-specifically labeled in its carboxyl terminus with C-13 amino acids. Using expressed protein ligation, 9- mer peptides were ligated to recombinant G alpha(i1) subunits lacking the corresponding carboxyl- terminal residues. In a receptor- G protein reconstitution assay, the truncated G alpha(i1) subunit could not be activated by receptor; whereas the semisynthetic protein demonstrated functionality that was comparable with recombinant G alpha(i1). To study the conformation of the carboxyl terminus of the semisynthetic G protein, we applied high resolution solution NMR to G alpha subunits containing 13C labels at the corresponding sites in G alpha(i1): Leu- 348 ( uniform), Gly-352 ( alpha carbon), and Phe- 354 ( ring). In the GDP- bound state, the spectra of the ligated carboxyl terminus appeared similar to the spectra obtained for C-13- labeled free peptide. Upon titration with increasing concentrations of AlF4-, the C-13 resonances demonstrated a marked loss of signal intensity in the semisynthetic G alpha subunit but not in free peptide subjected to the same conditions. Because AlF4- complexes with GDP to stabilize an activated state of the G alpha subunit, these results suggest that the G alpha carboxyl terminus is highly mobile in its GDP- bound state but adopts an ordered conformation upon activation by AlF4-.