Motion of Carboxyl Terminus of Gα Is Restricted upon G Protein Activation: A SOLUTION NMR STUDY USING SEMISYNTHETIC Gα SUBUNITS

• Published in: The Journal of biological chemistry 2005-09, Vol.280 (35), p.31019-31026
• Main Authors: Anderson, Lori L., Marshall, Garland R., Crocker, Evan, Smith, Steven O., Baranski, Thomas J.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M503690200

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 13 C 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 13 C 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 13 C-labeled free peptide. Upon titration with increasing concentrations of AlF 4 − , the 13 C resonances demonstrated a marked loss of signal intensity in the semisynthetic Gα subunit but not in free peptide subjected to the same conditions. Because AlF 4 − 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 AlF 4 − .