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

Lori L. Anderson, Garland R. Marshall, Evan Crocker, Steven O. Smith, Thomas J. Baranski

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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 Gαi1 subunits lacking the corresponding carboxyl-terminal residues. In a receptor-G protein reconstitution assay, the truncated Gαi1 subunit could not be activated by receptor; whereas the semisynthetic protein demonstrated functionality that was comparable with recombinant Gαi1. 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 Gαi1: 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-.

Original languageEnglish
Pages (from-to)31019-31026
Number of pages8
JournalJournal of Biological Chemistry
Issue number35
StatePublished - Sep 2 2005


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