Motion of carboxyl terminus of Galpha is restricted upon G protein activation. A solution NMR study using semisynthetic Galpha subunits.
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 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 Galpha subunits upon G protein activation in a more native system, we generated a semisynthetic Galpha subunit, site-specifically labeled in its carboxyl terminus with 13C amino acids. Using expressed protein ligation, 9-mer peptides were ligated to recombinant Galpha(i1) subunits lacking the corresponding carboxyl-terminal residues. In a receptor-G protein reconstitution assay, the truncated Galpha(i1) subunit could not be activated by receptor; whereas the semisynthetic protein demonstrated functionality that was comparable with recombinant Galpha(i1). To study the conformation of the carboxyl terminus of the semisynthetic G protein, we applied high resolution solution NMR to Galpha subunits containing 13C labels at the corresponding sites in Galpha(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 13C-labeled free peptide. Upon titration with increasing concentrations of AlF4-, the 13C resonances demonstrated a marked loss of signal intensity in the semisynthetic Galpha subunit but not in free peptide subjected to the same conditions. Because AlF4- complexes with GDP to stabilize an activated state of the Galpha subunit, these results suggest that the Galpha carboxyl terminus is highly mobile in its GDP-bound state but adopts an ordered conformation upon activation by AlF4-.
Department of Biochemistry and Molecular Biophysics, Washington University, St. Louis, Missouri 63110, USA.
Lori L
LL
Anderson
Garland R
GR
Marshall
Evan
E
Crocker
Steven O
SO
Smith
Thomas J
TJ
Baranski
D017607:Aluminum Compounds / Q000737:chemistry; D000818:Animals; D006224:Cricetinae; D003412:Cricetulus; D005459:Fluorides / Q000737:chemistry; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D044087:Receptor, Anaphylatoxin C5a / Q000378:metabolism
D017607:Aluminum Compounds; D044385:GTP-Binding Protein alpha Subunits; D010455:Peptides; D044087:Receptor, Anaphylatoxin C5a; C050992:tetrafluoroaluminate; D005459:Fluorides