Trp fluorescence reveals an activation-dependent cation-pi interaction in the Switch II region of Galphai proteins.
Crystal structures of Galpha(i) (and closely related family member Galpha(t)) reveal much of what we currently know about G protein structure, including changes which occur in Switch regions. Galpha(t) exhibits a low rate of basal (uncatalyzed) nucleotide exchange and an ordered Switch II region in the GDP-bound state, unlike Galpha(i), which exhibits higher basal exchange and a disordered Switch II region in Galpha(i)GDP structures. Using purified Galpha(i) and Galpha(t), we examined the intrinsic tryptophan fluorescence of these proteins, which reports conformational changes associated with activation and deactivation of Galpha proteins. In addition to the expected enhancement in tryptophan fluorescence intensity, activation of GalphaGDP proteins was accompanied by a modest but notable red shift in tryptophan emission maxima. We identified a cation-pi interaction between tryptophan and arginine residues in the Switch II of Galpha(i) family proteins that mediates the observed red shift in emission maxima. Furthermore, amino-terminal myristoylation of Galpha(i) resulted in a less polar environment for tryptophan residues in the GTPase domain, consistent with an interaction between the myristoylated amino terminus and the GTPase domain of Galpha proteins. These results reveal unique insights into conformational changes which occur upon activation and deactivation of G proteins in solution.
Hamm
Heidi E
HE
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
Meier
Scott M
SM
Liao
Guihua
G
Preininger
Anita M
AM
D001120:Arginine / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D019814:Myristic Acid / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics*; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan / Q000378:metabolism*
D019814:Myristic Acid; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014364:Tryptophan; D001120:Arginine; D019206:GTP-Binding Protein alpha Subunits, Gi-Go