Folding of G_α Subunits: Implications for Disease States

G-proteins play a central role in signal transduction by fluctuating between "on" and "off" phases that are determined by a conformational change. cAMP is a secondary messenger whose formation is inhibited or stimulated by activated G_iα1 or G_sα subunit. We used tryptophan fluorescence, UV/vis spectrophotometry, and circular dichroism to probe distinct structural features within active and inactive conformations from wild-type and tryptophan mutants of G_iα1 and G_sα. For all proteins studied, we found that the active conformations were more stable than the inactive conformations, and upon refolding from higher temperatures, activated wild-type subunits recovered significantly more native structure. We also observed that the wild-type subunits partially regained the ability to bind nucleotide. The increased compactness observed upon activation was consistent with the calculated decrease in solvent accessible surface area for wild-type G_iα1. We found that as the temperature increased, G_α subunits, which are known to be rich in α-helices, converted to proteins with increased content of β-sheets and random coil. For active conformations from wild-type and tryptophan mutants of G_iα1, melting temperatures indicated that denaturation starts around hydrophobic tryptophan microenvironments and then radiates toward tyrosine residues at the surface, followed by alteration of the secondary structure. For G_sα, however, disruption of secondary structure preceded unfolding around tyrosine residues. In the active conformations, a π-cation interaction between essential arginine and tryptophan residues, which was characterized by a fluorescence-measured red shift and modeled by molecular dynamics, was also shown to be a contributor to the stability of G_α subunits. The folding properties of G_α subunits reported here are discussed in the context of diseases associated to G-proteins.