Abstract
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.