Rapid irreversible G protein alpha subunit misfolding due to intramolecular kinetic bottleneck that precedes Mg2+ "lock" after GTP/GDP exchange

Stoichiometric exchange of GTP for GDP on heterotrimeric G protein alpha (G (alpha)) subunits is essential to most hormone and neurotransmitter initiat ed signal transduction. G(alpha)s are stably activated in a Mg2+ complex wi th GTP gammaS, a nonhydrolyzable GTP analogue that is reported to bind G(al pha) with very high affinity. Yet, it is common to find that substantial am ounts (30-90%) of purified G proteins cannot be activated. Inactivatable G protein has heretofore been thought to have become "denatured" during forma tion of the obligatory nucleotide-free or empty (MT) G(alpha)-state that is intermediary to GDP/GTP exchange at a single binding site. We find G(alpha ) native secondary and tertiary structure to persist during formation of th e irreversibly inactivatable state of transducin. MT G(alpha) is therefore irreversibly misfolded rather than denatured. Inactivation by misfolding is found to compete kinetically with protective but weak preequilibrium nucle otide binding at micromolar ambient GTP gammaS concentrations. Because of t he weak preequilibrium, quantitative protection against G(alpha) aggregatio n is only achieved at free nucleotide concentrations 10- 100 times higher t han those commonly employed in G protein radio-nucleotide binding studies. Initial GTP protection is also poor because of the extreme slowness of an i ntramolecular G refolding step (isomerization) necessary for GTP sequestrat ion after its weak preequilibrium binding. Of the two slowly interconvertin g G(alpha). GTP isomers described here, only the second can bind Mg2+, "loc king" GTP in place with a large net rise in GTP binding affinity. A compani on G(alpha). GDP isomerization reaction is identified as the cause of the v ery slow spontaneous GDP dissociation that characterizes G protein nucleoti de exchange and low spontaneous background activity in the absence of GPCR activation. G(alpha). GDP and G(alpha). GTP isomerization reactions are pro posed as the dual target for GPCR catalysis of nucleotide exchange.