The function of guanine nucleotide binding (G) proteins is Mg2+ dependent with guanine nucleotide exchange requiring higher metal ion concentration than guanosine 5'-triphosphate hydrolysis. it is unclear whether two Mg2+ binding sites are present or if one Mg2+ binding site exhibits different affinities for the inactive GDP-bound or the active GTP-bound conformations. We used furaptra, a Mg2+-specific fluorophore, to investigate Mg2+ binding to alpha subunits in both conformations of the stimulatory (G(s)alpha) and inhibitory (G(i)alpha(1)) regulators of adenylyl cyclase. Regardless of the conformation or alpha protein studied, we found that two distinct Mg2+ sites were present with dissimilar affinities. With the exception of G(s)alpha(1) in the active conformation, cooperativity, between the two Mg2+ sites was also observed. Whereas the high affinity Mg2+ site corresponds to that observed in published X-ray structures of G proteins, the low affinity Mg2+ site may involve coordination to the terminal phosphate of the nucleotide. (c) 2008 Elsevier Inc. All rights reserved.