Designing Point Mutants to Detect Structural Coupling in a Heterotrimeric G Protein α-subunit by NMR Spectroscopy

To better understand the mechanism by which the activating signal is transmitted from the receptor‐interacting regions on the G protein α‐subunit (Gα) to the guanine nucleotide‐binding pocket, we generated and characterized mutant forms of Gα with alterations in switch II (Trp‐207→Phe) and the carboxyl‐terminus (Phe‐350→Ala). Previously reported bacterial expression methods for the high‐level production of a uniformly isotope‐labeled Gtα/Gi1α chimera, ChiT, were successfully used to isolate milligram quantities of 15N‐labeled mutant protein. NMR analysis showed that while the GDP/Mg2+‐bound state of both mutants shared an overall conformation similar to that of the GDP/Mg2+‐bound state of ChiT, formation of the “transition/activated” state in the presence of aluminum fluoride (AlF4−) revealed distinct differences between the wild‐type and mutant Gα subunits, particularly in the response of the 1HN, 15N cross‐peak for the Trp‐254 indole in the Trp‐207→Phe mutant and the 1HN, 15N cross‐peak for Ala‐350 in the Phe‐350→Ala mutant. Consistent with the NMR data, the F350→Ala mutant showed an increase in intrinsic fluorescence that was similar to Gtα and ChiT upon formation of the “transition/activated” state in the presence of AlF4−, whereas the intrinsic fluorescence of the Trp‐207→Phe mutant decreased. These results show that the substitution of key amino acid positions in Gα can effect structural changes that may compromise receptor interactions and GDP/GTP exchange.