Designing Point Mutants to Detect Structural Coupling in a Heterotrimeric G Protein α‐subunit by NMR Spectroscopy† |
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Authors: | Najmoutin G Abdulaev Xiang Mao Eva Ramon Tony Ngo Justyna Mysliwy John P Marino Kevin D Ridge |
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Institution: | 1. Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute and National Institute of Standards and Technology, Rockville, MD;2. Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center—Houston, Houston, TX |
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Abstract: | 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. |
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