Designing Point Mutants to Detect Structural Coupling in a Heterotrimeric G Protein [alpha]-subunit by NMR Spectroscopy[dagger]

• Published in: Photochemistry and photobiology 2009-03, Vol.85 (2), p.431
• Main Authors: Abdulaev, Najmoutin G, Mao, Xiang, Ramon, Eva, Ngo, Tony, Mysliwy, Justyna, Marino, John P, Ridge, Kevin D
• Format: Article
• Language: English
• Subjects: Acids; Changes; Data analysis; Fluorescence; Fusion; Proteins
• ISSN: 0031-8655; 1751-1097

To better understand the mechanism by which the activating signal is transmitted from the receptor-interacting regions on the G protein α-subunit (G^sub α^) to the guanine nucleotide-binding pocket, we generated and characterized mutant forms of G^sub α^ with alterations in switch II (Trp-207 [arrow right] Phe) and the carboxylterminus (Phe-350 [arrow right] Ala). Previously reported bacterial expression methods for the high-level production of a uniformly isotope-labeled G^sub ta^/G^sub i1α^ chimera, ChiT, were successfully used to isolate milligram quantities of ^sup 15^N-labeled mutant protein. NMR analysis showed that while the GDP/Mg^sup 2+^-bound state of both mutants shared an overall conformation similar to that of the GDP/Mg^sup 2+^-bound state of ChiT, formation of the "transition/activated" state in the presence of aluminum fluoride (AlF^sub 4^^sup -^) revealed distinct differences between the wild-type and mutant G^sub α^ subunits, particularly in the response of the ^sup 1^HN, ^sup 15^N cross-peak for the Trp-254 indole in the Trp-207 [arrow right] Phe mutant and the ^sup 1^HN, ^sup 15^N cross-peak for Ala-350 in the Phe-350 [arrow right] Ala mutant. Consistent with the NMR data, the F350 [arrow right] Ala mutant showed an increase in intrinsic fluorescence that was similar to G^sub tα^ and ChiT upon formation of the "transition/activated" state in the presence of AlF^sub 4^^sup -^, whereas the intrinsic fluorescence of the Trp-207 [arrow right] Phe mutant decreased. These results show that the substitution of key amino acid positions in G^sub α^ can effect structural changes that may compromise receptor interactions and GDP/GTP exchange. [PUBLICATION ABSTRACT]