Premium
Designing Point Mutants to Detect Structural Coupling in a Heterotrimeric G Protein α‐subunit by NMR Spectroscopy †
Author(s) -
Abdulaev Najmoutin G.,
Mao Xiang,
Ramon Eva,
Ngo Tony,
Mysliwy Justyna,
Marino John P.,
Ridge Kevin D.
Publication year - 2009
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.2008.00522.x
Subject(s) - chemistry , mutant , heterotrimeric g protein , g protein , guanine , protein subunit , stereochemistry , gtp' , nuclear magnetic resonance spectroscopy , biochemistry , nucleotide , receptor , gene , enzyme
To better understand the mechanism by which the activating signal is transmitted from the receptor‐interacting regions on the G protein α‐subunit (G α ) to the guanine nucleotide‐binding pocket, we generated and characterized mutant forms of G α 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 G tα /G i1α chimera, ChiT, were successfully used to isolate milligram quantities of 15 N‐labeled mutant protein. NMR analysis showed that while the GDP/Mg 2+ ‐bound state of both mutants shared an overall conformation similar to that of the GDP/Mg 2+ ‐bound state of ChiT, formation of the “transition/activated” state in the presence of aluminum fluoride (AlF 4 − ) revealed distinct differences between the wild‐type and mutant G α subunits, particularly in the response of the 1 HN, 15 N cross‐peak for the Trp‐254 indole in the Trp‐207→Phe mutant and the 1 HN, 15 N 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 G tα and ChiT upon formation of the “transition/activated” state in the presence of AlF 4 − , whereas the intrinsic fluorescence of the Trp‐207→Phe mutant decreased. These results show that the substitution of key amino acid positions in G α can effect structural changes that may compromise receptor interactions and GDP/GTP exchange.