Designing point mutants to detect structural coupling in a heterotrimeric G protein alpha-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 alpha-subunit (G(alpha)) to the guanine nucleotide-binding pocket, we generated and characterized mutant forms of G(alpha) 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(talpha)/G(i1alpha) 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(alpha) 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(talpha) 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(alpha) can effect structural changes that may compromise receptor interactions and GDP/GTP exchange.
Abdulaev
Najmoutin G
NG
National Institute of Standards and Technology, Rockville, MD, USA.
Mao
Xiang
X
Ramon
Eva
E
Ngo
Tony
T
Mysliwy
Justyna
J
Marino
John P
JP
Ridge
Kevin D
KD
D017607:Aluminum Compounds; D005456:Fluorescent Dyes / Q000737:chemistry; D005459:Fluorides; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D017354:Point Mutation / Q000235:genetics*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry
D017607:Aluminum Compounds; D005456:Fluorescent Dyes; D044385:GTP-Binding Protein alpha Subunits; D011994:Recombinant Proteins; D014364:Tryptophan; D005459:Fluorides; C032311:aluminum fluoride