Biochemical characteristics of guanine nucleotide binding protein alpha-subunit recombinant protein and three mutants: investigation of a domain motion involved in GDP-GTP exchange.
Our previous studies using molecular dynamics have shown a hinge bending motion between the helical and the GTPase domains of GalphaT (Mello et al., 1998). The hypothesis that this motion is allowed by residues Gly56 and Gly179 and that this motion may affect the ligand exchange was tested in this work. Mutations of Gly 56 were carried out and the mutant proteins were expressed in Sf9 cells using the Baculovirus expression system. The recombinant proteins were purified using Ni-NTA affinity chromatography. The results for the (GDP/GTP) exchange assays showed that G56S and double mutants (D55G/G56S) proteins differ significantly from the wild type and D55G mutant forms. The Kd values for GTPgammaS binding of those mutants have decreased by approximately 10-fold. No difference in the GTPase activity was detected for the mutants. Thus, the biochemical results obtained support the conclusions of the computational studies.
Mello
L V
LV
School of Biochemistry and Molecular Biology, University of Leeds, UK. mello@cenargen.embrapa.br
Millner
P A
PA
Findlay
J B C
JB
D000818:Animals; D002417:Cattle; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D009154:Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D015293:Transducin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D015293:Transducin