Tryptophan W207 in transducin T alpha is the fluorescence sensor of the G protein activation switch and is involved in the effector binding.
We have produced a recombinant transducin alpha subunit (rT alpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N-terminal increased the solubility and allowed the purification of rT alpha. When reconstituted with excess T beta gamma on retinal membrane, rT alpha displayed functional characteristics of wild-type T alpha vis à vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]T alpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor-catalysed GTP gamma S binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector-activating interaction we measured the affinity of [W207F]T alpha GDP-AlFx for PDE gamma, the effector subunit that binds most tightly to T alpha. [W207F]T alpha still bound in an activation-dependent way to PDE gamma, but with a 100-fold lower affinity than rT alpha. This suggests that W207 contributes to the G protein effector binding.
Faurobert
E
E
CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France.
Otto-Bruc
A
A
Chardin
P
P
Chabre
M
M
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism; D017607:Aluminum Compounds / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D005453:Fluorescence; D005459:Fluorides / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D015293:Transducin / Q000235:genetics / Q000378:metabolism*; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*
D017607:Aluminum Compounds; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D014364:Tryptophan; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D019204:GTP-Binding Proteins; D015293:Transducin; D005459:Fluorides; C032311:aluminum fluoride