Isolation and functional characterization of a stable complex between photoactivated rhodopsin and the G protein, transducin.
Transitory binding between photoactivated rhodopsin (Rho* or Meta II) and the G protein transducin (Gt-GDP) is the first step in the visual signaling cascade. Light causes photoisomerization of the 11-cis-retinylidene chromophore in rhodopsin (Rho) to all-trans-retinylidene, which induces conformational changes that allow Gt-GDP to dock onto the Rho* surface. GDP then dissociates from Gt, leaving a transient nucleotide-empty Rho*-Gt(e) complex before GTP becomes bound, and Gt-GTP then dissociates from Rho*. Further biochemical advances are required before structural studies of the various Rho*-Gt complexes can be initiated. Here, we describe the isolation of n-dodecyl-beta-maltoside solubilized, stable, functionally active, Rho*-Gt(e), Rho(e)*-Gt(e), and 9-cis-retinal/11-cis-retinal regenerated Rho-Gt(e) complexes by sucrose gradient centrifugation. In these complexes, Rho* spectrally remained in its Meta II state, and Gt(e) retained its ability to interact with GTPgammaS. Removal of all-trans-retinylidene from Rho*-Gt(e) had no effect on the stability of the Rho(e)*-Gt(e) complex. Moreover, opsin in the Rho(e)*-Gt(e) complex with an empty nucleotide-binding pocket in Gt and an empty retinoid-binding pocket in Rho was regenerated up to 75% without complex dissociation. These results indicate that once Rho* couples with Gt, the chromophore plays a minor role in stabilizing this complex. Moreover, in complexes regenerated with 9-cis-retinal/11-cis-retinal, Rho retains a conformation similar to Rho* that is stabilized by Gt(e) apo-protein.
Jastrzebska
Beata
B
Department of Pharmacology, School of Medicine, Case Western Reserve University, Wood Bldg., 10900 Euclid Ave., Cleveland, OH 44106-4965, USA. bxj27@case.edu
Golczak
Marcin
M
Fotiadis
Dimitrios
D
Engel
Andreas
A
Palczewski
Krzysztof
K
D000818:Animals; D002417:Cattle; D046529:Microscopy, Electron, Transmission; D010777:Photochemistry; D011485:Protein Binding; D012243:Rhodopsin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000648:ultrastructure; D012995:Solubility; D015293:Transducin / Q000302:isolation & purification* / Q000378:metabolism* / Q000648:ultrastructure
D012243:Rhodopsin; D015293:Transducin