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Isolation and functional characterization of a stable complex between photoactivated rhodopsin and the G protein, transducin

Beata Jastrzebska^*,1, Marcin Golczak^*, Dimitrios Fotiadis^,, Andreas Engel and Krzysztof Palczewski

^* Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA;

M.E. Müller Institute for Microscopy, Biozentrum, University of Basel, Basel, Switzerland; and

Institute of Biochemistry and Molecular Medicine, University of Berne, Berne, Switzerland

¹Correspondence: Department of Pharmacology, School of Medicine, Case Western Reserve University, Wood Bldg., 10900 Euclid Ave., Cleveland, OH 44106-4965, USA. E-mail: bxj27{at}case.edu

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-β-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 GTPS. 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, B., Golczak, M., Fotiadis, D., Engel, A., and Palczewski, K.. Isolation and functional characterization of a stable complex between photoactivated rhodopsin and the G protein, transducin.

Key Words: sucrose gradient • detergent • rhodopsin regeneration

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