期刊
FASEB JOURNAL
卷 23, 期 2, 页码 371-381出版社
WILEY
DOI: 10.1096/fj.07-114835
关键词
sucrose gradient; detergent; rhodopsin regeneration
资金
- U. S. National Institutes of Health [EY09339, GM 079191, P30 EY11373]
- Swiss National Foundation (SNF) [3100A0 - 108299]
- Amgen Inc.
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), Rhoe*-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 GTP gamma S. 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 Gte 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. FASEB J. 23, 371-381 (2009)
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