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Conformations of the rhodopsin third cytoplasmic loop grafted onto bacteriorhodopsin

Heymann, J. B. and Pfeiffer, M. and Hildebrandt, V. and Kaback, H. R. and Fotiadis, D. and Groot, B. and Engel, A. and Oesterhelt, D. and Muller, D. J.. (2000) Conformations of the rhodopsin third cytoplasmic loop grafted onto bacteriorhodopsin. Structure: with folding and design, Vol. 8, H. 6. pp. 643-653.

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Official URL: http://edoc.unibas.ch/dok/A5257666

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Abstract

BACKGROUND: The third cytoplasmic loop of rhodopsin (Rho EF) is important in signal transduction from the retinal in rhodopsin to its G protein, transducin. This loop also interacts with rhodopsin kinase, which phosphorylates light-activated rhodopsin, and arrestin, which displaces transducin from light-activated phosphorylated rhodopsin. RESULTS: We replaced eight residues of the EF loop of bacteriorhodopsin (BR) with 24 residues from the third cytoplasmic loop of bovine Rho EF. The surfaces of purple membrane containing the mutant BR (called IIIN) were imaged by atomic force microscopy (AFM) under physiological conditions to a resolution of 0.5-0.7 nm. The crystallinity and extracellular surface of IIIN were not perturbed, and the cytoplasmic surface of IIIN increased in height compared with BR, consistent with the larger loop. Ten residues of Rho EF were excised by V8 protease, revealing helices E and F in the AFM topographs. Rho EF was modeled onto the BR structure, and the envelope derived from the AFM data of IIIN was used to select probable models. CONCLUSIONS: A likely conformation of Rho EF involves some extension of helices E and F, with the tip of the loop lying over helix C and projecting towards the C terminus. This is consistent with mutagenesis data showing the TTQ transducin-binding motif close to loop CD, and cysteine cross-linking data indicating the C-terminal part of Rho EF to be close to the CD loop.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Structural Biology (Engel)
UniBasel Contributors:Engel, Andreas H
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:Current Biology
ISSN:0969-2126
Note:Publication type according to Uni Basel Research Database: Journal article
Last Modified:22 Mar 2012 14:20
Deposited On:22 Mar 2012 13:18

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