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Protein Science (2006), 15:1679-1690. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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Bacteriorhodopsin chimeras containing the third cytoplasmic loop of bovine rhodopsin activate transducin for GTP/GDP exchange

Andrew H. Geiser1, Michael K. Sievert1, Lian-Wang Guo1, Jennifer E. Grant1,5, Mark P. Krebs2,4, Dimitrios Fotiadis3, Andreas Engel3 and Arnold E. Ruoho1

1 Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA
2 Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA
3 M.E. Müller Institute for Structural Biology, Biozentrum, University of Basel, CH-4056 Basel, Switzerland

(RECEIVED March 2, 2006; FINAL REVISION April 13, 2006; ACCEPTED April 13, 2006)

The mechanisms by which G-protein-coupled receptors (GPCRs) activate G-proteins are not well understood due to the lack of atomic structures of GPCRs in an active form or in GPCR/G-protein complexes. For study of GPCR/G-protein interactions, we have generated a series of chimeras by replacing the third cytoplasmic loop of a scaffold protein bacteriorhodopsin (bR) with various lengths of cytoplasmic loop 3 of bovine rhodopsin (Rh), and one such chimera containing loop 3 of the human beta2-adrenergic receptor. The chimeras expressed in the archaeon Halobacterium salinarum formed purple membrane lattices thus facilitating robust protein purification. Retinal was correctly incorporated into the chimeras, as determined by spectrophotometry. A 2D crystal (lattice) was evidenced by circular dichroism analysis, and proper organization of homotrimers formed by the bR/Rh loop 3 chimera Rh3C was clearly illustrated by atomic force microscopy. Most interestingly, Rh3C (and Rh3G to a lesser extent) was functional in activation of GTP{gamma}35S/GDP exchange of the transducin {alpha} subunit (G{alpha}t) at a level 3.5-fold higher than the basal exchange. This activation was inhibited by GDP and by a high-affinity peptide analog of the G{alpha}t C terminus, indicating specificity in the exchange reaction. Furthermore, a specific physical interaction between the chimera Rh3C loop 3 and the G{alpha}t C terminus was demonstrated by cocentrifugation of transducin with Rh3C. This G{alpha}t-activating bR/Rh chimera is highly likely to be a useful tool for studying GPCR/G-protein interactions.

Keywords: G-protein-coupled receptors; chimera; bacteriorhodopsin; rhodopsin; transducin; GTP exchange


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