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J. Biol. Chem., Vol. 281, Issue 51, 39708-39718, December 22, 2006

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Influence of Agonists and Antagonists on the Segmental Motion of Residues near the Agonist Binding Pocket of the Acetylcholine-binding Protein^*

Ryan E. Hibbs, Zoran Radi, Palmer Taylor, and David A. Johnson^¶1

From the Department of Pharmacology and the Biomedical Sciences Graduate Program, University of California San Diego, La Jolla, California 92093 and the ^¶Division of Biomedical Sciences, University of California Riverside, Riverside, Calfornia 92521

Using the Lymnaea acetylcholine-binding protein as a surrogate of the extracellular domain of the nicotinic receptor, we combined site-directed labeling with fluorescence spectroscopy to assess possible linkages between ligand binding and conformational dynamics. Specifically, 2-[(5-fluoresceinyl)aminocarbonyl]ethyl methanethiosulfonate was conjugated to a free cysteine on loop C and to five substituted cysteines at strategic locations in the subunit sequence, and the backbone flexibility around each site of conjugation was measured with time-resolved fluorescence anisotropy. The sites examined were in loop C (Cys-188 using a C187S mutant), in the 9 strand (T177C), in the 10 strand (D194C), in the 8-9 loop (N158C and Y164C), and in the 7 strand (K139C). Conjugated fluorophores at these locations show distinctive anisotropy decay patterns indicating different degrees of segmental fluctuations near the agonist binding pocket. Ligand occupation and decay of anisotropy were assessed for one agonist (epibatidine) and two antagonists (-bungarotoxin and d-tubocurarine). The Y164C and Cys-188 conjugates were also investigated with additional agonists (nicotine and carbamylcholine), partial agonists (lobeline and 4-hydroxy,2-methoxy-benzylidene anabaseine), and an antagonist (methyllycaconitine). With the exception of the T177C conjugate, both agonists and antagonists perturbed the backbone flexibility of each site; however, agonist-selective changes were only observed at Y164C in loop F where the agonists and partial agonists increased the range and/or rate of the fast anisotropy decay processes. The results reveal that agonists and antagonists produced distinctive changes in the flexibility of a portion of loop F.

Received for publication, May 17, 2006 , and in revised form, October 24, 2006.

^* This work was supported by National Institutes of Health Grant R37-GM18360 (to P. T.) and a pre-doctoral fellowship from the Pharmaceutical Research Manufacturers Association Foundation (to R. E. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Division of Biomedical Sciences, B605 Statistics Rd., University of California Riverside, Riverside, CA 92521-0121. Tel.: 951-827-3831; Fax: 951-827-5504; E-mail: david.johnson{at}ucr.edu.

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