Tryptophan fluorescence reveals conformational changes in the acetylcholine binding protein.
The recent characterization of an acetylcholine binding protein (AChBP) from the fresh water snail, Lymnaea stagnalis, shows it to be a structural homolog of the extracellular domain of the nicotinic acetylcholine receptor (nAChR). To ascertain whether the AChBP exhibits the recognition properties and functional states of the nAChR, we have expressed the protein in milligram quantities from a synthetic cDNA transfected into human embryonic kidney (HEK) cells. The protein secreted into the medium shows a pentameric rosette structure with ligand stoichiometry approximating five sites per pentamer. Surprisingly, binding of acetylcholine, selective agonists, and antagonists ranging from small alkaloids to larger peptides results in substantial quenching of the intrinsic tryptophan fluorescence. Using stopped-flow techniques, we demonstrate rapid rates of association and dissociation of agonists and slow rates for the alpha-neurotoxins. Since agonist binding occurs in millisecond time frames, and the alpha-neurotoxins may induce a distinct conformational state for the AChBP-toxin complex, the snail protein shows many of the properties expected for receptor recognition of interacting ligands. Thus, the marked tryptophan quenching not only documents the importance of aromatic residues in ligand recognition, but establishes that the AChBP will be a useful functional as well as structural surrogate of the nicotinic receptor.
Hansen
Scott B
SB
Department of Pharmacology, University of California, San Diego, La Jolla 92093-0636, USA.
Radic'
Zoran
Z
Talley
Todd T
TT
Molles
Brian E
BE
Deerinck
Tom
T
Tsigelny
Igor
I
Taylor
Palmer
P
D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008195:Lymnaea / Q000737:chemistry*; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry*
D011978:Receptors, Nicotinic; D014364:Tryptophan; D000109:Acetylcholine