DIRECT MEASUREMENT OF AGONIST BINDING TO GENETICALLY ENGINEERED PEPTIDES OF THE ACETYLCHOLINE-RECEPTOR BY SELECTIVE T1 NMR RELAXATION

Interactions of four ligands of the nicotinic acetylcholine receptor with genetically engineered peptides have been studied by NMR. A recombinant cholinergic binding site was prepared as a fusion protein between a truncated form of the bacterial protein trpE and a peptide corresponding to the sequence α 184–200 from the Torpedo califomica receptor. This construct binds α-bungarotoxin while the trpE protein alone does not, and thus serves as a negative control [Aronheim, A., Eshel, Y., Mosckovitz, R., & Gershoni, j. M. (1988) J. Biol. Chem. 263, 9933–9937]. In this study agonist binding to α184-200 is demonstrated by monitoring the T1, relaxation of the ligand’s protons in the presence and absence of the recombinant binding site. This binding is specific as it can be competed with α-bungarotoxin. Quantitative analyses of such competitions yielded the concentration of binding sites, which corresponded to 3.3% and 16.5% of the total protein, for partially purified and affinity-purified α 184–200 constructs, respectively. The KD values for the binding of acetylcholine, nicotine, d-tubocurarine, and gallamine to the affinity-purified construct were 1.4, 1.4, 0.20, and 0.21 mM, respectively, while KD’s with the nontoxin binding protein were all above 10 mM. Thus, this is a direct demonstration that the toxin binding domain α 184–200 may comprise a major component of the cholinergic agonist site. © 1990, American Chemical Society. All rights reserved.