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 K_D 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 K_D’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.