Acrylodan-conjugated Cysteine Side Chains Reveal Conformational State and Ligand Site Locations of the Acetylcholine-binding Protein *

摘要: We undertook cysteine substitution mutagenesis and fluorophore conjugation at selected residue positions to map sites of ligand binding changes in solvent exposure the acetylcholine-binding protein from Lymnaea stagnalis, a nicotinic receptor surrogate. Acrylodan fluorescence emission is highly sensitive its local environment, when bound protein, exhibits both intensity wavelength that are reflected degree exclusion effective dielectric constant environment fluorophore. Hence, mutants were generated based on crystal structure predicted sites, parameters assayed acrylodan-conjugated proteins. This approach allows one analyze around conjugated side chain induced by ligand. Introduction an acrylodan-cysteine conjugate position 178 yields large blue shift with α-bungarotoxin association, whereas agonists alkaloid antagonists induce red shifts reflecting this position. Such residue-selective suggest certain ligands can distinct conformational states mutually exclusive results disparate portals entry orientations α-toxin smaller acetylcholine congeners pocket. Labeling other pocket also reveals distinctive spectral for α-bungarotoxin, agonists, antagonists.