Agonist-mediated Conformational Changes in Acetylcholine-binding Protein Revealed by Simulation and Intrinsic Tryptophan Fluorescence

• Published in: The Journal of biological chemistry Vol. 280; no. 9; pp. 8443 - 8451
• Main Authors: Gao, Fan, Bren, Nina, Burghardt, Thomas P, Hansen, Scott, Henchman, Richard H, Taylor, Palmer, McCammon, J Andrew, Sine, Steven M
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 04.03.2005
• Subjects: Acetylcholine - chemistry; Acrylamide - chemistry; Animals; Binding Sites; Carrier Proteins - agonists; Carrier Proteins - chemistry; Crystallography, X-Ray; Humans; Ligands; Models, Molecular; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Snails; Spectrometry, Fluorescence; Time Factors; Tryptophan - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M412389200

We delineated acetylcholine (ACh)-dependent conformational changes in a prototype of the nicotinic receptor ligand binding domain by molecular dynamics simulation and changes in intrinsic tryptophan (Trp) fluorescence. Prolonged molecular dynamics simulation of ACh-binding protein showed that binding of ACh establishes close register of Trps from adjacent subunits, Trp 143 and Trp 53 , and draws the peripheral C-loop inward to occlude the entrance to the binding cavity. Close register of Trp 143 and Trp 53 was demonstrated by ACh-mediated quenching of intrinsic Trp fluorescence, elimination of quenching by mutation of one or both Trps to Phe, and decreased lifetime of Trp fluorescence by bound ACh. Occlusion of the binding cavity by the C-loop was demonstrated by restricted access of an extrinsic quencher of binding site Trp fluorescence by ACh. The collective findings showed that ACh initially establishes close register of conserved Trps from adjacent subunits and then draws the C-loop inward to occlude the entrance to the binding cavity.