@article {Gao1230, author = {Fan Gao and Georges Mer and Marco Tonelli and Scott B. Hansen and Thomas P. Burghardt and Palmer Taylor and Steven M. Sine}, title = {Solution NMR of Acetylcholine Binding Protein Reveals Agonist-Mediated Conformational Change of the C-Loop}, volume = {70}, number = {4}, pages = {1230--1235}, year = {2006}, doi = {10.1124/mol.106.027185}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {Previous X-ray crystallography, molecular dynamics simulation, fluorescence spectroscopy, and deuterium-hydrogen exchange of acetylcholine binding protein (AChBP) suggest that after binding of the agonist, the C-loop at the periphery of the binding site draws inward to cap the site and envelop the agonist. In this study, we use high-resolution solution NMR to monitor changes in the chemical environment of the C-loop without and with acetylcholine (ACh) bound. Substitution of [15N]cysteine for the native cysteines 123, 136, 187, and 188 provided intrinsic monitors of the chemical environments of the Cys- and C-loops, respectively. Two-dimensional transverse relaxation-optimized spectroscopy 15N-1H HSQC spectroscopy of apo-AChBP revealed seven well resolved cross-peaks for the group of cysteines. The spectrum of AChBP with Ser substituted for Cys 187 and 188 shows only two main cross-peaks, corresponding to Cys 123 and 136 from the Cys-loop, enabling resonance assignments. After binding of ACh, the five cross-peaks associated with cysteines from the C-loop condense into two predominant cross-peaks not observed in the spectrum from the apo protein, indicating a restricted range of conformations and change in chemical environment of the C-loop. The results show that isotopic cysteine can be incorporated into specified positions of AChBP expressed from a eukaryotic source, that the C-loop assumes multiple conformations without ACh, but that its conformation becomes restricted with ACh bound. The collective findings suggest a structural mechanism for agonist recognition in AChBP and related Cys-loop receptors. The American Society for Pharmacology and Experimental Therapeutics}, issn = {0026-895X}, URL = {https://molpharm.aspetjournals.org/content/70/4/1230}, eprint = {https://molpharm.aspetjournals.org/content/70/4/1230.full.pdf}, journal = {Molecular Pharmacology} }