DETECTION OF ACH-MEDIATED CONFORMATIONAL CHANGES OF ACHBP

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Acetylcholine receptor (AChR) is a member of ligand-gated ion channel family. Small agonists like ACh trigger the channel opening by changing its conformation from the closed state to the open state. Here we want to study ACh-mediated conformational changes to AChBP, a soluble protein that is homologous to the ligand binding domain of AChR. Our molecular dynamics simulation (JBC in press) indicates ACh triggers the collapse of a peripheral loop region called C-loop on the binding site. At the tip of the C-loop, two vicinal cysteines have dramatic conformational changes. We isotopically-labeled only cysteine residues (U-13C3, 15N) in the protein and plan to see change of chemical shift in NMR spectra following addition of ACh. Since there are other cysteines in the protein, the spectra of a mutant protein with the vicinal cyteines mutated to Serine will be compared.