pK_a Determination of Histidine Residues in α-Conotoxin MII Peptides by ¹H NMR and Constant pH Molecular Dynamics Simulation

α-Conotoxin MII (α-CTxMII) is a potent and selective peptide antagonist of neuronal nicotinic acetylcholine receptors (nAChR’s). Studies have shown that His9 and His12 are significant determinants of toxin binding affinity for nAChR, while Glu11 may dictate differential toxin affinity between nAChR isoforms. The protonation state of these histidine residues and therefore the charge on the α-CTx may contribute to the observed differences in binding affinity and selectivity. In this study, we assess the pH dependence of the protonation state of His9 and His12 by ¹H NMR spectroscopy and constant pH molecular dynamics (CpHMD) in α-CTxMII, α-CTxMII[E11A], and the triple mutant, α-CTxMII- [N5R:E11A:H12K]. The E11A mutation does not significantly perturb the pK_a of His9 or His12, while N5R:E11A:H12K results in a significant decrease in the pK_a value of His9. The pK_a values predicted by CpHMD simulations are in good agreement with ¹H NMR spectroscopy, with a mean absolute deviation from experiment of 0.3 pK_a units. These results support the use of CpHMD as an efficient and inexpensive predictive tool to determine pK_a values and structural features of small peptides critical to their function.