Structural dynamics of the M4 transmembrane segment during acetylcholine receptor gating

The transition state structures that link the stable end states of allosteric proteins are largely unresolved. We used single-molecule kinetic analysis to probe the dynamics of the M4 transmembrane segments during the closedopen isomerization of the neuromuscular acetylcholine receptor ion channel (AChR). We measured the slopes (φ) of the free energy relationships for 87 mutants, which reveal the open- versus closed-like characters of the mutated residues at the transition state and hence the sequence and organization of gating molecular motions. φ was constant throughout the length of the α subunit M4 segment with an average value of 0.54, suggesting that this domain moves as a unit, approximately midway through the reaction. Analysis of a hybrid construct indicates that the two α subunits move synchronously. Between subunits, the sequence of M4 motions is α-ε-β. The AChR ion channel emerges as a dynamic nanomachine with many moving parts.