Mapping heat exchange in an allosteric protein

Abstract

Nicotinic acetylcholine receptors (AChRs) are synaptic ion channels that spontaneously isomerize (i.e., gate) between resting and active conformations. We used single-molecule electrophysiology to measure the temperature dependencies of mouse neuromuscular AChR gating rate and equilibrium constants. From these we estimated free energy, enthalpy, and entropy changes caused by mutations of amino acids located between the transmitter binding sites and the middle of the membrane domain. The range of equilibrium enthalpy change (13.4 kcal/mol) was larger than for free energy change (5.5 kcal/mol at 25°C). For two residues, the slope of the rate-equilibrium free energy relationship (Φ) was approximately constant with temperature. Mutant cycle analysis showed that both free energies and enthalpies are additive for energetically independent mutations. We hypothesize that changes in energy associated with changes in structure mainly occur close to the site of the mutation, and, hence, that it is possible to make a residue-by-residue map of heat exchange in the AChR gating isomerization. The structural correlates of enthalpy changes are discussed for 12 different mutations in the protein.

Figure 1

Locations of the mutations. The unliganded Torpedo AChR (accession number 2bg9.pdb) Only the α_δ-and δ-subunits are displayed; horizontal lines mark approximately the membrane. αW149 is at a transmitter binding site. The four transmembrane helices are labeled M1–M4 in the δ-subunit. The mutated residues are dark gray.

Figure 2

Temperature dependence of the gating equilibrium constant. The van 't Hoff plots are shown for 15 mutant AChRs (Table S3). (A) No agonist present (unliganded gating); αDYP is αD97A+αY127F+αP272A; DYS is αD97A+αY127F+αS269I. (B) Unliganded gating of αW149 mutants expressed on the DYS background (example currents, Fig. S3). (C) Mutants expressed on the wt background and activated by agonists. αG153S was activated by Cho, and the others were activated by ACh. αP272G and αG153S are heat-activated and the αY127 mutants are cold-activated. (D) Unliganded gating of δV269 mutants expressed on the DYS background.

Figure 3

Rate-equilibrium analyses. (A and B) Log-log plots of opening rate constant (f₀) versus gating equilibrium constant (E₀) for families of mutations of a single residue (αW149 or δV269), at different temperatures (no agonist present). The slope (±SD) of the linear fit, Φ, is shown at the bottom right of each panel. (C) For both amino-acid positions, Φ is approximately independent of temperature. (D) Enthalpy rate-equilibrium plots for αW149 and δV269. The y axis is the change in the activation enthalpy of the opening (forward) rate constant (ΔE_a^f) and the x axis is the change in enthalpy difference between the ground states (ΔΔH) for a family of mutations at a single position. For these two positions the slope of this relationship was the same as Φ.

Figure 4

Enthalpy mutant-cycle analysis. (A) Example clusters from αDYS and αW149F, δV269L and the double-mutant αW149F+δV269L expressed on the αDYS background (no agonist present; 25°C). (B) (Solid circles and solid line) The van 't Hoff plot for the αW149F+δV269L construct. (Dashed lines) Single mutant constructs. The change in enthalpy for the double mutant (ΔH =+3.2 kcal/mol) is approximately equal to the sum of the individual enthalpy changes for the single-mutant construct (+5.0 kcal/mol for αW149F and −1.5 kcal/mol for δV269L).

Figure 5

Summary of thermodynamic parameters for mutant AChRs. (A) Mutation-induced changes in enthalpy (ΔΔH; black) and free energy (ΔΔG; white) relative to the background construct (Table 1). The range of values for ΔΔH is greater than for ΔΔG because of compensating entropy changes. (B) A map of enthalpy changes in AChR gating. αW149F and αP272G are heat-activated (ΔΔH>+2 kcal/mol); αY127C and δV269A are cold-activated (ΔΔH<−2 kcal/mol); αG153S, −1<ΔΔH<1 kcal/mol. Not shown, mutations of αW149 and δV269 could result in either positive or negative ΔΔH values.