Mapping heat exchange in an allosteric protein.

Area of Science:

• Biophysics
• Molecular Biology
• Neuroscience

Background:

• Nicotinic acetylcholine receptors (AChRs) are crucial ion channels.
• AChRs gate between resting and active states via spontaneous isomerization.
• Understanding the energetics of AChR gating is key to their function.

Purpose of the Study:

• To investigate the temperature dependencies of mouse neuromuscular AChR gating kinetics.
• To quantify free energy, enthalpy, and entropy changes associated with specific mutations.
• To develop a residue-by-residue map of heat exchange during AChR gating isomerization.

Main Methods:

• Single-molecule electrophysiology was employed to measure gating rates and equilibrium constants.
• Temperature dependence analysis was used to calculate thermodynamic parameters.
• Mutant cycle analysis was performed to assess the additivity of energetic contributions.

Main Results:

• Enthalpy changes ranged up to 13.4 kcal/mol, exceeding free energy changes (5.5 kcal/mol at 25°C).
• Rate-equilibrium free energy relationships (Φ) remained temperature-independent for two key residues.
• Mutant cycle analysis confirmed additivity for energetically independent mutations.

Conclusions:

• The study provides insights into the energetic landscape of AChR gating.
• Results suggest that energy changes are localized near mutation sites, enabling residue-specific mapping.
• Structural correlates of enthalpy changes were elucidated for 12 distinct mutations.