A designed four-alpha-helix bundle that binds the volatile general anesthetic halothane with high affinity.

Area of Science:

• Biochemistry
• Structural Biology
• Pharmacology

Background:

• Understanding volatile anesthetic binding sites on proteins is crucial for elucidating their mechanism of action.
• Previous studies indicated that cavities in the hydrophobic core enhance anesthetic binding affinity.

Purpose of the Study:

• To investigate the role of enhanced dispersion forces and altered binding site accessibility in volatile anesthetic binding.
• To explore the structural basis for improved anesthetic affinity using a four-alpha-helix bundle model.

Main Methods:

• Utilized a four-alpha-helix bundle protein model with a hydrophobic core.
• Substituted leucine with methionine to increase polarizability and enhance dispersion forces.
• Employed photoaffinity labeling with (14)C-halothane and measured amide hydrogen exchange rates.
• Performed molecular dynamics simulations to visualize the protein-ligand interactions.

Main Results:

• The methionine-containing bundle showed significantly improved halothane binding affinity (K(d) = 0.20 mM) compared to the leucine bundle (K(d) = 0.69 mM).
• Photoaffinity labeling identified W15 residue as a key site for halothane binding in both variants.
• Halothane binding stabilized the folded conformations of the four-alpha-helix bundle proteins.

Conclusions:

• Enhanced dispersion forces, potentially via methionine substitution, contribute to higher affinity anesthetic binding sites.
• Improved access to the binding site or allosteric optimization of the binding pocket dimensions may also play a role.
• Stabilization of folded protein conformations by anesthetics could be a fundamental mechanism underlying their action.