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

The structural features of volatile anesthetic binding sites on proteins ar e being examined with the use of a defined model system consisting of a fou r-a-helix bundle scaffold with a hydrophobic core. Previous work has sugges ted that introducing a cavity into the hydrophobic core improves anesthetic binding affinity. The more polarizable methionine side chain was substitut ed for a leucine, in an attempt to enhance the dispersion forces between th e ligand and the protein. The resulting bundle variant has an improved affi nity (K-d = 0.20 +/- 0.01 mM) for halothane binding, compared with the leuc ine-containing bundle (K-d = 0.69 +/- 0.06 mM). Photoaffinity labeling with C-14-halothane reveals preferential labeling of the W15 residue in both pe ptides, supporting the view that fluorescence quenching by bound anesthetic reports both the binding energetics and the location of the ligand in the hydrophobic core. The rates of amide hydrogen exchange were similar for the two bundles, suggesting that differences in binding affinity were not due to changes in protein stability. Binding of halothane to both four-alpha-he lix bundle proteins stabilized the native folded conformations. Molecular d ynamics simulations of the bundles illustrate the existence of the hydropho bic core, containing both W15 residues. These results suggest that in addit ion to packing defects, enhanced dispersion forces may be important in prov iding higher affinity anesthetic binding sites. Alternatively, the effect o f the methionine substitution on halothane binding energetics may reflect e ither improved access to the binding site or allosteric optimization of the dimensions of the binding pocket. Finally, preferential stabilization of f olded protein conformations may represent a fundamental mechanism of inhale d anesthetic action.