Probing the structural features of volatile anesthetic binding sites with synthetic peptides.

The structural features of volatile anesthetic binding sites on proteins were explored using a model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. This system serves as a model for the lipid-spanning portions of several membrane proteins. Two hydrophobic core designs were compared: H10A24 consisting mainly of leucine residues, and (Aalpha2)2 which has four leucine and two histidine residues replaced by smaller alanines with the intent of forming a cavity. Halothane binds to (Aalpha2)2 with a Kd of 0.71 +/- 0.04 mM as monitored by the quenching of tryptophan fluorescence. This is a 3.2-fold higher affinity compared with binding to H10A24 (Kd = 2.3 +/- 0.4 mM). The presence of a preexisting protein hydrophobic cavity may favor volatile anesthetic binding. Guanidinium chloride denaturation studies reveal that bound anesthetic favors the native folded form of (Aalpha2)2 by 1.8 kcal/mol. The use of synthetic peptides should allow predictions to be made concerning the structural composition of in vivo anesthetic binding sites and may provide clues to how anesthetics alter protein function.