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

The structural features of volatile anesthetic binding sites on proteins we re explored using a model system consisting of a four-a-helix bundle scaffo ld with a hydrophobic core. This system serves as a model for the lipid-spa nning portions of several membrane proteins. Two hydrophobic core designs w ere compared: H10A24 consisting mainly of leucine residues, and (Acc,), whi ch has four leucine and two histidine residues replaced by smaller alanines with the intent of forming a cavity. Halothane binds to (A alpha(2))(2) wi th a K-d of 0.71 +/- 0.04 mM as monitored by the quenching of tryptophan fl uorescence. This is a 3.2-fold higher affinity compared with binding to H10 A24 (K-d = 2.3 +/- 0.4 mM). The presence of a preexisting protein hydrophob ic cavity may favor volatile anesthetic binding. Guanidinium chloride denat uration studies reveal that bound anesthetic favors the native folded form of (A alpha(2))(2) by 1.8 kcal/mol. The use of synthetic peptides should al low predictions to be made concerning the structural composition of in vivo anesthetic binding sites and may provide clues to how anesthetics alter pr otein function. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.