Binding of the volatile anesthetic halothane to the hydrophobic core of a tetra-alpha-helix-bundle protein

JS Johansson, F Rabanal and PL Dutton

Department of Anesthesia, University of Pennsylvania, Philadelphia, USA.

Although volatile general anesthetics interact with several proteins, little is known about the location or characteristics of the binding sites at the molecular level. A detailed structural description of how anesthetics associate with macromolecules is necessary for understanding anesthetic mechanisms of action. The recent introduction of designed synthetic proteins provides new opportunities for obtaining structural and functional information on anesthetic-protein interactions. A synthetic tetra-alpha-helix-bundle protein was used to examine the interaction of halothane with a designed protein interior. The tetra-alpha-helix-bundle comprises 124 residues in the form of two identical 62-residue di-alpha-helical peptides, held together in an all- parallel bundle by hydrophobic forces. Steady-state and time-resolved tryptophan fluorescence and circular dichroism spectroscopy were used to study the anesthetic-protein interaction. Halothane quenches bundle tryptophan fluorescence with a dissociation constant of 2.3 +/- 0.4 mM and a Hill number of 0.9 +/- 0.1. Tryptophan fluorescence decay analysis indicates that halothane quenches the protein fluorescence by a static mechanism. Circular dichroism spectroscopy revealed no change in protein secondary structure on exposure to halothane. Dissociation of the tetra-alpha-helix-bundle into 62-residue di-alpha-helical peptides by trifluoroethanol eliminated the halothane-protein interaction. The results suggest that halothane binds to the hydrophobic interior of the tetra-alpha-helix-bundle, close to the tryptophan residues. The protein tertiary and quaternary structures are required for anesthetic binding. This study demonstrates the feasibility of using synthetic tetra-alpha-helix-bundles as model anesthetic-binding proteins. The use of de novo designed bundle proteins should allow structural, energetic and functional descriptions of anesthetic-protein interactions.

Volume 279, Issue 1, pp. 56-61, 10/01/1996
Copyright © 1996 by American Society for Pharmacology and Experimental Therapeutics

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