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Binding of Four General Anesthetics to the Hydrophobic Core of a Four-Alpha-Helix Bundle Protein
Jonas S. Johansson, M.D., Ph.D.; Gavin A. Manderson, Ph.D.; Ravindernath Pidikiti, M.D.; Roderic G. Eckenhoff, M.D.
Department of Anesthesia and the Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania
INTRODUCTION: A molecular understanding of volatile anesthetic mechanisms of action requires structural and dynamic descriptions of their protein complexes. Because the in vivo sites of action remain to be determined, the structural features of anesthetic binding sites on proteins are being explored using well-defined model systems, consisting of the common α-helical bundle motif. Halogenated alkanes have been shown to bind to this four-α-helix bundle protein using tyrosine fluorescence quenching (1). In this study, the interaction of additional anesthetic molecules with the four-α-helix bundle is examined, along with the effects of binding on protein dynamics.

METHODS: The four-α-helix bundle (Aα2-L38M/W15Y)2 was assembled on an Applied Biosystems model 433A synthesizer and purified to homogeneity as described (1). Photoaffinity labeling of the four-α-helix bundle with 14C-halothane was carried out by exposing the complex to 254 nm light for 60 s in 5 mm pathlength quartz cuvettes (2). The location of the now covalently bound anesthetic was determined by microsequencing on an Applied Biosystems model 473A sequencer (2). Hydrogen exchange was used to determine the effect of bound anesthetic on global dynamics, initially exchanging-in 3HOH, and switching to an exchange-out buffer of 50 mM sodium phosphate, at pH 6.0 (2).

RESULTS: After exposure to 60 s of UV light, the four-α-helix bundle had incorporated halothane at a stoichiometry of 1:1. Microsequencing revealed that the dominant release occurred coincidentally with the Y15 residue. The folded conformation of the four-α-helix bundle was stabilized in the presence of halothane, isoflurane, chloroform, and trichloroethanol, with ΔΔG values of 0.70 ± 0.05, 0.62 ± 0.07, 0.81 ± 0.04, and 1.31 ± 0.06 kcal/mol, respectively (mean ± SD, n = 3 or 4).

CONCLUSIONS: The tyrosine fluorescence quenching results (1) indicate that halothane binds to the four-α-helix bundle close to the Y15 residue. This follows because heavy atom perturbation (the mechanism whereby halothane quenches fluorescence) is a short-range phenomenon, occurring over distances of ≤ 3-5 angstroms. The results obtained with photoaffinity labeling followed by microsequencing of the protein provide support for this view. The hydrogen exchange results suggest that binding of halothane, isoflurane, chloroform, and trichloroethanol to the hydrophobic core of the protein all lead to a stabilization of the native folded conformation. This is in agreement with prior studies (2-4) and adds further support for the hypothesis that general anesthetics alter protein function by perturbing the equilibrium between different protein conformations.


1. Manderson GA, Johansson JS. Biochemistry 41:4080-4087, 2002.

2. Johansson JS, Scharf D, Davies LA, Reddy KS, Eckenhoff RG. Biophys J 78:982-993, 2000.

3. Eckenhoff RG. Mol Pharmacol 54:610-615, 1998.

4. Johansson JS, Zou H, Tanner TW. Anesthesiology 90:235-245, 1999.

Anesthesiology 2002; 96: A79