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Biophys. J. BioFAST: First Published February 29, 2008. doi:10.1529/biophysj.107.117853
© 2008 by the Biophysical Society.

A more recent version of this article appeared on June 1, 2008.
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PROTEINS

Four-{alpha}-Helix Bundle with Designed Anesthetic Binding Pockets II: Halothane Effects on Structure and Dynamics

Tanxing Cui 1, Vasyl Bondarenko 1, Dejian Ma 1, Christian G. Canlas 1, Nicole R. Brandon 1, Jonas Johansson 2, Yan Xu 1* and Pei Tang 3

1 University of Pittsburgh School of Medicine
2 University of Pennsylvania
3 University of PIttsburgh School of Medicine

* To whom correspondence should be addressed. E-mail: xuy{at}anes.upmc.edu.

Submitted on July 19, 2007
Revised on August 19, 2007
Accepted on 18 January 2008


  Abstract
As a model of the protein targets for volatile anesthetics, the dimeric four-{alpha}-helix bundle, (A{alpha}2-L1M/L38M)2, was designed to contain a long hydrophobic core, enclosed by four amphipathic {alpha}-helices, for specific anesthetic binding. The structural and dynamical analyses of (A{alpha}2-L1M/L38M)2 in the absence of anesthetics (see companion paper) showed a highly dynamic anti-parallel dimer with an asymmetric arrangement of the four helices and a lateral accessing pathway from the aqueous phase to the hydrophobic core. In this study, we determined the high-resolution NMR structure of (A{alpha}2-L1M/L38M)2 in the presence of halothane, a clinically used volatile anesthetic. The high-solution NMR structure, with a backbone RMSD of 1.72-Å (2JST), and the NMR binding measurements revealed that the primary halothane binding site is located between two side-chains of W15 from each monomer, different from the initially designed anesthetic binding sites. Hydrophobic interactions with residues A44 and L18 also contribute to stabilizing the bound halothane. While halothane produces minor changes in the monomer structure, the quaternary arrangement of the dimer is shifted by about half a helical turn and twists relative to each other, leading to the closure of the lateral access pathway to the hydrophobic core. Quantitative dynamics analyses, including Model-Free analysis of the relaxation data and the CPMG transverse relaxation dispersion measurements, suggest that the most profound anesthetic effect is the suppression of the conformational exchange term (Rex) both near and remote from the binding site. Our results revealed a novel mechanism of an induced fit between anesthetic molecule and its protein target, with the direct consequence being the change in protein dynamics on a global rather than a local scale. This mechanism may be universal to anesthetic action on neuronal proteins.

Key Words: anesthesia mechanisms, four-alpha-helix bundle, general anesthetics, global dynamics, nuclear magnetic resonance (NMR), protein quaternary structure






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Copyright © 2008 by the Biophysical Society.