As a model of the protein targets for volatile anesthetics, the dimeric four-α-helix bundle, (Aα2-L1M/L38M)2, was designed to contain a long hydrophobic core, enclosed by four amphipathic α-helices, for specific anesthetic binding. The structural and dynamical analyses of (Aα2-L1M/L38M)2 in the absence of anesthetics (another study) showed a highly dynamic antiparallel 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 (Aα2-L1M/L38M)2 in the presence of halothane, a clinically used volatile anesthetic. The high-solution NMR structure, with a backbone root mean-square deviation 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. Whereas 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, which leads to the closure of the lateral access pathway to the hydrophobic core. Quantitative dynamics analyses, including Modelfree analysis of the relaxation data and the Carr-Purcell-Meiboom-Gill transverse relaxation dispersion measurements, suggest that the most profound anesthetic effect is the suppression of the conformational exchange 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 of protein dynamics changing on a global rather than a local scale. This mechanism may be universal to anesthetic action on neuronal proteins.
Bibliographical noteFunding Information:
This work was supported in part by grants from the National Institutes of Health (R37GM049202 to Y.X. and P.T., R01GM056257 to PT, and P01GM055876 to Y.X. and J.S.J.).
ASJC Scopus subject areas