Towards a three-)alpha)-helix bundle protein that binds volatile general anesthetics

The general anesthetics halothane and chloroform are capable of binding to synthetic water-soluble four-(alpha)-helix bundles, which model the putative in vivo receptors. In this study, we investigate the binding of these anesthetics to synthetic water-soluble three-(alpha)-helix bundles. A series of variants containing up to four X-to-Ala and up to four X-to-Met substitutions was made; and the effect of these substitutions on structure, stability and anesthetic binding affinity was examined. Generally, the amount of (alpha)-helix and the stability of the three-(alpha)-helix bundles decreased as the number of X-to-Ala substitutions increased. A concomitant red-shift in tryptophan fluorescence (lambda)max was seen, suggesting an increased flexibility of the native structure. Up to four X-to-Met substitutions had little effect on the amount of (alpha)-helix, but an increase in tryptophan (lambda)max was seen for the variants with three and four methionine substitutions. The exceptions were a) a variant with a clustering of alanine and methionine residues at one end of the three-(alpha)-helix bundle, suggesting a gate structure that can admit ligand molecules; and b) a variant with a single Leu35Ala substitution, suggesting that at select positions, the size of the side chain is important for defining anesthetic binding affinity.