A designed cavity in the hydrophobic core of a four-alpha-helix bundle improves volatile anesthetic binding affinity.

The structural features of protein binding sites for volatile anesthetics are being explored using a defined model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. Earlier work has demonstrated that a prototype hydrophobic core is capable of binding the volatile anesthetic halothane. Exploratory work on the design of an improved affinity anesthetic binding site is presented, based upon the introduction of a simple cavity into a prototype (alpha 2)2 four-alpha-helix bundle by replacing six core leucines with smaller alanines. The presence of such a cavity increases the affinity (Kd = 0.71 +/- 0.04 mM) of volatile anesthetic binding to the designed bundle core by a factor of 4.4 as compared to an analogous bundle core lacking such a cavity (Kd = 3.1 +/- 0.4 mM). This suggests that such packing defects present on natural proteins are likely to be occupied by volatile general anesthetics in vivo. Replacing six hydrophobic core leucine residues with alanines results in a destabilization of the folded bundle by 1.7-2.7 kcal/mol alanine, although the alanine-substituted bundle still exhibits a high degree of thermodynamic stability with an overall folded conformational delta GH2O = 14.3 +/- 0.8 kcal/mol. Covalent attachment of the spin label MTSSL to cysteine residues in the alanine-substituted four-alpha-helix bundle indicates that the di-alpha-helical peptides dimerize in an anti orientation. The rotational correlation time of the four-alpha-helix bundle is 8.1 +/- 0.5 ns, in line with earlier work on similar peptides. Fluorescence, far-UV circular dichroism, and Fourier transform infrared spectroscopies verified the hydrophobic core location of the tryptophan and cysteine residues, showing good agreement between experiment and design. These small synthetic proteins may prove useful for the study of the structural features of small molecule binding sites.