Organic ligand binding by a hydrophobic cavity in a designed tetrameric coiled-coil protein.

The design and characterization of a hydrophobic cavity in de novo designed proteins provides a wide range of information about the functions of de novo proteins. We designed a de novo tetrameric coiled-coil protein with a hydrophobic pocketlike cavity. Tetrameric coiled coils with hydrophobic cavities have previously been reported. By replacing one Leu residue at the a position with Ala, hydrophobic cavities that did not flatten out due to loose peptide chains were reliably created. To perform a detailed examination of the ligand-binding characteristics of the cavities, we originally designed two other coiled-coil proteins: AM2, with eight Ala substitutions at the adjacent a and d positions at the center of a bundled structure, and AM2W, with one Trp and seven Ala substitutions at the same positions. To increase the association of the helical peptides, each helical peptide was connected with flexible linkers, which resulted in a single peptide chain. These proteins exhibited CD spectra corresponding to superhelical structures, despite weakened hydrophobic packing. AM2W exhibited binding affinity for size-complementary organic compounds. The dissociation constants, K(d), of AM2W were 220 nM for adamantane, 81 microM for 1-adamantanol, and 294 microM for 1-adamantaneacetic acid, as measured by fluorescence titration analyses. Although it was contrary to expectations, AM2 did not exhibit any binding affinity, probably due to structural defects around the designed hydrophobic cavity. Interestingly, AM2W exhibited incremental structure stability through ligand binding. Plugging of structural defects with organic ligands would be expected to facilitate protein folding.