Design and synthesis of a globin fold

We propose a simple method to find an amino acid sequence that is foldable into a globular protein with a desired structure based on a knowledge-based 3D-1D compatibility function. An asymmetric alpha-helical single-domain st ructure of sperm whale myoglobin consisting of 153 amino acid residues was chosen for the design target. The optimal sequence to fit the main-chain fr amework has been searched by recursive generation of the protein 3D profile . The heme-binding site was designed by fixing His64 and His93 at the dista l and proximal positions, respectively, and by penalizing residues that pro trude into the space with a repulsive function. The apparent bumps among si de chains in the computer model of the converged, self-consistent sequence were removed by replacing some of the bumping residues with smaller ones ac cording to the final 3D profile. The finally obtained sequence shares 26% o f sequence with the natural myoglobin. The designed globin-1 (DG1) with the artificial sequence was obtained by expression of the synthetic gene in Es cherichia coli. Analyses using size-exclusion chromatography, circular dich roism spectroscopy, and solution X-ray scattering showed that DG1 folds int o a monomeric, compact, highly helical, and globular form with an overall m olecular shape similar to the target structure in an aqueous solution. Furt hermore, it binds a single heme per protein molecule, which exhibited well- defined spectroscopic properties. The radius of gyration of DG1 was determi ned to be 20.6 Angstrom, slightly larger than that of natural apoMb, and de creased to 19.5 Angstrom upon heme binding based on X-ray scattering analys is. However, the heme-bound DG1 did notstably bind molecular oxygen as natu ral globins do, possibly due to high conformational diversity of side-chain structures observed in the NMR and denaturation experiments. These results give insight into the relationship between the sequence selection and the structural uniqueness of natural proteins to achieve biological functions.