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.