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 structure of sperm whale myoglobin consisting of 153 amino acid residues was chosen for the design target. The optimal sequence to fit the main-chain framework has been searched by recursive generation of the protein 3D profile. The heme-binding site was designed by fixing His64 and His93 at the distal and proximal positions, respectively, and by penalizing residues that protrude into the space with a repulsive function. The apparent bumps among side chains in the computer model of the converged, self-consistent sequence were removed by replacing some of the bumping residues with smaller ones according to the final 3D profile. The finally obtained sequence shares 26% of sequence with the natural myoglobin. The designed globin-1 (DG1) with the artificial sequence was obtained by expression of the synthetic gene in Escherichia coli. Analyses using size-exclusion chromatography, circular dichroism spectroscopy, and solution X-ray scattering showed that DG1 folds into a monomeric, compact, highly helical, and globular form with an overall molecular shape similar to the target structure in an aqueous solution. Furthermore, it binds a single heme per protein molecule, which exhibited well-defined spectroscopic properties. The radius of gyration of DG1 was determined to be 20.6 A, slightly larger than that of natural apoMb, and decreased to 19.5 A upon heme binding based on X-ray scattering analysis. However, the heme-bound DG1 did not stably bind molecular oxygen as natural 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.
Isogai
Y
Y
The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. yisogai@postman.riken.go.jp
Ota
M
M
Fujisawa
T
T
Izuno
H
H
Mukai
M
M
Nakamura
H
H
Iizuka
T
T
Nishikawa
K
K
D000595:Amino Acid Sequence; D000818:Animals; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013059:Spectrum Analysis, Raman; D013816:Thermodynamics; D014907:Whales
D009211:Myoglobin; D006418:Heme