title: Creation and structure determination of an artificial protein with three complete sequence repeats.
abstract: Symfoil-4P is a de novo protein exhibiting the threefold symmetrical β-trefoil fold designed based on the human acidic fibroblast growth factor. First three asparagine-glycine sequences of Symfoil-4P are replaced with glutamine-glycine (Symfoil-QG) or serine-glycine (Symfoil-SG) sequences protecting from deamidation, and His-Symfoil-II was prepared by introducing a protease digestion site into Symfoil-QG so that Symfoil-II has three complete repeats after removal of the N-terminal histidine tag. The Symfoil-QG and SG and His-Symfoil-II proteins were expressed in Eschericha coli as soluble protein, and purified by nickel affinity chromatography. Symfoil-II was further purified by anion-exchange chromatography after removing the HisTag by proteolysis. Both Symfoil-QG and Symfoil-II were crystallized in 0.1 M Tris-HCl buffer (pH 7.0) containing 1.8 M ammonium sulfate as precipitant at 293 K; several crystal forms were observed for Symfoil-QG and II. The maximum diffraction of Symfoil-QG and II crystals were 1.5 and 1.1 Å resolution, respectively. The Symfoil-II without histidine tag diffracted better than Symfoil-QG with N-terminal histidine tag. Although the crystal packing of Symfoil-II is slightly different from Symfoil-QG and other crystals of Symfoil derivatives having the N-terminal histidine tag, the refined crystal structure of Symfoil-II showed pseudo-threefold symmetry as expected from other Symfoils. Since the removal of the unstructured N-terminal histidine tag did not affect the threefold structure of Symfoil, the improvement of diffraction quality of Symfoil-II may be caused by molecular characteristics of Symfoil-II such as molecular stability.
authors.affiliation: Molecular Structural Biology Group, Quantum Beam Science Directorate, Japan Atomic Energy Agency, Shirakatashirane 2-4, Nakagun Tokaimura, Ibaraki 319-1195, Japan.
authors.forename: Motoyasu
authors.initials: M
authors.lastname: Adachi
authors.forename: Rumi
authors.initials: R
authors.lastname: Shimizu
authors.forename: Ryota
authors.initials: R
authors.lastname: Kuroki
authors.forename: Michael
authors.initials: M
authors.lastname: Blaber
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D020449:Repetitive Sequences, Amino Acid; D017386:Sequence Homology, Amino Acid
chemical_list: D011506:Proteins
keywords: Symfoil; acidic FGF; protein design