title: Computational de novo design and characterization of a four-helix bundle protein that selectively binds a nonbiological cofactor.
abstract: We report the complete de novo design of a four-helix bundle protein that selectively binds the nonbiological DPP-Fe(III) metalloporphyrin cofactor (DPP-Fe(III) = 5, 15-Di[(4-carboxymethyleneoxy)phenyl]porphinato iron(III)). A tetrameric, D2-symmetric backbone scaffold was constructed to encapsulate two DPP-Fe(III) units through bis(His) coordination. The complete sequence was determined with the aid of the statistical computational design algorithm SCADS. The 34-residue peptide was chemically synthesized. UV-vis and CD spectroscopy, size-exclusion chromatography, and analytical ultracentrifugation indicated the peptide undergoes a transition from a predominantly random coil monomer to an alpha-helical tetramer upon binding DPP-Fe(III). EPR spectroscopy studies indicated the axial imidazole ligands were oriented in a perpendicular fashion, as defined by second-shell interactions that were included in the design. The 1-D 1H NMR spectrum of the assembled protein displayed features of a well-packed interior. The assembled protein possessed functional redox properties different from those of structurally similar systems containing the heme cofactor. The designed peptide demonstrated remarkable cofactor selectivity with a significantly weaker binding affinity for the natural heme cofactor. These findings open a path for the selective incorporation of more elaborate cofactors into designed scaffolds for constructing molecularly well-defined nanoscale materials.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Johnson Foundation, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Frank V
authors.initials: FV
authors.lastname: Cochran
authors.forename: Sophia P
authors.initials: SP
authors.lastname: Wu
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Vikas
authors.initials: V
authors.lastname: Nanda
authors.forename: Jeffery G
authors.initials: JG
authors.lastname: Saven
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Therien
authors.forename: William F
authors.initials: WF
authors.lastname: DeGrado
mesh_terms: D000595:Amino Acid Sequence; D004578:Electron Spin Resonance Spectroscopy; D005290:Ferric Compounds / Q000138:chemical synthesis / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D007093:Imidazoles / Q000138:chemical synthesis / Q000737:chemistry; D008667:Metalloproteins / Q000138:chemical synthesis / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011199:Potentiometry; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D013056:Spectrophotometry, Ultraviolet
chemical_list: D005290:Ferric Compounds; D007093:Imidazoles; D008667:Metalloproteins; D006418:Heme