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 α-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.