Computational De Novo Design and Characterization of a Four-Helix Bundle Protein that Selectively Binds a Nonbiological Cofactor Frank V. Cochran, ² Sophia P. Wu, ‡ Wei Wang, ‡ Vikas Nanda, ² Jeffery G. Saven,* ,‡ Michael J. Therien,* ,‡ and William F. DeGrado* ,²,‡ Department of Biochemistry and Molecular Biophysics, Johnson Foundation, School of Medicine, UniVersity of PennsylVania, Philadelphia, PennsylVania 19104, and Department of Chemistry, UniVersity of PennsylVania, Philadelphia, PennsylVania 19104 Received September 27, 2004; E-mail: wdegrado@mail.med.upenn.edu De novo design tests current understanding of metalloproteins 1,2 and offers the potential to construct novel biomaterials, such as catalysts 3,4 and bioelectrochemical devices. 5 The use of nonbio- logical cofactors in these efforts offers the possibility of creating proteins with unusual properties. We have developed computational design methodology 6,7 to derive a unique protein framework for a specified cofactor of interest as an alternative to re-engineering natural protein scaffolds. 8 Selective cofactor recognition is a hallmark of achieving this goal and a significant challenge, especially in the absence of covalent attachment. 9 Previously designed heme proteins bound various metalloporphyrins with relatively low specificity, which is likely attributable to molten globule character. 2 In contrast, natural proteins generally bind cofactors in well-structured environments with precisely positioned amino acid side chains. Here, we report the complete de novo design of a nativelike protein that selectively binds a nonbiological cofactor. The designed protein serves as a scaffold for future electron- transfer studies by encapsulating a symmetrical dyad of DPP-Fe units (Figure 1a) through bis(His) coordination. DPP-Fe(III)Cl was used as the precursor complex for incorporating the DPP-Fe unit in the protein matrix. 10 Requirements for efficient cofactor binding dictated the precise protein backbone geometry. A single low-energy structure (Figure 1b) was computed with a Monte Carlo simulated annealing protocol that considered the following constraints: (1) a metal-metal distance between 17 and 19 Å, (2) optimal His N(ǫ) to Fe bonding interactions, (3) second-shell hydrogen bonds between His N(δ) and Thr O(γ) (Figure 1c), (4) minimal steric clashes, (5) maintenance of D 2 symmetry. This process led to imidazole rings in near-perpendicular alignment as a result of the second-shell hydrogen bonds. The identities of the remaining positions were determined by recursive calculations with the computational design algorithm SCADS, 6,7 which provided site-dependent side chain probabilities. In particular, SCADS located sites appropriate for complementary charge patterning to enforce an antiparallel bundle topology 11 (E7, E14, K21, K28). Ala side chains at critical sites promoted efficient helix-helix and helix-cofactor packing (A4, A8, A12, A15, A18, A25). A suitable position for tyrosine was found to aid peptide concentration measurements (Y32). Finally, S0 and G33 were added as N- and C-terminal capping residues 12 (Figure 1d). The 34-residue peptide was prepared by Fmoc-based solid-phase synthesis. 13 UV-vis spectroscopy demonstrated that the peptide binds DPP- Fe via bis(His) coordination to low-spin Fe(III) and with a peptide/ cofactor stoichiometry consistent with the design. An increase in Soret band absorbance at 408 nm and a Q-band blue shift from 580 to 530 nm was observed upon adding four equivalents of peptide to two equivalents of DPP-Fe(III) (Figure 2). A titration showed a linear increase in Soret band intensity with added peptide ² Department of Biochemistry and Molecular Biophysics, Johnson Foundation, School of Medicine. ‡ Department of Chemistry. Figure 1. (a) DPP-Fe cofactor. (b) Model of designed four-helix bundle protein containing two DPP-Fe cofactors. (c) Axial ligand interactions. (d) Peptide sequence. Figure 2. UV-vis spectra demonstrating cofactor selectivity. Published on Web 01/15/2005 1346 9 J. AM. CHEM. SOC. 2005, 127, 1346-1347 10.1021/ja044129a CCC: $30.25 © 2005 American Chemical Society