Computational De Novo Design and Characterization of a Four-Helix Bundle Protein that Selectively Binds a Nonbiological Cofactor - Analytical Methods & Tools for Lyophilization

Computational De Novo Design and Characterization of a Four-Helix Bundle Protein that Selectively Binds a Nonbiological Cofactor
Received September 27, 2004
Web Release Date: January 15, 2005
Frank V. Cochran, Sophia P. Wu, Wei Wang, Vikas Nanda, Jeffery G. Saven,* Michael J. Therien,* and William F. DeGrado*
J. Am. Chem. Soc.
ACS Publications
Copyright ? 2005 American Chemical Society
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
wdegrado@mail.med.upenn.edu
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 -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.
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