title: Computational design and elaboration of a de novo heterotetrameric alpha-helical protein that selectively binds an emissive abiological (porphinato)zinc chromophore.
abstract: The first example of a computationally de novo designed protein that binds an emissive abiological chromophore is presented, in which a sophisticated level of cofactor discrimination is pre-engineered. This heterotetrameric, C(2)-symmetric bundle, A(His):B(Thr), uniquely binds (5,15-di[(4-carboxymethyleneoxy)phenyl]porphinato)zinc [(DPP)Zn] via histidine coordination and complementary noncovalent interactions. The A(2)B(2) heterotetrameric protein reflects ligand-directed elements of both positive and negative design, including hydrogen bonds to second-shell ligands. Experimental support for the appropriate formulation of [(DPP)Zn:A(His):B(Thr)](2) is provided by UV/visible and circular dichroism spectroscopies, size exclusion chromatography, and analytical ultracentrifugation. Time-resolved transient absorption and fluorescence spectroscopic data reveal classic excited-state singlet and triplet PZn photophysics for the A(His):B(Thr):(DPP)Zn protein (k(fluorescence) = 4 x 10(8) s(-1); tau(triplet) = 5 ms). The A(2)B(2) apoprotein has immeasurably low binding affinities for related [porphinato]metal chromophores that include a (DPP)Fe(III) cofactor and the zinc metal ion hemin derivative [(PPIX)Zn], underscoring the exquisite active-site binding discrimination realized in this computationally designed protein. Importantly, elements of design in the A(His):B(Thr) protein ensure that interactions within the tetra-alpha-helical bundle are such that only the heterotetramer is stable in solution; corresponding homomeric bundles present unfavorable ligand-binding environments and thus preclude protein structural rearrangements that could lead to binding of (porphinato)iron cofactors.
authors.affiliation: Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323, USA.
authors.forename: H Christopher
authors.initials: HC
authors.lastname: Fry
authors.forename: Andreas
authors.initials: A
authors.lastname: Lehmann
authors.forename: Jeffery G
authors.initials: JG
authors.lastname: Saven
authors.forename: William F
authors.initials: WF
authors.lastname: DeGrado
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Therien
mesh_terms: D000042:Absorption; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003198:Computer Simulation; D015195:Drug Design; D004583:Electrons; D008665:Metalloporphyrins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D014461:Ultracentrifugation; D015032:Zinc / Q000737:chemistry*
chemical_list: D008665:Metalloporphyrins; D010455:Peptides; D011506:Proteins; D015032:Zinc