Saven, Jeffery G.Fry, H ChristopherDeGrado, William FLehmann, AndreasTherien, Michael J2023-05-222023-05-222010-03-242016-06-19https://repository.upenn.edu/handle/20.500.14332/6109The 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.This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of the American Chemical Society, copyright © 2011 American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see doi: http://dx.doi.org/10.1021%2Fja907407mAbsorptionAmino Acid SequenceCircular DichroismComputer SimulationDrug DesignElectronsMetalloporphyrinsModels, MolecularMolecular Sequence DataPeptidesProtein MultimerizationProtein Structure, QuaternaryProtein Structure, SecondaryProteinsSubstrate SpecificityUltracentrifugationZincAbsorptionAmino Acid SequenceCircular DichroismComputer SimulationDrug DesignElectronsMetalloporphyrinsModelsMolecularMolecular Sequence DataPeptidesProtein MultimerizationProtein StructureQuaternaryProtein StructureSecondaryProteinsSubstrate SpecificityUltracentrifugationZincBiochemistryOrganic ChemistryArticle