Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 132, Issue 11, Pages 3997-4005Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ja907407m
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Funding
- National Institutes of Health [R01 GM-071628]
- U.S. Department of Energy Biomolecular Materials Program [DE-FG02-04ER46156]
- MRSEC [DMR-00-79909]
- NSEC [DMR-0425780]
- National Science Foundation
- Francqui Foundation (Belgium)
- VLAC (Vlaams Academisch Centrum)
- Centre for Advanced Studies of the Royal Flemish Academy of Belgium for Science and the Arts
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The first example of a computationally de novo designed protein that binds an emissive abiological chromohore 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.
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