Computational design and elaboration of a de novo heterotetrameric alpha-helical protein that selectively binds an emissive abiological (porphinato)zinc chromophore.

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Fry, HC; Lehmann, A; Saven, JG; DeGrado, WF; Therien, MJ

Published Date

  • March 24, 2010

Published In

Volume / Issue

  • 132 / 11

Start / End Page

  • 3997 - 4005

PubMed ID

  • 20192195

Electronic International Standard Serial Number (EISSN)

  • 1520-5126

Digital Object Identifier (DOI)

  • 10.1021/ja907407m

Language

  • eng

Conference Location

  • United States