Computational Design and Elaboration of a De Novo Heterotetrameric α-Helical Protein that Selectively Binds an Emissive Abiological (Porphinato)zinc Chromophore
Date of this Version
Journal of the American Chemical Society
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.
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: 10.1021/ja907407m
Absorption, Amino Acid Sequence, Circular Dichroism, Computer Simulation, Drug Design, Electrons, Metalloporphyrins, Models, Molecular, Molecular Sequence Data, Peptides, Protein Multimerization, Protein Structure, Quaternary, Protein Structure, Secondary, Proteins, Substrate Specificity, Ultracentrifugation, Zinc
Fry, H. C., Lehmann, A., Saven, J. G., DeGrado, W. F., & Therien, M. J. (2010). Computational Design and Elaboration of a De Novo Heterotetrameric α-Helical Protein that Selectively Binds an Emissive Abiological (Porphinato)zinc Chromophore. Journal of the American Chemical Society, 132 (11), 3997-4005. http://dx.doi.org/10.1021/ja907407m
Date Posted: 07 December 2016
This document has been peer reviewed.