Computational Design and Elaboration of a de Novo Heterotetrameric α-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, C2-symmetric bundle, AHis:BThr, uniquely binds (5,15-di[(4-carboxymethyleneoxy)phenyl]porphinato)zinc [(DPP)Zn] via histidine coordination and complementary noncovalent interactions. The A2B2 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:AHis:BThr]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 AHis:BThr:(DPP)Zn protein (kfluorescence = 4 × 108 s−1; τriplet = 5 ms). The A2B2 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 AHis:Bthr protein ensure that interactions within the tetra-α-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.

Computational Enzyme Design

“…Boas and Harbury applied computational design to periplasmic binding proteins and found that native site recapitulation required high-resolution rotamer sampling, continuous minimization, and accurate electrostatic calculations. [147,148] DeGrado and co-workers were able to create an a-helical bundle which was able to bind a heme-like cofactor. [148] Recent attempts at redesigning a dipeptide binder, on the other hand, were unsuccessful, presumably because of inadequately accounting for the binding site flexibility.…”

“…[147,148] DeGrado and co-workers were able to create an a-helical bundle which was able to bind a heme-like cofactor. [148] Recent attempts at redesigning a dipeptide binder, on the other hand, were unsuccessful, presumably because of inadequately accounting for the binding site flexibility. [149] …”

Protein Design: Toward Functional Metalloenzymes

“…888 The peptide PA SC was prepared by taking a previous design (PA TET ), which was the same base as for PRIME (vide supra), and computationally linking it with loop regions and numerous designed H-bonds to orient the coordinating His residues. In addition to incorporating heme into this construct, a Zn(II)DPP version was also prepared 889 because Zn(II)-porphyrins do not favor bis-His coordination. This resulted in a less symmetric construct [A His :B Thr : (DPP)Zn].…”

Engineering a zinc binding site into the de novo designed protein DS119 with a βαβ structure

“…Various approaches can be used to incorporate functions in the designed protein scaffolds (Smith and Hecht, 2011), and metal binding sites have been a particularly interesting target for this purpose because the biological chemistry of metals is extremely rich (Holm et al, 1996;Lu et al, 2009). For example, by engineering different cofactors like Zn 2+ (Handel et al, 1993), Fe 2+ /Fe 3+ (Kaplan and DeGrado, 2004), heme (Choma et al, 1994) and abiological chromophore (DPP) Zn (Fry et al, 2010) into the de novo designed four-helix bundles, functions like phenol oxidation, electron transfer, and nonlinear optical properties have been obtained.…”