Elementary tetrahelical protein design for diverse oxidoreductase functions

Abstract

Emulating functions of natural enzymes in man-made constructs has proven challenging. Here we describe a man-made protein platform that reproduces many of the diverse functions of natural oxidoreductases without importing the complex and obscure interactions common to natural proteins. Our design is founded on an elementary, structurally stable 4-α-helix protein monomer with a minimalist interior malleable enough to accommodate various light- and redox-active cofactors and with an exterior tolerating extensive charge patterning for modulation of redox cofactor potentials and environmental interactions. Despite its modest size, the construct offers several independent domains for functional engineering that targets diverse natural activities, including dioxygen binding and superoxide and peroxide generation, interprotein electron transfer to natural cytochrome c and light-activated intraprotein energy transfer and charge separation approximating the core reactions of photosynthesis, cryptochrome and photolyase. The highly stable, readily expressible and biocompatible characteristics of these open-ended designs promise development of practical in vitro and in vivo applications.

Principles of light harvesting from single photosynthetic complexes

“…The molecular machinery behind this large-scale power conversion process can serve as a guide for the development of solar energy sources [3][4][5][6]. The efforts towards artificial photosynthesis can be considered in three categories: (i) create elements inspired by photosynthetic systems [7][8][9][10][11]; (ii) incorporate components from photosynthetic organisms [12][13][14][15]; and (iii) use living organisms, either wild-type or genetically modified [16][17][18][19]. Recent work upregulating light harvesting through downstream processes has been particularly promising.…”

Maquette Strategy for Creation of Light- and Redox-Active Proteins

“…The most prominent and promising is the identification of the presence of operationally distinct domains that lend themselves to separate engineering. As best stated in the original paper [Farid et al, 2013], "…these domains include the interior of paired helices 1 and 3 and the interior of paired helices 2 and 4 [see numbered helices in Figure 15 Part E], as well as the external charged surfaces; loop regions also operate as mostly independent domains. The functional evidence for independent domains is well complemented by CD monitored thermal stabilities of the secondary a-helices and NMR views of the tertiary structuring.…”

“…The physical-chemical characteristics of disulfide-linked homodimeric predecessors were carried forward into the single chain structure without significant alterations [Farid et al, 2013]. For example, the rotational strain on the histidine ligations to the heme iron promoted by locally acting interfacial glutamates in helical column [Koder et al, 2009] continued to support the formation, dynamics and stability of the oxyferrous state.…”

“…With a central magnesium, the chlorins become the light-harvesting and redox chargeseparation units of photosynthesis. For maquette design and engineering, this cofactor class offers many of light-and redox-active cofactor possibilities from natural sources (for recent work see [Farid et al, 2013;Solomon et al, 2014;Goparaju et al, 2016]) and an enormous resource of synthetic variants (see for example ) that on satisfying simple binding requirements, ligate to all the apo-maquettes except 1 and 4. However while a great deal is learned from synthetic tetrapyrroles, the very significant advantage regarding the use of natural heme cofactors such as hemes A, B and C in functionalizing maquettes, as will be described later, is their potential for co-production in vivo with the single-chain maquette proteins such as 5 [Anderson et al, 2014;Watkins et al, 2016]).…”

“…Pairs of helices of this anti-configured helical pair were repositioned and joined by a loop; these helix pairs were linked in turn by a di-cystenyl disulfide link either between the N-terminus of each di-helix peptide or as shown in the figure, between the loops to produce the "candelabra" maquette 3 [Koder et al, 2009]. The next step involved eliminating the disulfide link and adding a third loop to 3 to produce a native-like single chain four-a-helix maquette protein 5 reflecting size and a common motif of natural proteins enabling, importantly, one-step expression in E. coli (see [Farid et al, 2013;Solomon et al, 2014]). This water-soluble single-chain maquette has since been patterned externally with hydrophobic residues to produce the amphiphilic structure that incorporates into and spans a membrane bilayer 6 [Goparaju et al, Auman and G. Kodali unpublished] or helices lengthened from about 45 Å to 62 Å (8) for which crystals are revealing a complete structure [Ennist, 2017].…”