Constructing a man-made c-type cytochrome maquette in vivo:electron transfer, oxygen transport and conversion to a photoactive light harvesting maquette

The successful use of man-made proteins to advance synthetic biology requires both the fabrication of functional artificial proteins in a living environment, and the ability of these proteins to interact productively with other proteins and substrates in that environment. Proteins made by the maquette method integrate sophisticated oxidoreductase function into evolutionarily naive, non-computationally designed protein constructs with sequences that are entirely unrelated to any natural protein. Nevertheless, we show here that we can efficiently interface with the natural cellular machinery that covalently incorporates heme into natural cytochromes c to produce in vivo an artificial c-type cytochrome maquette. Furthermore, this c-type cytochrome maquette is designed with a displaceable histidine heme ligand that opens to allow functional oxygen binding, the primary event in more sophisticated functions ranging from oxygen storage and transport to catalytic hydroxylation. To exploit the range of functions that comes from the freedom to bind a variety of redox cofactors within a single maquette framework, this c-type cytochrome maquette is designed with a second, non-heme C, tetrapyrrole binding site, enabling the construction of an elementary electron transport chain, and when the heme C iron is replaced with zinc to create a Zn porphyrin, a light-activatable artificial redox protein. The work we describe here represents a major advance in de novo protein design, offering a robust platform for new c-type heme based oxidoreductase designs and an equally important proof-of-principle that cofactor-equipped man-made proteins can be expressed in living cells, paving the way for constructing functionally useful man-made proteins in vivo

Engineering an artificial, multifunctional oxidoreductase protein maquette

Redox reactions drive the energy transduction processes that are the cornerstone of biology. Oxidoreductases are of great importance not only for their biological role, but also because their charge-transferring, potential-generating functions may someday be harnessed in the inexpensive generation of clean fuels. Detailed study of natural oxidoreductases is often limited by complexity resulting from millennia of evolution. Empirical observation, however, suggests a few basic design criteria necessary to attain function. The application of these criteria was tested in the design of a minimal, hydrophilic, monomeric protein, HM-1, capable of supporting multiple redox processes, including oxygen transport, light harvesting, and electron transport, with properties predicted by the empirical models. This success demonstrates that relatively simple engineering guidelines are sufficient to design a functional protein. Sophisticated function does not come at the price of specialization. Despite its simple design, HM-1 supports diverse functions seen in natural proteins with dramatically different structures. Thus, a minimalist approach to protein design has yielded a general oxidoreductase scaffold that spontaneously folds into a helical bundle and carries out diverse biological functions without mimicking natural proteins in structure or sequence. The monomeric construction overcomes the design limitations of earlier symmetric dimer and tetramer conformations, enabling flexible modifications for varying uses. It is easily modified by changing cofactors, ligation modes, or the peptide sequence itself. The utility of an iterative, intuition-driven design process for both structural and functional improvements is demonstrated. Observed functions include covalent and non-covalent cofactor incorporation, reversible oxygen binding, and photo-induced intramolecular electron transfer. The remarkable adaptability of the scaffold suggests that nature tunes oxidoreductases principally by the adjustment of a few key parameters, knowledge of which can be applied to design synthetic and novel enzymes

Electron tunneling chains of mitochondria

AbstractThe single, simple concept that natural selection adjusts distances between redox cofactors goes a long way towards encompassing natural electron transfer protein design. Distances are short or long as required to direct or insulate promiscuously tunneling single electrons. Along a chain, distances are usually 14 Å or less. Shorter distances are needed to allow climbing of added energetic barriers at paired-electron catalytic centers in which substrate and the required number of cofactors form a compact cluster. When there is a short-circuit danger, distances between shorting centers are relatively long. Distances much longer than 14Å will support only very slow electron tunneling, but could act as high impedance signals useful in regulation. Tunneling simulations of the respiratory complexes provide clear illustrations of this simple engineering

Engineering oxidoreductases:maquette proteins designed from scratch

The study of natural enzymes is complicated by the fact that only the most recent evolutionary progression can be observed. In particular, natural oxidoreductases stand out as profoundly complex proteins in which the molecular roots of function, structure and biological integration are collectively intertwined and individually obscured. In the present paper, we describe our experimental approach that removes many of these often bewildering complexities to identify in simple terms the necessary and sufficient requirements for oxidoreductase function. Ours is a synthetic biology approach that focuses on from-scratch construction of protein maquettes designed principally to promote or suppress biologically relevant oxidations and reductions. The approach avoids mimicry and divorces the commonly made and almost certainly false ascription of atomistically detailed functionally unique roles to a particular protein primary sequence, to gain a new freedom to explore protein-based enzyme function. Maquette design and construction methods make use of iterative steps, retraceable when necessary, to successfully develop a protein family of sturdy and versatile single-chain three- and four-α-helical structural platforms readily expressible in bacteria. Internally, they prove malleable enough to incorporate in prescribed positions most natural redox cofactors and many more simplified synthetic analogues. External polarity, charge-patterning and chemical linkers direct maquettes to functional assembly in membranes, on nanostructured titania, and to organize on selected planar surfaces and materials. These protein maquettes engage in light harvesting and energy transfer, in photochemical charge separation and electron transfer, in stable dioxygen binding and in simple oxidative chemistry that is the basis of multi-electron oxidative and reductive catalysis

Elementary tetrahelical protein design for diverse oxidoreductase functions

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