Tailorable Tetrahelical Bundles as a Toolkit for Redox Studies.

Oxidoreductases have evolved over millions of years to perform a variety of metabolic tasks crucial for life. Understanding how these tasks are engineered relies on delivering external electron donors or acceptors to initiate electron transfer reactions. This is a challenge. Small-molecule redox reagents can act indiscriminately, poisoning the cell. Natural redox proteins are more selective, but finding the right partner can be difficult due to the limited number of redox potentials and difficulty tuning them. De novo proteins offer an alternative path. They are robust and can withstand mutations that allow for tailorable changes. They are also devoid of evolutionary artifacts and readily bind redox cofactors. However, no reliable set of engineering principles have been developed that allow for these proteins to be fine-tuned so their redox midpoint potential (Em) can form donor/acceptor pairs with any natural oxidoreductase. This work dissects protein-cofactor interactions that can be tuned to modulate redox potentials of acceptors and donors using a mutable de novo designed tetrahelical protein platform with iron tetrapyrrole cofactors as a test case. We show a series of engineered heme b-binding de novo proteins and quantify their resulting effect on Em. By focusing on the surface charge and buried charges, as well as cofactor placement, chemical modification, and ligation of cofactors, we are able to achieve a broad range of Em values spanning a range of 330 mV. We anticipate this work will guide the design of proteinaceous tools that can interface with natural oxidoreductases inside and outside the cell while shedding light on how natural proteins modulate Em values of bound cofactors.

Rational design of photosynthetic reaction center protein maquettes.

New technologies for efficient solar-to-fuel energy conversion will help facilitate a global shift from dependence on fossil fuels to renewable energy. Nature uses photosynthetic reaction centers to convert photon energy into a cascade of electron-transfer reactions that eventually produce chemical fuel. The design of new reaction centers de novo deepens our understanding of photosynthetic charge separation and may one day allow production of biofuels with higher thermodynamic efficiency than natural photosystems. Recently, we described the multi-step electron-transfer activity of a designed reaction center maquette protein (the RC maquette), which can assemble metal ions, tyrosine, a Zn tetrapyrrole, and heme into an electron-transport chain. Here, we detail our modular strategy for rational protein design and show that the intended RC maquette design agrees with crystal structures in various states of assembly. A flexible, dynamic apo-state collapses by design into a more ordered holo-state upon cofactor binding. Crystal structures illustrate the structural transitions upon binding of different cofactors. Spectroscopic assays demonstrate that the RC maquette binds various electron donors, pigments, and electron acceptors with high affinity. We close with a critique of the present RC maquette design and use electron-tunneling theory to envision a path toward a designed RC with a substantially higher thermodynamic efficiency than natural photosystems.

De novo protein design of photochemical reaction centers.

Natural photosynthetic protein complexes capture sunlight to power the energetic catalysis that supports life on Earth. Yet these natural protein structures carry an evolutionary legacy of complexity and fragility that encumbers protein reengineering efforts and obfuscates the underlying design rules for light-driven charge separation. De novo development of a simplified photosynthetic reaction center protein can clarify practical engineering principles needed to build new enzymes for efficient solar-to-fuel energy conversion. Here, we report the rational design, X-ray crystal structure, and electron transfer activity of a multi-cofactor protein that incorporates essential elements of photosynthetic reaction centers. This highly stable, modular artificial protein framework can be reconstituted in vitro with interchangeable redox centers for nanometer-scale photochemical charge separation. Transient absorption spectroscopy demonstrates Photosystem II-like tyrosine and metal cluster oxidation, and we measure charge separation lifetimes exceeding 100 ms, ideal for light-activated catalysis. This de novo-designed reaction center builds upon engineering guidelines established for charge separation in earlier synthetic photochemical triads and modified natural proteins, and it shows how synthetic biology may lead to a new generation of genetically encoded, light-powered catalysts for solar fuel production.

The aprotic electrochemistry of quinones.

Quinones play important roles in biological electron transfer reactions in almost all organisms, with specific roles in many physiological processes and chemotherapy. Quinones participate in two-electron, two-proton reactions in aqueous solution at equilibrium near neutral pH, but protons often lag behind the electron transfers. The relevant reactions in proteins are often sequential one electron redox processes without involving protons. Here we report the aprotic electrochemistry of the two half-couples, Q/Q.- and Q.-/Q=, of 11 parent quinones and 118 substituted 1,4-benzoquinones, 91 1,4-naphthoquinones, and 107 9,10-anthraquinones. The measured redox potentials are fit quite well with the Hammett para sigma (σpara) parameter. Occasional exceptions can involve important groups, such as methoxy substituents in ubiquinone and hydroxy substituents in therapeutics. These can generally be explained by reasonable conjectures involving steric clashes and internal hydrogen bonds. We also provide data for 25 other quinones, 2 double quinones and 15 non-quinones, all measured under similar conditions.

A Thermostable Protein Matrix for Spectroscopic Analysis of Organic Semiconductors.

Advances in protein design and engineering have yielded peptide assemblies with enhanced and non-native functionalities. Here, various molecular organic semiconductors (OSCs), with known excitonic up- and down-conversion properties, are attached to a de novo-designed protein, conferring entirely novel functions on the peptide scaffolds. The protein-OSC complexes form similarly sized, stable, water-soluble nanoparticles that are robust to cryogenic freezing and processing into the solid-state. The peptide matrix enables the formation of protein-OSC-trehalose glasses that fix the proteins in their folded states under oxygen-limited conditions. The encapsulation dramatically enhances the stability of protein-OSC complexes to photodamage, increasing the lifetime of the chromophores from several hours to more than 10 weeks under constant illumination. Comparison of the photophysical properties of astaxanthin aggregates in mixed-solvent systems and proteins shows that the peptide environment does not alter the underlying electronic processes of the incorporated materials, exemplified here by singlet exciton fission followed by separation into weakly bound, localized triplets. This adaptable protein-based approach lays the foundation for spectroscopic assessment of a broad range of molecular OSCs in aqueous solutions and the solid-state, circumventing the laborious procedure of identifying the experimental conditions necessary for aggregate generation or film formation. The non-native protein functions also raise the prospect of future biocompatible devices where peptide assemblies could complex with native and non-native systems to generate novel functional materials.

Ultrafast flavin/tryptophan radical pair kinetics in a magnetically sensitive artificial protein.

Radical pair formation and decay are implicated in a wide range of biological processes including avian magnetoreception. However, studying such biological radical pairs is complicated by both the complexity and relative fragility of natural systems. To resolve open questions about how natural flavin-amino acid radical pair systems are engineered, and to create new systems with novel properties, we developed a stable and highly adaptable de novo artificial protein system. These protein maquettes are designed with intentional simplicity and transparency to tolerate aggressive manipulations that are impractical or impossible in natural proteins. Here we characterize the ultrafast dynamics of a series of maquettes with differing electron-transfer distance between a covalently ligated flavin and a tryptophan in an environment free of other potential radical centers. We resolve the spectral signatures of the cysteine-ligated flavin singlet and triplet states and reveal the picosecond formation and recombination of singlet-born radical pairs. Magnetic field-sensitive triplet-born radical pair formation and recombination occurs at longer timescales. These results suggest that both triplet- and singlet-born radical pairs could be exploited as biological magnetic sensors.

Rational Construction of Compact de Novo-Designed Biliverdin-Binding Proteins.

We report the rational construction of de novo-designed biliverdin-binding proteins by first principles of protein design, informed by energy minimization modeling in Rosetta. The self-assembling tetrahelical bundles bind biliverdin IXa (BV) cofactor autocatalytically in vitro, like photosensory proteins that bind BV (and related bilins or linear tetrapyrroles) despite lacking sequence and structural homology to the natural counterparts. Upon identification of a suitable site for ligation of the cofactor to the protein scaffold, stepwise placement of residues stabilized BV within the hydrophobic core. Rosetta modeling was used in the absence of a high-resolution structure to inform the structure-function relationships of the cofactor binding pocket. Holoprotein formation stabilized BV, resulting in increased far-red BV fluorescence. Via removal of segments extraneous to cofactor stabilization or bundle stability, the initial 15 kDa de novo-designed fluorescence-activating protein was truncated without any change to its optical properties, down to a miniature 10 kDa "mini", in which the protein scaffold extends only a half-heptad repeat beyond the hypothetical position of the bilin D-ring. This work demonstrates how highly compact holoprotein fluorochromes can be rationally constructed using de novo protein design technology and natural cofactors.

A synthetic biological quantum optical system.

In strong plasmon-exciton coupling, a surface plasmon mode is coupled to an array of localized emitters to yield new hybrid light-matter states (plexcitons), whose properties may in principle be controlled via modification of the arrangement of emitters. We show that plasmon modes are strongly coupled to synthetic light-harvesting maquette proteins, and that the coupling can be controlled via alteration of the protein structure. For maquettes with a single chlorin binding site, the exciton energy (2.06 ± 0.07 eV) is close to the expected energy of the Qy transition. However, for maquettes containing two chlorin binding sites that are collinear in the field direction, an exciton energy of 2.20 ± 0.01 eV is obtained, intermediate between the energies of the Qx and Qy transitions of the chlorin. This observation is attributed to strong coupling of the LSPR to an H-dimer state not observed under weak coupling.

Magnetically Sensitive Radical Photochemistry of Non-natural Flavoproteins.

It is a remarkable fact that ∼50 µT magnetic fields can alter the rates and yields of certain free-radical reactions and that such effects might be the basis of the light-dependent ability of migratory birds to sense the direction of the Earth's magnetic field. The most likely sensory molecule at the heart of this chemical compass is cryptochrome, a flavin-containing protein that undergoes intramolecular, blue-light-induced electron transfer to produce magnetically sensitive radical pairs. To learn more about the factors that control the magnetic sensitivity of cryptochromes, we have used a set of de novo designed protein maquettes that self-assemble as four-α-helical proteins incorporating a single tryptophan residue as an electron donor placed approximately 0.6, 1.1, or 1.7 nm away from a covalently attached riboflavin as chromophore and electron acceptor. Using a specifically developed form of cavity ring-down spectroscopy, we have characterized the photochemistry of these designed flavoprotein maquettes to determine the identities and kinetics of the transient radicals responsible for the magnetic field effects. Given the gross structural and dynamic differences from the natural proteins, it is remarkable that the maquettes show magnetic field effects that are so similar to those observed for cryptochromes.

De novo synthetic biliprotein design, assembly and excitation energy transfer.

Bilins are linear tetrapyrrole chromophores with a wide range of visible and near-visible light absorption and emission properties. These properties are tuned upon binding to natural proteins and exploited in photosynthetic light-harvesting and non-photosynthetic light-sensitive signalling. These pigmented proteins are now being manipulated to develop fluorescent experimental tools. To engineer the optical properties of bound bilins for specific applications more flexibly, we have used first principles of protein folding to design novel, stable and highly adaptable bilin-binding four-α-helix bundle protein frames, called maquettes, and explored the minimal requirements underlying covalent bilin ligation and conformational restriction responsible for the strong and variable absorption, fluorescence and excitation energy transfer of these proteins. Biliverdin, phycocyanobilin and phycoerythrobilin bind covalently to maquette Cys in vitro A blue-shifted tripyrrole formed from maquette-bound phycocyanobilin displays a quantum yield of 26%. Although unrelated in fold and sequence to natural phycobiliproteins, bilin lyases nevertheless interact with maquettes during co-expression in Escherichia coli to improve the efficiency of bilin binding and influence bilin structure. Bilins bind in vitro and in vivo to Cys residues placed in loops, towards the amino end or in the middle of helices but bind poorly at the carboxyl end of helices. Bilin-binding efficiency and fluorescence yield are improved by Arg and Asp residues adjacent to the ligating Cys on the same helix and by His residues on adjacent helices.

Probing the quality control mechanism of the Escherichia coli twin-arginine translocase with folding variants of a de novo-designed heme protein.

Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin-arginine translocase (Tat). The Tat machinery exports folded and cofactor-containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality-control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model Escherichia coli Tat system to recognize and translocate de novo-designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one, or no heme b cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional 1H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the E. coli trimethylamine-N-oxide (TMAO) reductase (TorA) to the N terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme b-induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system's quality-control mechanism.

Design and engineering of water-soluble light-harvesting protein maquettes.

Natural selection in photosynthesis has engineered tetrapyrrole based, nanometer scale, light harvesting and energy capture in light-induced charge separation. By designing and creating nanometer scale artificial light harvesting and charge separating proteins, we have the opportunity to reengineer and overcome the limitations of natural selection to extend energy capture to new wavelengths and to tailor efficient systems that better meet human as opposed to cellular energetic needs. While tetrapyrrole cofactor incorporation in natural proteins is complex and often assisted by accessory proteins for cofactor transport and insertion, artificial protein functionalization relies on a practical understanding of the basic physical chemistry of protein and cofactors that drive nanometer scale self-assembly. Patterning and balancing of hydrophobic and hydrophilic tetrapyrrole substituents is critical to avoid natural or synthetic porphyrin and chlorin aggregation in aqueous media and speed cofactor partitioning into the non-polar core of a man-made water soluble protein designed according to elementary first principles of protein folding. This partitioning is followed by site-specific anchoring of tetrapyrroles to histidine ligands strategically placed for design control of rates and efficiencies of light energy and electron transfer while orienting at least one polar group towards the aqueous phase.

Multi-step excitation energy transfer engineered in genetic fusions of natural and synthetic light-harvesting proteins.

Synthetic proteins designed and constructed from first principles with minimal reference to the sequence of any natural protein have proven robust and extraordinarily adaptable for engineering a range of functions. Here for the first time we describe the expression and genetic fusion of a natural photosynthetic light-harvesting subunit with a synthetic protein designed for light energy capture and multi-step transfer. We demonstrate excitation energy transfer from the bilin of the CpcA subunit (phycocyanin α subunit) of the cyanobacterial photosynthetic light-harvesting phycobilisome to synthetic four-helix-bundle proteins accommodating sites that specifically bind a variety of selected photoactive tetrapyrroles positioned to enhance energy transfer by relay. The examination of combinations of different bilin, chlorin and bacteriochlorin cofactors has led to identification of the preconditions for directing energy from the bilin light-harvesting antenna into synthetic protein-cofactor constructs that can be customized for light-activated chemistry in the cell.

Engineering an Artificial Flavoprotein Magnetosensor.

Migratory birds use the Earth's magnetic field as a source of navigational information. This light-dependent magnetic compass is thought to be mediated by cryptochrome proteins in the retina. Upon light activation, electron transfer between the flavin adenine dinucleotide cofactor and tryptophan residues leads to the formation of a spin-correlated radical pair, whose subsequent fate is sensitive to external magnetic fields. To learn more about the functional requirements of this complex chemical compass, we have created a family of simplified, adaptable proteins-maquettes-that contain a single tryptophan residue at different distances from a covalently bound flavin. Despite the complete absence of structural resemblance to the native cryptochrome fold or sequence, the maquettes exhibit a strong magnetic field effect that rivals those observed in the natural proteins in vitro. These novel maquette designs offer unprecedented flexibility to explore the basic requirements for magnetic sensing in a protein environment.

Strong Coupling of Localized Surface Plasmons to Excitons in Light-Harvesting Complexes.

Gold nanostructure arrays exhibit surface plasmon resonances that split after attaching light harvesting complexes 1 and 2 (LH1 and LH2) from purple bacteria. The splitting is attributed to strong coupling between the localized surface plasmon resonances and excitons in the light-harvesting complexes. Wild-type and mutant LH1 and LH2 from Rhodobacter sphaeroides containing different carotenoids yield different splitting energies, demonstrating that the coupling mechanism is sensitive to the electronic states in the light harvesting complexes. Plasmon-exciton coupling models reveal different coupling strengths depending on the molecular organization and the protein coverage, consistent with strong coupling. Strong coupling was also observed for self-assembling polypeptide maquettes that contain only chlorins. However, it is not observed for monolayers of bacteriochlorophyll, indicating that strong plasmon-exciton coupling is sensitive to the specific presentation of the pigment molecules.

Designing Light-Activated Charge-Separating Proteins with a Naphthoquinone Amino Acid.

The first principles design of manmade redox-protein maquettes is used to clarify the physical/chemical engineering supporting the mechanisms of natural enzymes with a view to recapitulate and surpass natural performance. Herein, we use intein-based protein semisynthesis to pair a synthetic naphthoquinone amino acid (Naq) with histidine-ligated photoactive metal-tetrapyrrole cofactors, creating a 100 µs photochemical charge separation unit akin to photosynthetic reaction centers. By using propargyl groups to protect the redox-active para-quinone during synthesis and assembly while permitting selective activation, we gain the ability to employ the quinone amino acid redox cofactor with the full set of natural amino acids in protein design. Direct anchoring of quinone to the protein backbone permits secure and adaptable control of intraprotein electron-tunneling distances and rates.

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 the assembly of heme cofactors in man-made proteins.

Timely ligation of one or more chemical cofactors at preselected locations in proteins is a critical preamble for catalysis in many natural enzymes, including the oxidoreductases and allied transport and signaling proteins. Likewise, ligation strategies must be directly addressed when designing oxidoreductase and molecular transport functions in man-made, first-principle protein constructs intended to operate in vitro or in vivo. As one of the most common catalytic cofactors in biology, we have chosen heme B, along with its chemical analogues, to determine the kinetics and barriers to cofactor incorporation and bishistidine ligation in a range of 4-α-helix proteins. We compare five elementary synthetic designs (maquettes) and the natural cytochrome b562 that differ in oligomeric forms, apo- and holo-tertiary structural stability; qualities that we show can either assist or hinder assembly. The cofactor itself also imposes an assembly barrier if amphiphilicity ranges toward too hydrophobic or hydrophilic. With progressive removal of identified barriers, we achieve maquette assembly rates as fast as native cytochrome b562, paving the way to in vivo assembly of man-made hemoprotein maquettes and integration of artificial proteins into enzymatic pathways.

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.

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.