Bohdana M. Discher

BIOMIMETIC CHEMICAL SENSORS USING NANOELECTRONIC READOUT OF OLFACTORY RECEPTORS

We have designed and implemented a practical nanoelectronic interface to G-protein coupled receptors (GPCRs), a large family of membrane proteins whose roles in the detection of molecules outside eukaryotic cells make them important pharmaceutical targets. Specifically, we have coupled olfactory receptor proteins (ORs) with carbon nanotube transistors. The resulting devices transduce signals associated with odorant binding to ORs in the gas phase under ambient conditions and show responses that are in excellent agreement with results from established assays for OR-ligand binding. The work represents significant progress on a path toward a bioelectronic nose that can be directly compared to biological olfactory systems as well as a general method for the study of GPCR function in multiple domains using electronic readout.

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.

Functionalizing Nanocrystalline Metal Oxide Electrodes With Robust Synthetic Redox Proteins

De novo designed synthetic redox proteins (maquettes) are structurally simpler, working counterparts of natural redox proteins. The robustness and adaptability of the maquette protein scaffold are ideal for functionalizing electrodes. A positive amino acid patch has been designed into a maquette surface for strong electrostatic anchoring to the negatively charged surfaces of nanocrystalline, mesoporous TiO2 and SnO2 films. Such mesoporous metal oxide electrodes offer a major advantage over conventional planar gold electrodes by facilitating formation of high optical density, spectroelectrochemically active thin films with protein loading orders of magnitude greater (up to 8 nmol cm−2) than that achieved with gold electrodes. The films are stable for weeks, essentially all immobilized‐protein display rapid, reversible electrochemistry. Furthermore, carbon monoxide ligand binding to the reduced heme group of the protein is maintained, can be sensed optically and reversed electrochemically. Pulsed UV excitation of the metal oxide results in microsecond or faster photoreduction of an immobilized cytochrome and millisecond reoxidation. Upon substitution of the heme‐group Fe by Zn, the light‐activated maquette injects electrons from the singlet excited state of the Zn protoporphyrin IX into the metal oxide conduction band. The kinetics of cytochrome/metal oxide interfacial electron transfer obtained from the electrochemical and photochemical data obtained are discussed in terms of the free energies of the observed reactions and the electronic coupling between the protein heme group and the metal oxide surface.

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.

Direct probe of molecular polarization in de novo protein-electrode interfaces.

A novel approach to energy harvesting and biosensing devices would exploit optoelectronic processes found in proteins that occur in nature. However, in order to design such systems, the proteins need to be attached to electrodes and the optoelectronic properties in nonliquid (ambient) environments must be understood at a fundamental level. Here we report the simultaneous detection of electron transport and the effect of optical absorption on dielectric polarizability in oriented peptide single molecular layers. This characterization requires a peptide design strategy to control protein/electrode interface interactions, to allow peptide patterning on a substrate, and to induce optical activity. In addition, a new method to probe electronic, dielectric, and optical properties at the single molecular layer level is demonstrated. The combination enables a quantitative comparison of the change in polarization volume between the ground state and excited state in a single molecular layer in a manner that allows spatial mapping relevant to ultimate device design.

First principles design of a core bioenergetic transmembrane electron-transfer protein.

Here we describe the design, Escherichia coli expression and characterization of a simplified, adaptable and functionally transparent single chain 4-α-helix transmembrane protein frame that binds multiple heme and light activatable porphyrins. Such man-made cofactor-binding oxidoreductases, designed from first principles with minimal reference to natural protein sequences, are known as maquettes. This design is an adaptable frame aiming to uncover core engineering principles governing bioenergetic transmembrane electron-transfer function and recapitulate protein archetypes proposed to represent the origins of photosynthesis. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

De Novo Construction of Redox Active Proteins.

Relatively simple principles can be used to plan and construct de novo proteins that bind redox cofactors and participate in a range of electron-transfer reactions analogous to those seen in natural oxidoreductase proteins. These designed redox proteins are called maquettes. Hydrophobic/hydrophilic binary patterning of heptad repeats of amino acids linked together in a single-chain self-assemble into 4-alpha-helix bundles. These bundles form a robust and adaptable frame for uncovering the default properties of protein embedded cofactors independent of the complexities introduced by generations of natural selection and allow us to better understand what factors can be exploited by man or nature to manipulate the physical chemical properties of these cofactors. Anchoring of redox cofactors such as hemes, light active tetrapyrroles, FeS clusters, and flavins by His and Cys residues allow cofactors to be placed at positions in which electron-tunneling rates between cofactors within or between proteins can be predicted in advance. The modularity of heptad repeat designs facilitates the construction of electron-transfer chains and novel combinations of redox cofactors and new redox cofactor assisted functions. Developing de novo designs that can support cofactor incorporation upon expression in a cell is needed to support a synthetic biology advance that integrates with natural bioenergetic pathways.

Cellular mimics engineered from diblock copolymers

Cell-size vesicles were made from amphiphilic diblock copolymers and characterized by micromanipulation. The average molecular weight of the base, synthetic polymer studied, polyethyleneoxide-polyethylethylene (EO/sub 40/-EE/sub 37/), is several-fold greater than that of usual phospholipids of biomembranes. Both the membrane bending and area expansion moduli of electroformed polymersomes (polymer-based liposomes) fell within the range of lipid membrane measurements, but the giant polymersomes membrane proved to be almost an order of magnitude tougher, sustaining far greater areal strain before rupture. The polymersome membrane was also at least tenfold less permeable to water than common phospholipid bilayers. A rich range of vesicle shapes, as well as encapsulation of oxygen-binding proteins, and capabilities to mix the polymer with other amphiphiles and crosslink it, all suggest a new class of functional, biomimetic capsules based on block copolymer chemistry.

Converting Signalling Pathway of Olfactory Receptor Proteins Into Electronic Read Out

Integration of modern nanoelectronic technology with the potent molecular machines of living organisms offers a pathway to advanced chemical sensing and high throughput screening of ligand binding. Integration of amphiphilic membrane proteins remains a challenging problem despite their vital and varied functionality in living organisms. We have created a nanoelectronic interface to G-protein coupled receptors (GPCRs), a large family of membrane proteins whose roles in the detection of molecules outside eukaryotic cells and initiation of cascades of intracellular responses make them important pharmaceutical targets. Olfactory receptor proteins (ORs) are the most numerous class of GPCRs, representing transcription products of ∼ 3% of the mammalian genome. We report a method to integrate ORs with carbon nanotube (NT) transistors. The resulting devices transduce signals associated with odorant binding to ORs in the gas phase under ambient conditions and show responses that are in excellent agreement with results from established assays for OR-ligand binding. The work represents significant progress towards an electronic nose that can be directly compared to biological olfactory systems as well as a general method for the study of GPCR function in multiple domains using electronic readout.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Design of Transmembrane Electron Transport Chain within Amphiphilic Protein Maquettes

Electron transport chains are fundamental to both photosynthesis and oxidative phosphorylation. Protein-based electron transport chains transfer electrons from high-energy donors to lower-energy acceptors and are commonly coupled to the translocation of protons across a membrane, producing a transmembrane electrochemical potential gradient. Electron transfer rates within these chains are governed primarily by the distance between redox centers and by the driving force that originates from the redox mid-point potentials or coupled catalytic reactions. The complexity of natural redox protein structures contrasts the relatively simple rules of cofactor placement that, in principle, govern the electron transfer behavior. Rather than focusing on the structural details of a specific natural protein, we are designing general protein structural scaffolds (“maquettes”) to accommodate a variety of functions. Here we will present transmembrane electron transfer via AP6, an amphiphilic tetra-helical maquette that binds up to 6 hemes. We demonstrate that AP6 self-assembles with phospholipids into vesicles. Our stop flow experiments confirm that the AP6 maquette significantly increases the electron transfer rates between oxidizing interior and an external redox mediator dye, as shown below.View Large Image | View Hi-Res Image | Download PowerPoint Slide