Structure of Microbial Nanowires Reveals Stacked Hemes that Transport Electrons over Micrometers.

Long-range (>10 µm) transport of electrons along networks of Geobacter sulfurreducens protein filaments, known as microbial nanowires, has been invoked to explain a wide range of globally important redox phenomena. These nanowires were previously thought to be type IV pili composed of PilA protein. Here, we report a 3.7 Å resolution cryoelectron microscopy structure, which surprisingly reveals that, rather than PilA, G. sulfurreducens nanowires are assembled by micrometer-long polymerization of the hexaheme cytochrome OmcS, with hemes packed within ∼3.5-6 Å of each other. The inter-subunit interfaces show unique structural elements such as inter-subunit parallel-stacked hemes and axial coordination of heme by histidines from neighboring subunits. Wild-type OmcS filaments show 100-fold greater conductivity than other filaments from a ΔomcS strain, highlighting the importance of OmcS to conductivity in these nanowires. This structure explains the remarkable capacity of soil bacteria to transport electrons to remote electron acceptors for respiration and energy sharing.

Rapid quantification of alcohol production in microorganisms based on nanostructure-initiator mass spectrometry (NIMS).

We described a mass spectrometry-based assay to rapidly quantify the production of primary alcohols directly from cell cultures. This novel assay used the combination of TEMPO-based oxidation chemistry and oxime ligation, followed by product analysis based on Nanostructure-Initiator Mass Spectrometry. This assay enables quantitative monitor both C5 to C18 alcohols as well as glucose and gluconate in the growth medium to support strain characterization and optimization. We find that this assay yields similar results to gas chromatography for isoprenol production but required much less acquisition time per sample. We applied this assay to gain new insights into P. Putida's utilization of alcohols and find that this strain largely could not grow on heptanol and octanol.

A structural and kinetic survey of GH5_4 endoglucanases reveals determinants of broad substrate specificity and opportunities for biomass hydrolysis.

Broad-specificity glycoside hydrolases (GHs) contribute to plant biomass hydrolysis by degrading a diverse range of polysaccharides, making them useful catalysts for renewable energy and biocommodity production. Discovery of new GHs with improved kinetic parameters or more tolerant substrate-binding sites could increase the efficiency of renewable bioenergy production even further. GH5 has over 50 subfamilies exhibiting selectivities for reaction with ß-(1,4)-linked oligo- and polysaccharides. Among these, subfamily 4 (GH5_4) contains numerous broad-selectivity endoglucanases that hydrolyze cellulose, xyloglucan, and mixed-linkage glucans. We previously surveyed the whole subfamily and found over 100 new broad-specificity endoglucanases, although the structural origins of broad specificity remained unclear. A mechanistic understanding of GH5_4 substrate specificity would help inform the best protein design strategies and the most appropriate industrial application of broad-specificity endoglucanases. Here we report structures of 10 new GH5_4 enzymes from cellulolytic microbes and characterize their substrate selectivity using normalized reducing sugar assays and MS. We found that GH5_4 enzymes have the highest catalytic efficiency for hydrolysis of xyloglucan, glucomannan, and soluble ß-glucans, with opportunistic secondary reactions on cellulose, mannan, and xylan. The positions of key aromatic residues determine the overall reaction rate and breadth of substrate tolerance, and they contribute to differences in oligosaccharide cleavage patterns. Our new composite model identifies several critical structural features that confer broad specificity and may be readily engineered into existing industrial enzymes. We demonstrate that GH5_4 endoglucanases can have broad specificity without sacrificing high activity, making them a valuable addition to the biomass deconstruction toolset.

Correction: Geobacter sulfurreducens pili support ohmic electronic conduction in aqueous solution.

Correction for 'Geobacter sulfurreducens pili support ohmic electronic conduction in aqueous solution' by Nicole L. Ing et al., Phys. Chem. Chem. Phys., 2017, 19, 21791-21799.

Geobacter sulfurreducens pili support ohmic electronic conduction in aqueous solution.

The bacterium Geobacter sulfurreducens is a model biological catalyst in microbial electrochemical devices. G. sulfurreducens forms electrically conductive, electrode-associated biofilms, but the biological structures mediating electrical conduction from cells to the electrodes are a matter of debate. Bacteria in these communities produce a network of fiber-like Type IV pili, which have been proposed to act either as inherent, protein-based electronic conductors, or as electronically inert scaffolds for cytochromes mediating long-range charge transport. Previous studies have examined pilus conduction mechanisms under vacuum and in dry conditions, but their conduction mechanism under physiologically relevant conditions has yet to be characterized. In this work, we isolate G. sulfurreducens pili, and compare the electronic conduction mechanism of both live biofilms and purified pili networks under dry and aqueous conditions. Solid-state I-V characteristics indicate that electronic transport in films of purified pili is representative of conduction in a fiber percolation network. Electrochemical gating measurements in a bipotentiostat device configuration confirm previous results suggesting redox currents dominate live biofilm conduction. Purified pili films, however, exhibit non-redox electronic conduction under aqueous, buffered conditions, and their conductivity increases with decreasing temperature. These findings show that isolated pili possess inherent, non-redox-mediated conductivity consistent with a metallic-like model of charge carrier transport. The results demonstrate an experimental platform for studying electronic transport in biomaterials and suggest that pili serve as an exemplary model for designing bioelectronic interfaces.

Electronic Structure of de Novo Peptide ACC-Hex from First Principles.

Proteins are promising components for bioelectronic devices due in part to their biocompatibility, flexibility, and chemical diversity, which enable tuning of material properties. Indeed, an increasingly broad range of conductive protein supramolecular materials have been reported. However, due to their structural and environmental complexity, the electronic structure, and hence conductivity, of protein assemblies is not well-understood. Here we perform an all-atom simulation of the physical and electronic structure of a recently synthesized self-assembled peptide antiparallel coiled-coil hexamer, ACC-Hex. Using classical molecular dynamics and first-principles density functional theory, we examine the interactions of each peptide, containing phenylalanine residues along a hydrophobic core, to form a hexamer structure. We find that while frontier electronic orbitals are composed of phenylalanine, the peptide backbone and remaining residues, including those influenced by solvent, also contribute to the electronic density. Additionally, by studying dimers extracted from the hexamer, we show that structural distortions due to atomic fluctuations significantly impact the electronic structure of the peptide bundle. These results indicate that it is necessary to consider the full atomistic picture when using the electronic structure of supramolecular protein complexes to predict electronic properties.

A multiplexed nanostructure-initiator mass spectrometry (NIMS) assay for simultaneously detecting glycosyl hydrolase and lignin modifying enzyme activities.

Lignocellulosic biomass is composed of three major biopolymers: cellulose, hemicellulose and lignin. Analytical tools capable of quickly detecting both glycan and lignin deconstruction are needed to support the development and characterization of efficient enzymes/enzyme cocktails. Previously we have described nanostructure-initiator mass spectrometry-based assays for the analysis of glycosyl hydrolase and most recently an assay for lignin modifying enzymes. Here we integrate these two assays into a single multiplexed assay against both classes of enzymes and use it to characterize crude commercial enzyme mixtures. Application of our multiplexed platform based on nanostructure-initiator mass spectrometry enabled us to characterize crude mixtures of laccase enzymes from fungi Agaricus bisporus (Ab) and Myceliopthora thermophila (Mt) revealing activity on both carbohydrate and aromatic substrates. Using time-series analysis we determined that crude laccase from Ab has the higher GH activity and that laccase from Mt has the higher activity against our lignin model compound. Inhibitor studies showed a significant reduction in Mt GH activity under low oxygen conditions and increased activities in the presence of vanillin (common GH inhibitor). Ultimately, this assay can help to discover mixtures of enzymes that could be incorporated into biomass pretreatments to deconstruct diverse components of lignocellulosic biomass.

Structural Determination of a Filamentous Chaperone to Fabricate Electronically Conductive Metalloprotein Nanowires.

The transfer of electrons through protein complexes is central to cellular respiration. Exploiting proteins for charge transfer in a controllable fashion has the potential to revolutionize the integration of biological systems and electronic devices. Here we characterize the structure of an ultrastable protein filament and engineer the filament subunits to create electronically conductive nanowires under aqueous conditions. Cryoelectron microscopy was used to resolve the helical structure of gamma-prefoldin, a filamentous protein from a hyperthermophilic archaeon. Conjugation of tetra-heme c3-type cytochromes along the longitudinal axis of the filament created nanowires capable of long-range electron transfer. Electrochemical transport measurements indicated networks of the nanowires capable of conducting current between electrodes at the redox potential of the cytochromes. Functionalization of these highly engineerable nanowires with other molecules, such as redox enzymes, may be useful for bioelectronic applications.

Electrical Conductivity, Selective Adhesion, and Biocompatibility in Bacteria-Inspired Peptide-Metal Self-Supporting Nanocomposites.

Bacterial type IV pili (T4P) are polymeric protein nanofibers that have diverse biological roles. Their unique physicochemical properties mark them as a candidate biomaterial for various applications, yet difficulties in producing native T4P hinder their utilization. Recent effort to mimic the T4P of the metal-reducing Geobacter sulfurreducens bacterium led to the design of synthetic peptide building blocks, which self-assemble into T4P-like nanofibers. Here, it is reported that the T4P-like peptide nanofibers efficiently bind metal oxide particles and reduce Au ions analogously to their native counterparts, and thus give rise to versatile and multifunctional peptide-metal nanocomposites. Focusing on the interaction with Au ions, a combination of experimental and computational methods provides mechanistic insight into the formation of an exceptionally dense Au nanoparticle (AuNP) decoration of the nanofibers. Characterization of the thus-formed peptide-AuNPs nanocomposite reveals enhanced thermal stability, electrical conductivity from the single-fiber level up, and substrate-selective adhesion. Exploring its potential applications, it is demonstrated that the peptide-AuNPs nanocomposite can act as a reusable catalytic coating or form self-supporting immersible films of desired shapes. The films scaffold the assembly of cardiac cells into synchronized patches, and present static charge detection capabilities at the macroscale. The study presents a novel T4P-inspired biometallic material.

Going the Distance: Long-Range Conductivity in Protein and Peptide Bioelectronic Materials.

Bioelectronic materials interface biomolecules, cells, organs, or organisms with electronic devices, and they represent an active and growing field of materials research. Protein and peptide nanostructures are ideal bioelectronic materials. They possess many of the properties required for biocompatibility across scales from enzymatic to organismal interfaces, and recent examples of supramolecular protein and peptide nanostructures exhibit impressive electronic properties. The ability of such natural and synthetic protein and peptide materials to conduct electricity over micrometer to centimeter length scales, however, is not readily understood from a conventional view of their amino acid building blocks. Distinct in structure and properties from solid-state inorganic and synthetic organic metals and semiconductors, supramolecular conductive proteins and peptides require careful theoretical treatment and experimental characterization methods to understand their electronic structure. In this review, we discuss theory and experimental evidence from recent literature describing the long-range conduction of electronic charge in protein and peptide materials. Electron transfer across proteins has been studied extensively, but application of models for such short-range charge transport to longer distances relevant to bioelectronic materials are less well-understood. Implementation of electronic band structure and electron transfer formulations in extended biomolecular systems will be covered in the context of recent materials discoveries and efforts at characterization of electronic transport mechanisms.

Amino-acid-encoded biocatalytic self-assembly enables the formation of transient conducting nanostructures.

Aqueous compatible supramolecular materials hold promise for applications in environmental remediation, energy harvesting and biomedicine. One remaining challenge is to actively select a target structure from a multitude of possible options, in response to chemical signals, while maintaining constant, physiological conditions. Here, we demonstrate the use of amino acids to actively decorate a self-assembling core molecule in situ, thereby controlling its amphiphilicity and consequent mode of assembly. The core molecule is the organic semiconductor naphthalene diimide, functionalized with D- and L- tyrosine methyl esters as competing reactive sites. In the presence of α-chymotrypsin and a selected encoding amino acid, kinetic competition between ester hydrolysis and amidation results in covalent or non-covalent amino acid incorporation, and variable supramolecular self-assembly pathways. Taking advantage of the semiconducting nature of the naphthalene diimide core, electronic wires could be formed and subsequently degraded, giving rise to temporally regulated electro-conductivity.

Electronic Conductivity in Biomimetic α-Helical Peptide Nanofibers and Gels.

Examples of long-range electronic conductivity are rare in biological systems. The observation of micrometer-scale electronic transport through protein wires produced by bacteria is therefore notable, providing an opportunity to study fundamental aspects of conduction through protein-based materials and natural inspiration for bioelectronics materials. Borrowing sequence and structural motifs from these conductive protein fibers, we designed self-assembling peptides that form electronically conductive nanofibers under aqueous conditions. Conductivity in these nanofibers is distinct for two reasons: first, they support electron transport over distances orders of magnitude greater than expected for proteins, and second, the conductivity is mediated entirely by amino acids lacking extended conjugation, π-stacking, or redox centers typical of existing organic and biohybrid semiconductors. Electrochemical transport measurements show that the fibers support ohmic electronic transport and a metallic-like temperature dependence of conductance in aqueous buffer. At higher solution concentrations, the peptide monomers form hydrogels, and comparisons of the structure and electronic properties of the nanofibers and gels highlight the critical roles of α-helical secondary structure and supramolecular ordering in supporting electronic conductivity in these materials. These findings suggest a structural basis for long-range electronic conduction mechanisms in peptide and protein biomaterials.