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1.
Sci Adv ; 8(19): eabm7193, 2022 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-35544567

RESUMO

Although proteins are considered as nonconductors that transfer electrons only up to 1 to 2 nanometers via tunneling, Geobacter sulfurreducens transports respiratory electrons over micrometers, to insoluble acceptors or syntrophic partner cells, via nanowires composed of polymerized cytochrome OmcS. However, the mechanism enabling this long-range conduction is unclear. Here, we demonstrate that individual nanowires exhibit theoretically predicted hopping conductance, at rate (>1010 s-1) comparable to synthetic molecular wires, with negligible carrier loss over micrometers. Unexpectedly, nanowires show a 300-fold increase in their intrinsic conductance upon cooling, which vanishes upon deuteration. Computations show that cooling causes a massive rearrangement of hydrogen bonding networks in nanowires. Cooling makes hemes more planar, as revealed by Raman spectroscopy and simulations, and lowers their reduction potential. We find that the protein surrounding the hemes acts as a temperature-sensitive switch that controls charge transport by sensing environmental perturbations. Rational engineering of heme environments could enable systematic tuning of extracellular respiration.

2.
Nat Commun ; 13(1): 829, 2022 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-35149672

RESUMO

Advances in synthetic biology permit the genetic encoding of synthetic chemistries at monomeric precision, enabling the synthesis of programmable proteins with tunable properties. Bacterial pili serve as an attractive biomaterial for the development of engineered protein materials due to their ability to self-assemble into mechanically robust filaments. However, most biomaterials lack electronic functionality and atomic structures of putative conductive proteins are not known. Here, we engineer high electronic conductivity in pili produced by a genomically-recoded E. coli strain. Incorporation of tryptophan into pili increased conductivity of individual filaments >80-fold. Computationally-guided ordering of the pili into nanostructures increased conductivity 5-fold compared to unordered pili networks. Site-specific conjugation of pili with gold nanoparticles, facilitated by incorporating the nonstandard amino acid propargyloxy-phenylalanine, increased filament conductivity ~170-fold. This work demonstrates the sequence-defined production of highly-conductive protein nanowires and hybrid organic-inorganic biomaterials with genetically-programmable electronic functionalities not accessible in nature or through chemical-based synthesis.


Assuntos
Condutividade Elétrica , Nanopartículas Metálicas/química , Nanofios , Proteínas/metabolismo , Fenômenos Químicos , Escherichia coli/genética , Proteínas de Fímbrias , Fímbrias Bacterianas/metabolismo , Ouro/química , Nanoestruturas , Nanofios/química , Fenilalanina/metabolismo , Engenharia de Proteínas , Triptofano/metabolismo
3.
Nature ; 597(7876): 430-434, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34471289

RESUMO

Extracellular electron transfer by Geobacter species through surface appendages known as microbial nanowires1 is important in a range of globally important environmental phenomena2, as well as for applications in bio-remediation, bioenergy, biofuels and bioelectronics. Since 2005, these nanowires have been thought to be type 4 pili composed solely of the PilA-N protein1. However, previous structural analyses have demonstrated that, during extracellular electron transfer, cells do not produce pili but rather nanowires made up of the cytochromes OmcS2,3 and OmcZ4. Here we show that Geobacter sulfurreducens binds PilA-N to PilA-C to assemble heterodimeric pili, which remain periplasmic under nanowire-producing conditions that require extracellular electron transfer5. Cryo-electron microscopy revealed that C-terminal residues of PilA-N stabilize its copolymerization with PilA-C (to form PilA-N-C) through electrostatic and hydrophobic interactions that position PilA-C along the outer surface of the filament. PilA-N-C filaments lack π-stacking of aromatic side chains and show a conductivity that is 20,000-fold lower than that of OmcZ nanowires. In contrast with surface-displayed type 4 pili, PilA-N-C filaments show structure, function and localization akin to those of type 2 secretion pseudopili6. The secretion of OmcS and OmcZ nanowires is lost when pilA-N is deleted and restored when PilA-N-C filaments are reconstituted. The substitution of pilA-N with the type 4 pili of other microorganisms also causes a loss of secretion of OmcZ nanowires. As all major phyla of prokaryotes use systems similar to type 4 pili, this nanowire translocation machinery may have a widespread effect in identifying the evolution and prevalence of diverse electron-transferring microorganisms and in determining nanowire assembly architecture for designing synthetic protein nanowires.


Assuntos
Fímbrias Bacterianas/química , Fímbrias Bacterianas/metabolismo , Geobacter , Nanofios , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biopolímeros , Condutividade Elétrica , Proteínas de Fímbrias/química , Proteínas de Fímbrias/metabolismo , Geobacter/citologia , Geobacter/metabolismo , Multimerização Proteica
4.
Nat Chem Biol ; 16(10): 1136-1142, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32807967

RESUMO

Multifunctional living materials are attractive due to their powerful ability to self-repair and replicate. However, most natural materials lack electronic functionality. Here we show that an electric field, applied to electricity-producing Geobacter sulfurreducens biofilms, stimulates production of cytochrome OmcZ nanowires with 1,000-fold higher conductivity (30 S cm-1) and threefold higher stiffness (1.5 GPa) than the cytochrome OmcS nanowires that are important in natural environments. Using chemical imaging-based multimodal nanospectroscopy, we correlate protein structure with function and observe pH-induced conformational switching to ß-sheets in individual nanowires, which increases their stiffness and conductivity by 100-fold due to enhanced π-stacking of heme groups; this was further confirmed by computational modeling and bulk spectroscopic studies. These nanowires can transduce mechanical and chemical stimuli into electrical signals to perform sensing, synthesis and energy production. These findings of biologically produced, highly conductive protein nanowires may help to guide the development of seamless, bidirectional interfaces between biological and electronic systems.


Assuntos
Proteínas de Bactérias/metabolismo , Estimulação Elétrica , Geobacter/fisiologia , Nanofios/química , Proteínas de Bactérias/genética , Condutividade Elétrica , Fenômenos Eletrofisiológicos
5.
Cell ; 177(2): 361-369.e10, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30951668

RESUMO

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.


Assuntos
Transporte de Elétrons/fisiologia , Geobacter/metabolismo , Heme/metabolismo , Biofilmes , Condutividade Elétrica , Elétrons , Proteínas de Fímbrias/química , Fímbrias Bacterianas/química , Nanofios , Oxirredução
6.
Curr Protoc Microbiol ; 43: A.4K.1-A.4K.27, 2016 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-27858972

RESUMO

Anaerobic microorganisms play a central role in several environmental processes and regulate global biogeochemical cycling of nutrients and minerals. Many anaerobic microorganisms are important for the production of bioenergy and biofuels. However, the major hurdle in studying anaerobic microorganisms in the laboratory is the requirement for sophisticated and expensive gassing stations and glove boxes to create and maintain the anaerobic environment. This appendix presents a simple design for a gassing station that can be used readily by an inexperienced investigator for cultivation of anaerobic microorganisms. In addition, this appendix also details the low-cost assembly of bioelectrochemical systems and outlines a simplified procedure for cultivating and analyzing bacterial cell cultures and biofilms that produce electric current, using Geobacter sulfurreducens as a model organism. © 2016 by John Wiley & Sons, Inc.


Assuntos
Biofilmes , Técnicas de Cultura de Células/métodos , Técnicas Eletroquímicas/métodos , Geobacter/crescimento & desenvolvimento , Anaerobiose , Técnicas de Cultura de Células/economia , Eletricidade , Técnicas Eletroquímicas/economia , Geobacter/química , Geobacter/fisiologia
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