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Title: Combinatorial design of chemical-dependent protein switches for controlling intracellular electron transfer

Abstract

One concern with controlling electron flow in cells is the lack of biomolecules that directly couple the sensing of environmental conditions to electron transfer efficiency. To overcome this protein component limitation, we randomly inserted the ligand binding domain (LBD) from the human estrogen receptor (ER) into a thermostable 2Fe-2S ferredoxin (Fd) from Mastigocladus laminosus and used a bacterial selection to identify Fd-LBD fusion proteins that support electron transfer from a Fd-NADP reductase (FNR) to a Fd-dependent sulfite reductase (SIR). Mapping LBD insertion sites onto structure revealed that Fd tolerates domain insertion adjacent to or within the tetracysteine motif that coordinates the 2Fe-2S metallocluster. With both classes of the fusion proteins, cellular ET was enhanced by the ER antagonist 4-hydroxytamoxifen, albeit to different extents. In addition, one of Fds arising from ER-LBD insertion within the tetracysteine motif was found to acquire an oxygen-tolerant 2Fe-2S cluster, suggesting that ET is regulated through post-translational ligand binding. Furthermore, these findings show that domain insertion can be used to create protein electron carriers whose ET is regulated by environmental conditions. These metalloprotein switches are expected to be useful for achieving fast regulation of ET in engineered metabolic pathways and between electroactive bacteria and conductive materials.

Authors:
 [1]; ORCiD logo [1];  [1];  [1]; ORCiD logo [1]
  1. Rice Univ., Houston, TX (United States)
Publication Date:
Research Org.:
Rice Univ., Houston, TX (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); US Department of the Navy, Office of Naval Research (ONR)
OSTI Identifier:
1594060
Alternate Identifier(s):
OSTI ID: 1570514
Grant/Contract Number:  
SC0014462; N00014‐17‐1‐2639; DE‐SC0014462
Resource Type:
Accepted Manuscript
Journal Name:
AIChE Journal
Additional Journal Information:
Journal Volume: 66; Journal Issue: 3; Journal ID: ISSN 0001-1541
Publisher:
American Institute of Chemical Engineers
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 36 MATERIALS SCIENCE; metalloprotein; ferredoxin; electron transfer; microbiology; growth selection; allostery; domain insertion

Citation Formats

Wu, Bingyan, Atkinson, Joshua T., Kahanda, Dimithree, Bennett, George N., and Silberg, Jonathan J. Combinatorial design of chemical-dependent protein switches for controlling intracellular electron transfer. United States: N. p., 2019. Web. doi:10.1002/aic.16796.
Wu, Bingyan, Atkinson, Joshua T., Kahanda, Dimithree, Bennett, George N., & Silberg, Jonathan J. Combinatorial design of chemical-dependent protein switches for controlling intracellular electron transfer. United States. doi:10.1002/aic.16796.
Wu, Bingyan, Atkinson, Joshua T., Kahanda, Dimithree, Bennett, George N., and Silberg, Jonathan J. Tue . "Combinatorial design of chemical-dependent protein switches for controlling intracellular electron transfer". United States. doi:10.1002/aic.16796.
@article{osti_1594060,
title = {Combinatorial design of chemical-dependent protein switches for controlling intracellular electron transfer},
author = {Wu, Bingyan and Atkinson, Joshua T. and Kahanda, Dimithree and Bennett, George N. and Silberg, Jonathan J.},
abstractNote = {One concern with controlling electron flow in cells is the lack of biomolecules that directly couple the sensing of environmental conditions to electron transfer efficiency. To overcome this protein component limitation, we randomly inserted the ligand binding domain (LBD) from the human estrogen receptor (ER) into a thermostable 2Fe-2S ferredoxin (Fd) from Mastigocladus laminosus and used a bacterial selection to identify Fd-LBD fusion proteins that support electron transfer from a Fd-NADP reductase (FNR) to a Fd-dependent sulfite reductase (SIR). Mapping LBD insertion sites onto structure revealed that Fd tolerates domain insertion adjacent to or within the tetracysteine motif that coordinates the 2Fe-2S metallocluster. With both classes of the fusion proteins, cellular ET was enhanced by the ER antagonist 4-hydroxytamoxifen, albeit to different extents. In addition, one of Fds arising from ER-LBD insertion within the tetracysteine motif was found to acquire an oxygen-tolerant 2Fe-2S cluster, suggesting that ET is regulated through post-translational ligand binding. Furthermore, these findings show that domain insertion can be used to create protein electron carriers whose ET is regulated by environmental conditions. These metalloprotein switches are expected to be useful for achieving fast regulation of ET in engineered metabolic pathways and between electroactive bacteria and conductive materials.},
doi = {10.1002/aic.16796},
journal = {AIChE Journal},
number = 3,
volume = 66,
place = {United States},
year = {2019},
month = {9}
}

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Works referenced in this record:

Distinct Physiological Roles of the Three Ferredoxins Encoded in the Hyperthermophilic Archaeon Thermococcus kodakarensis
journal, March 2019

  • Burkhart, Brett W.; Febvre, Hallie P.; Santangelo, Thomas J.
  • mBio, Vol. 10, Issue 2
  • DOI: 10.1128/mBio.02807-18

Structural and mutational studies of an electron transfer complex of maize sulfite reductase and ferredoxin
journal, February 2016

  • Kim, Ju Yaen; Nakayama, Masato; Toyota, Hiroshi
  • Journal of Biochemistry, Vol. 160, Issue 2
  • DOI: 10.1093/jb/mvw016

Computational design of Candida boidinii xylose reductase for altered cofactor specificity
journal, October 2009

  • Khoury, George A.; Fazelinia, Hossein; Chin, Jonathan W.
  • Protein Science, Vol. 18, Issue 10
  • DOI: 10.1002/pro.227

Crystallographic comparison of the estrogen and progesterone receptor's ligand binding domains
journal, May 1998

  • Tanenbaum, D. M.; Wang, Y.; Williams, S. P.
  • Proceedings of the National Academy of Sciences, Vol. 95, Issue 11
  • DOI: 10.1073/pnas.95.11.5998

Biogenic Control of Manganese Doping in Zinc Sulfide Nanomaterial Using Shewanella oneidensis MR-1
journal, May 2019

  • Chellamuthu, Prithiviraj; Naughton, Kyle; Pirbadian, Sahand
  • Frontiers in Microbiology, Vol. 10
  • DOI: 10.3389/fmicb.2019.00938

Metalloproteins Containing Cytochrome, Iron–Sulfur, or Copper Redox Centers
journal, March 2014

  • Liu, Jing; Chakraborty, Saumen; Hosseinzadeh, Parisa
  • Chemical Reviews, Vol. 114, Issue 8
  • DOI: 10.1021/cr400479b

Protein Science by DNA Sequencing: How Advances in Molecular Biology Are Accelerating Biochemistry
journal, October 2017


Circular permutation profiling by deep sequencing libraries created using transposon mutagenesis
journal, April 2018

  • Atkinson, Joshua T.; Jones, Alicia M.; Zhou, Quan
  • Nucleic Acids Research, Vol. 46, Issue 13
  • DOI: 10.1093/nar/gky255

An arsenic-specific biosensor with genetically engineered Shewanella oneidensis in a bioelectrochemical system
journal, December 2014

  • Webster, Dylan P.; TerAvest, Michaela A.; Doud, Devin F. R.
  • Biosensors and Bioelectronics, Vol. 62
  • DOI: 10.1016/j.bios.2014.07.003

An ingestible bacterial-electronic system to monitor gastrointestinal health
journal, May 2018


A portable bioelectronic sensing system (BESSY) for environmental deployment incorporating differential microbial sensing in miniaturized reactors
journal, September 2017


Functional Evolution and Structural Conservation in Chimeric Cytochromes P450
journal, March 2004


Nanowire–Bacteria Hybrids for Unassisted Solar Carbon Dioxide Fixation to Value-Added Chemicals
journal, April 2015


Transforming exoelectrogens for biotechnology using synthetic biology: Synthetic Biology of Exoelectrogens
journal, September 2015

  • TerAvest, Michaela A.; Ajo-Franklin, Caroline M.
  • Biotechnology and Bioengineering, Vol. 113, Issue 4
  • DOI: 10.1002/bit.25723

Self-photosensitization of nonphotosynthetic bacteria for solar-to-chemical production
journal, December 2015


Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch
journal, May 2016

  • Oakes, Benjamin L.; Nadler, Dana C.; Flamholz, Avi
  • Nature Biotechnology, Vol. 34, Issue 6
  • DOI: 10.1038/nbt.3528

Extracellular Electron Transfer by Shewanella oneidensis Controls Palladium Nanoparticle Phenotype
journal, November 2018

  • Dundas, Christopher M.; Graham, Austin J.; Romanovicz, Dwight K.
  • ACS Synthetic Biology, Vol. 7, Issue 12
  • DOI: 10.1021/acssynbio.8b00218

Post-translational tools expand the scope of synthetic biology
journal, August 2012


CymA and Exogenous Flavins Improve Extracellular Electron Transfer and Couple It to Cell Growth in Mtr-Expressing Escherichia coli
journal, April 2016

  • Jensen, Heather M.; TerAvest, Michaela A.; Kokish, Mark G.
  • ACS Synthetic Biology, Vol. 5, Issue 7
  • DOI: 10.1021/acssynbio.5b00279

Molecular basis of agonism and antagonism in the oestrogen receptor
journal, October 1997

  • Brzozowski, Andrzej M.; Pike, Ashley C. W.; Dauter, Zbigniew
  • Nature, Vol. 389, Issue 6652
  • DOI: 10.1038/39645

Engineering allosteric protein switches by domain insertion
journal, July 2005


Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA
journal, June 2011

  • Tullman, Jennifer; Guntas, Gurkan; Dumont, Matthew
  • Biotechnology and Bioengineering, Vol. 108, Issue 11
  • DOI: 10.1002/bit.23224

Directed evolution of protein switches and their application to the creation of ligand-binding proteins
journal, August 2005

  • Guntas, G.; Mansell, T. J.; Kim, J. R.
  • Proceedings of the National Academy of Sciences, Vol. 102, Issue 32
  • DOI: 10.1073/pnas.0502673102

Photosynthetic electron partitioning between [FeFe]-hydrogenase and ferredoxin:NADP+-oxidoreductase (FNR) enzymes in vitro
journal, May 2011

  • Yacoby, I.; Pochekailov, S.; Toporik, H.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 23
  • DOI: 10.1073/pnas.1103659108

Engineering a Native Inducible Expression System in Shewanella oneidensis to Control Extracellular Electron Transfer
journal, June 2017

  • West, Elizabeth A.; Jain, Abhiney; Gralnick, Jeffrey A.
  • ACS Synthetic Biology, Vol. 6, Issue 9
  • DOI: 10.1021/acssynbio.6b00349

Biochemical and Crystallographic Characterization of Ferredoxin−NADP + Reductase from Nonphotosynthetic Tissues ,
journal, December 2001

  • Aliverti, Alessandro; Faber, Rick; Finnerty, Casey M.
  • Biochemistry, Vol. 40, Issue 48
  • DOI: 10.1021/bi011224c

Evolutionary Relationships Between Low Potential Ferredoxin and Flavodoxin Electron Carriers
journal, August 2019

  • Campbell, Ian J.; Bennett, George N.; Silberg, Jonathan J.
  • Frontiers in Energy Research, Vol. 7
  • DOI: 10.3389/fenrg.2019.00079

Rapid construction of metabolite biosensors using domain-insertion profiling
journal, July 2016

  • Nadler, Dana C.; Morgan, Stacy-Anne; Flamholz, Avi
  • Nature Communications, Vol. 7, Issue 1
  • DOI: 10.1038/ncomms12266

Engineering of a synthetic electron conduit in living cells
journal, October 2010

  • Jensen, H. M.; Albers, A. E.; Malley, K. R.
  • Proceedings of the National Academy of Sciences, Vol. 107, Issue 45
  • DOI: 10.1073/pnas.1009645107

Engineering redox balance through cofactor systems
journal, June 2014


Modular electron transfer circuits for synthetic biology: insulation of an engineered biohydrogen pathway
journal, November 2010


Enhancing hydrogen production of microalgae by redirecting electrons from photosystem I to hydrogenase
journal, January 2014

  • Rumpel, Sigrun; Siebel, Judith F.; Farès, Christophe
  • Energy Environ. Sci., Vol. 7, Issue 10
  • DOI: 10.1039/C4EE01444H

Interplay of SpkG kinase and the Slr0151 protein in the phosphorylation of ferredoxin 5 in Synechocystis sp. strain PCC 6803
journal, February 2018


Programming Post-Translational Control over the Metabolic Labeling of Cellular Proteins with a Noncanonical Amino Acid
journal, May 2017


Spectroscopic elucidation of energy transfer in hybrid inorganic–biological organisms for solar-to-chemical production
journal, October 2016

  • Kornienko, Nikolay; Sakimoto, Kelsey K.; Herlihy, David M.
  • Proceedings of the National Academy of Sciences, Vol. 113, Issue 42
  • DOI: 10.1073/pnas.1610554113

Exploring protein fitness landscapes by directed evolution
journal, December 2009

  • Romero, Philip A.; Arnold, Frances H.
  • Nature Reviews Molecular Cell Biology, Vol. 10, Issue 12
  • DOI: 10.1038/nrm2805

A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism
journal, January 2011

  • Barstow, Buz; Agapakis, Christina M.; Boyle, Patrick M.
  • Journal of Biological Engineering, Vol. 5, Issue 1
  • DOI: 10.1186/1754-1611-5-7

Combinatorial assembly of ferredoxin‐linked modules in Escherichia coli yields a testing platform for Rnf‐complexes
journal, May 2019

  • Schiffels, Johannes; Selmer, Thorsten
  • Biotechnology and Bioengineering, Vol. 116, Issue 9
  • DOI: 10.1002/bit.27079

Towards environmental systems biology of Shewanella
journal, July 2008

  • Fredrickson, James K.; Romine, Margaret F.; Beliaev, Alexander S.
  • Nature Reviews Microbiology, Vol. 6, Issue 8
  • DOI: 10.1038/nrmicro1947

Structural Basis for the Thermostability of Ferredoxin from the Cyanobacterium Mastigocladus laminosus
journal, July 2005

  • Fish, Alexander; Danieli, Tsafi; Ohad, Itzhak
  • Journal of Molecular Biology, Vol. 350, Issue 3
  • DOI: 10.1016/j.jmb.2005.04.071

Mesophilic and Hyperthermophilic Adenylate Kinases Differ in Their Tolerance to Random Fragmentation
journal, February 2011

  • Segall-Shapiro, Thomas H.; Nguyen, Peter Q.; Dos Santos, Edgardo D.
  • Journal of Molecular Biology, Vol. 406, Issue 1
  • DOI: 10.1016/j.jmb.2010.11.057

Identification of Global Ferredoxin Interaction Networks in Chlamydomonas reinhardtii
journal, October 2013

  • Peden, Erin A.; Boehm, Marko; Mulder, David W.
  • Journal of Biological Chemistry, Vol. 288, Issue 49
  • DOI: 10.1074/jbc.M113.483727

Identification of ferredoxin II as a major calcium binding protein in the nitrogen-fixing symbiotic bacterium Mesorhizobium loti
journal, January 2015


The In Vivo Potential-Regulated Protective Protein of Nitrogenase in Azotobacter vinelandii Supports Aerobic Bioelectrochemical Dinitrogen Reduction In Vitro
journal, June 2017

  • Milton, Ross D.; Cai, Rong; Sahin, Selmihan
  • Journal of the American Chemical Society, Vol. 139, Issue 26
  • DOI: 10.1021/jacs.7b04893

Deep sequencing methods for protein engineering and design
journal, August 2017

  • Wrenbeck, Emily E.; Faber, Matthew S.; Whitehead, Timothy A.
  • Current Opinion in Structural Biology, Vol. 45
  • DOI: 10.1016/j.sbi.2016.11.001

Pattern of Expression and Substrate Specificity of Chloroplast Ferredoxins from Chlamydomonas reinhardtii
journal, July 2009

  • Terauchi, Aimee M.; Lu, Shu-Fen; Zaffagnini, Mirko
  • Journal of Biological Chemistry, Vol. 284, Issue 38
  • DOI: 10.1074/jbc.M109.023622

Engineered PQQ-Glucose Dehydrogenase as a Universal Biosensor Platform
journal, August 2016

  • Guo, Zhong; Murphy, Lindy; Stein, Viktor
  • Journal of the American Chemical Society, Vol. 138, Issue 32
  • DOI: 10.1021/jacs.6b06342

Circular dichroism and magnetic circular dichroism of iron-sulfur proteins
journal, October 1978

  • Stephens, P. J.; Thomson, A. J.; Dunn, J. B. R.
  • Biochemistry, Vol. 17, Issue 22
  • DOI: 10.1021/bi00615a026

The long goodbye: the rise and fall of flavodoxin during plant evolution
journal, July 2014

  • Pierella Karlusich, J. J.; Lodeyro, A. F.; Carrillo, N.
  • Journal of Experimental Botany, Vol. 65, Issue 18
  • DOI: 10.1093/jxb/eru273

Extracellular electron transfer
journal, October 2001

  • Hernandez, M. E.; Newman, D. K.
  • Cellular and Molecular Life Sciences, Vol. 58, Issue 11
  • DOI: 10.1007/PL00000796

Metalloprotein switches that display chemical-dependent electron transfer in cells
journal, December 2018

  • Atkinson, Joshua T.; Campbell, Ian J.; Thomas, Emily E.
  • Nature Chemical Biology, Vol. 15, Issue 2
  • DOI: 10.1038/s41589-018-0192-3

Engineering PQQ-glucose dehydrogenase into an allosteric electrochemical Ca 2+ sensor
journal, January 2016

  • Guo, Zhong; Johnston, Wayne A.; Stein, Viktor
  • Chemical Communications, Vol. 52, Issue 3
  • DOI: 10.1039/C5CC07824E

Cofactor engineering for advancing chemical biotechnology
journal, December 2013


The Potential for Convergence between Synthetic Biology and Bioelectronics
journal, September 2018


Insulation of a synthetic hydrogen metabolism circuit in bacteria
journal, January 2010

  • Agapakis, Christina M.; Ducat, Daniel C.; Boyle, Patrick M.
  • Journal of Biological Engineering, Vol. 4, Issue 1
  • DOI: 10.1186/1754-1611-4-3

Cell-trappable fluorescent probes for endogenous hydrogen sulfide signaling and imaging H2O2-dependent H2S production
journal, April 2013

  • Lin, V. S.; Lippert, A. R.; Chang, C. J.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 18
  • DOI: 10.1073/pnas.1302193110

A Conformational Switch Triggers Nitrogenase Protection from Oxygen Damage by Shethna Protein II (FeSII)
journal, December 2015

  • Schlesier, Julia; Rohde, Michael; Gerhardt, Stefan
  • Journal of the American Chemical Society, Vol. 138, Issue 1
  • DOI: 10.1021/jacs.5b10341

Circular permutation and receptor insertion within green fluorescent proteins
journal, September 1999

  • Baird, G. S.; Zacharias, D. A.; Tsien, R. Y.
  • Proceedings of the National Academy of Sciences, Vol. 96, Issue 20
  • DOI: 10.1073/pnas.96.20.11241

DNA Transposition of Bacteriophage Mu: A QUANTITATIVE ANALYSIS OF TARGET SITE SELECTION
journal, November 2001

  • Haapa-Paananen, Saija; Rita, Hannu; Savilahti, Harri
  • Journal of Biological Chemistry, Vol. 277, Issue 4
  • DOI: 10.1074/jbc.M108044200

Microorganisms pumping iron: anaerobic microbial iron oxidation and reduction
journal, October 2006

  • Weber, Karrie A.; Achenbach, Laurie A.; Coates, John D.
  • Nature Reviews Microbiology, Vol. 4, Issue 10, p. 752-764
  • DOI: 10.1038/nrmicro1490

Linking the functions of unrelated proteins using a novel directed evolution domain insertion method
journal, June 2008

  • Edwards, Wayne R.; Busse, Kathy; Allemann, Rudolf K.
  • Nucleic Acids Research, Vol. 36, Issue 13
  • DOI: 10.1093/nar/gkn363

Proof of principle for an engineered microbial biosensor based on Shewanella oneidensis outer membrane protein complexes
journal, September 2013


A One Pot, One Step, Precision Cloning Method with High Throughput Capability
journal, November 2008


Cellular Assays for Ferredoxins: A Strategy for Understanding Electron Flow through Protein Carriers That Link Metabolic Pathways
journal, December 2016


A new fluorescence complementation biosensor for detection of estrogenic compounds
journal, August 2011

  • McLachlan, Michael J.; Katzenellenbogen, John A.; Zhao, Huimin
  • Biotechnology and Bioengineering, Vol. 108, Issue 12
  • DOI: 10.1002/bit.23254

A synthetic biology approach to engineering living photovoltaics
journal, January 2017

  • Schuergers, N.; Werlang, C.; Ajo-Franklin, C. M.
  • Energy & Environmental Science, Vol. 10, Issue 5
  • DOI: 10.1039/C7EE00282C