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:

Wu, Bingyan ^[1]; Atkinson, Joshua T.  ^[1];

• Search DOE PAGES for author "Atkinson, Joshua T."
• Search DOE PAGES for ORCID "0000-0001-9293-4123"
• Search orcid.org for ORCID "0000-0001-9293-4123"

Kahanda, Dimithree ^[1]; Bennett, George N. ^[1]; Silberg, Jonathan J.  ^[1]

• Search DOE PAGES for author "Silberg, Jonathan J."
• Search DOE PAGES for ORCID "0000-0001-5612-0667"
• Search orcid.org for ORCID "0000-0001-5612-0667"

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+ Show Author Affiliations

1. Rice Univ., Houston, TX (United States)

Publication Date:

2019-09-10

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