Engineering Strategy and Vector Library for the Rapid Generation of Modular Light-Controlled Protein–Protein Interactions

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Abstract

Optogenetics enables the spatio-temporally precise control of cell and animal behavior. Many optogenetic tools are driven by light-controlled protein–protein interactions (PPIs) that are repurposed from natural light-sensitive domains (LSDs). Applying light-controlled PPIs to new target proteins is challenging because it is difficult to predict which of the many available LSDs, if any, will yield robust light regulation. As a consequence, fusion protein libraries need to be prepared and tested, but methods and platforms to facilitate this process are currently not available. Here, we developed a genetic engineering strategy and vector library for the rapid generation of light-controlled PPIs. The strategy permits fusing a target protein to multiple LSDs efficiently and in two orientations. The public and expandable library contains 29 vectors with blue, green or red light-responsive LSDs, many of which have been previously applied ex vivo and in vivo. We demonstrate the versatility of the approach and the necessity for sampling LSDs by generating light-activated caspase-9 (casp9) enzymes. Collectively, this work provides a new resource for optical regulation of a broad range of target proteins in cell and developmental biology.
Original languageEnglish
Number of pages10
JournalJournal of Molecular Biology
DOIs
Publication statusAccepted/In press - 29 May 2019

Keywords

  • Optogenetics
  • light-sensitive domain
  • photoreceptor
  • protein-protein interaction
  • caspase9

Cite this

@article{f6d12fdbc83c47019f3614fda70c3575,
title = "Engineering Strategy and Vector Library for the Rapid Generation of Modular Light-Controlled Protein–Protein Interactions",
abstract = "Optogenetics enables the spatio-temporally precise control of cell and animal behavior. Many optogenetic tools are driven by light-controlled protein–protein interactions (PPIs) that are repurposed from natural light-sensitive domains (LSDs). Applying light-controlled PPIs to new target proteins is challenging because it is difficult to predict which of the many available LSDs, if any, will yield robust light regulation. As a consequence, fusion protein libraries need to be prepared and tested, but methods and platforms to facilitate this process are currently not available. Here, we developed a genetic engineering strategy and vector library for the rapid generation of light-controlled PPIs. The strategy permits fusing a target protein to multiple LSDs efficiently and in two orientations. The public and expandable library contains 29 vectors with blue, green or red light-responsive LSDs, many of which have been previously applied ex vivo and in vivo. We demonstrate the versatility of the approach and the necessity for sampling LSDs by generating light-activated caspase-9 (casp9) enzymes. Collectively, this work provides a new resource for optical regulation of a broad range of target proteins in cell and developmental biology.",
keywords = "Optogenetics, light-sensitive domain, photoreceptor, protein-protein interaction, caspase9",
author = "Alexandra-Madelaine Tichy and Gerrard, {Elliot J.} and Legrand, {Julien M.D.} and Hobbs, {Robin M.} and Harald Janovjak",
year = "2019",
month = "5",
day = "29",
doi = "10.1016/j.jmb.2019.05.033",
language = "English",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Elsevier",

}

TY - JOUR

T1 - Engineering Strategy and Vector Library for the Rapid Generation of Modular Light-Controlled Protein–Protein Interactions

AU - Tichy, Alexandra-Madelaine

AU - Gerrard, Elliot J.

AU - Legrand, Julien M.D.

AU - Hobbs, Robin M.

AU - Janovjak, Harald

PY - 2019/5/29

Y1 - 2019/5/29

N2 - Optogenetics enables the spatio-temporally precise control of cell and animal behavior. Many optogenetic tools are driven by light-controlled protein–protein interactions (PPIs) that are repurposed from natural light-sensitive domains (LSDs). Applying light-controlled PPIs to new target proteins is challenging because it is difficult to predict which of the many available LSDs, if any, will yield robust light regulation. As a consequence, fusion protein libraries need to be prepared and tested, but methods and platforms to facilitate this process are currently not available. Here, we developed a genetic engineering strategy and vector library for the rapid generation of light-controlled PPIs. The strategy permits fusing a target protein to multiple LSDs efficiently and in two orientations. The public and expandable library contains 29 vectors with blue, green or red light-responsive LSDs, many of which have been previously applied ex vivo and in vivo. We demonstrate the versatility of the approach and the necessity for sampling LSDs by generating light-activated caspase-9 (casp9) enzymes. Collectively, this work provides a new resource for optical regulation of a broad range of target proteins in cell and developmental biology.

AB - Optogenetics enables the spatio-temporally precise control of cell and animal behavior. Many optogenetic tools are driven by light-controlled protein–protein interactions (PPIs) that are repurposed from natural light-sensitive domains (LSDs). Applying light-controlled PPIs to new target proteins is challenging because it is difficult to predict which of the many available LSDs, if any, will yield robust light regulation. As a consequence, fusion protein libraries need to be prepared and tested, but methods and platforms to facilitate this process are currently not available. Here, we developed a genetic engineering strategy and vector library for the rapid generation of light-controlled PPIs. The strategy permits fusing a target protein to multiple LSDs efficiently and in two orientations. The public and expandable library contains 29 vectors with blue, green or red light-responsive LSDs, many of which have been previously applied ex vivo and in vivo. We demonstrate the versatility of the approach and the necessity for sampling LSDs by generating light-activated caspase-9 (casp9) enzymes. Collectively, this work provides a new resource for optical regulation of a broad range of target proteins in cell and developmental biology.

KW - Optogenetics

KW - light-sensitive domain

KW - photoreceptor

KW - protein-protein interaction

KW - caspase9

U2 - 10.1016/j.jmb.2019.05.033

DO - 10.1016/j.jmb.2019.05.033

M3 - Article

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

ER -