skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins

Abstract

The discovery of light-inducible protein–protein interactions has allowed for the spatial and temporal control of a variety of biological processes. To be effective, a photodimerizer should have several characteristics: it should show a large change in binding affinity upon light stimulation, it should not cross-react with other molecules in the cell, and it should be easily used in a variety of organisms to recruit proteins of interest to each other. In this study, to create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa. In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB. Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation. Here, we describe the use of computational protein design, phage display, and high-throughput binding assays to create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation. A crystal structure of iLIDmore » shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark. Finally, we demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.« less

Authors:
 [1];  [1];  [1];  [1];  [1];  [2];  [3]
  1. Univ. of North Carolina, Chapel Hill, NC (United States). Dept. of Biochemistry & Biophysics
  2. Univ. of North Carolina, Chapel Hill, NC (United States). Dept. of Cell Biology & Physiology. Lineberger Comprehensive Cancer Center. Howard Hughes Medical Inst.
  3. Univ. of North Carolina, Chapel Hill, NC (United States). Dept. of Biochemistry & Biophysics. Lineberger Comprehensive Cancer Center
Publication Date:
Research Org.:
Univ. of North Carolina, Chapel Hill, NC (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); National Inst. of Health (NIH) (United States); Human Frontier Science Program (HFSP) (France)
OSTI Identifier:
1168516
Grant/Contract Number:  
W-31-109-Eng-38; GM093208
Resource Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 112; Journal Issue: 1; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 61 RADIATION PROTECTION AND DOSIMETRY; optogenetic tool; PER-ARNT-SIM domain; computational library; phage display; Rosetta molecular modeling suite

Citation Formats

Guntas, Gurkan, Hallett, Ryan A., Zimmerman, Seth P., Williams, Tishan, Yumerefendi, Hayretin, Bear, James E., and Kuhlman, Brian. Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. United States: N. p., 2014. Web. doi:10.1073/pnas.1417910112.
Guntas, Gurkan, Hallett, Ryan A., Zimmerman, Seth P., Williams, Tishan, Yumerefendi, Hayretin, Bear, James E., & Kuhlman, Brian. Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. United States. doi:10.1073/pnas.1417910112.
Guntas, Gurkan, Hallett, Ryan A., Zimmerman, Seth P., Williams, Tishan, Yumerefendi, Hayretin, Bear, James E., and Kuhlman, Brian. Mon . "Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins". United States. doi:10.1073/pnas.1417910112. https://www.osti.gov/servlets/purl/1168516.
@article{osti_1168516,
title = {Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins},
author = {Guntas, Gurkan and Hallett, Ryan A. and Zimmerman, Seth P. and Williams, Tishan and Yumerefendi, Hayretin and Bear, James E. and Kuhlman, Brian},
abstractNote = {The discovery of light-inducible protein–protein interactions has allowed for the spatial and temporal control of a variety of biological processes. To be effective, a photodimerizer should have several characteristics: it should show a large change in binding affinity upon light stimulation, it should not cross-react with other molecules in the cell, and it should be easily used in a variety of organisms to recruit proteins of interest to each other. In this study, to create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa. In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB. Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation. Here, we describe the use of computational protein design, phage display, and high-throughput binding assays to create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation. A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark. Finally, we demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.},
doi = {10.1073/pnas.1417910112},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 1,
volume = 112,
place = {United States},
year = {2014},
month = {12}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 64 works
Citation information provided by
Web of Science

Save / Share: