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

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:
OSTI Identifier:
1168516
Grant/Contract Number:
W-31-109-Eng-38; GM093208
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)
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)
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