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Proc Natl Acad Sci U S A. 2015 Jan 6;112(1):112-7. doi: 10.1073/pnas.1417910112. Epub 2014 Dec 22.

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

Author information

1
Department of Biochemistry & Biophysics.
2
Department of Cell Biology & Physiology, University of North Carolina Lineberger Comprehensive Cancer Center, and Howard Hughes Medical Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
3
Department of Biochemistry & Biophysics, University of North Carolina Lineberger Comprehensive Cancer Center, and bkuhlman@email.unc.edu.

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. 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. We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.

KEYWORDS:

PER-ARNT-SIM domain; Rosetta molecular modeling suite; computational library; optogenetic tool; phage display

PMID:
25535392
PMCID:
PMC4291625
DOI:
10.1073/pnas.1417910112
[Indexed for MEDLINE]
Free PMC Article

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