Journal
BIOCHEMISTRY
Volume 57, Issue 18, Pages 2590-2596Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.biochem.8b00131
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Funding
- Cell and Molecular Biology National Institutes of Health (NIH) Training Grant [5-T32-GM-007229-39]
- NIH postdoctoral fellowship [F32GM119430]
- CASI grant from Burroughs Wellcome Fund
- Charles E. Kaufman Foundation
- National Science Foundation (NSF) Graduate Research Fellowship
- NSF [CHE-1404836]
- NIH [GM118510]
- U.S. Department of Energy [DE-SC0007063]
- U.S. Department of Energy (DOE) [DE-SC0007063] Funding Source: U.S. Department of Energy (DOE)
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We report inducible dimerization strategies for controlling protein positioning, enzymatic activity, and organelle assembly inside synthetic cell-like compartments upon photostimulation. Using a photocaged TMP-Haloligand compound, we demonstrate small molecule and light-induced dimerization of DHFR and Haloenzyme to localize proteins to a compartment boundary and reconstitute tripartite sfGFP assembly. Using photocaged rapamycin and fragments of split TEV protease fused to FRB and FKBP, we establish optical triggering of protease activity inside cell-size compartments. We apply light inducible protease activation to initiate assembly of membraneless organelles, demonstrating the applicability of these tools for characterizing cell biological processes in vitro. This modular toolkit, which affords spatial and temporal control of protein function in a minimal cell-like system, represents a critical step toward the reconstitution of a tunable synthetic cell, built from the bottom up.
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