Many biological processes occur at specific times and subcellular locations. Although technologies exist for descriptive analysis of molecular changes in living cells with high spatiotemporal resolution, only limited methods exist that permit experimental control of protein function in living cells. We propose to fulfill this unmet need with a flexible, genetically encoded system that uses light to locally induce the association of two proteins. Such complex formation is a widespread mechanism for protein activation in biology. Our proposed system consists of two adaptor proteins that have the capacity to heterodimerize with high affinity. A light sensing domain fused to one of the two partner proteins will inhibit heterodimerization in the dark state. Absorbance of light will trigger a reversible conformational change that will relieve this inhibition and allow dimerization. These adaptor proteins will be fused to proteins that trigger a biological response when colocalized. Hence, heterodimerization of the adaptor proteins will cause the fusion partners to associate with each other and activate the pathway of interest. Thus, we envisage a general system that will afford the ability to activate arbitrary biological pathways with high temporal and spatial resolution. Importantly, our strategy is genetically encoded and applicable to most cell types. These are the two features that have enabled the widespread use of green fluorescent protein (GFP) and we anticipate that our strategy will likewise be of broad utility and significantly impact biological research.

Public Health Relevance

This project proposes a generic strategy to spatially and temporally control the activation of many proteins and regulatory pathways using light. This strategy is genetically encoded and does not require exogenous cofactors, therefore it will be suitable for use in a wide range of cellular and developmental contexts as well as biochemical studies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZGM1-CBB-7 (EU))
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Smith, Ward
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University of Chicago
Schools of Medicine
United States
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Zayner, Josiah P; Sosnick, Tobin R (2014) Factors that control the chemistry of the LOV domain photocycle. PLoS One 9:e87074
Zayner, Josiah P; Antoniou, Chloe; French, Alexander R et al. (2013) Investigating models of protein function and allostery with a widespread mutational analysis of a light-activated protein. Biophys J 105:1027-36
Zayner, Josiah P; Antoniou, Chloe; Sosnick, Tobin R (2012) The amino-terminal helix modulates light-activated conformational changes in AsLOV2. J Mol Biol 419:61-74
Strickland, Devin; Lin, Yuan; Wagner, Elizabeth et al. (2012) TULIPs: tunable, light-controlled interacting protein tags for cell biology. Nat Methods 9:379-84
Strickland, Devin; Yao, Xiaolan; Gawlak, Grzegorz et al. (2010) Rationally improving LOV domain-based photoswitches. Nat Methods 7:623-6