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.
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.
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