Spatial and temporal control of cell behavior with a genetically-encoded photoswi

描述（由申请人提供）：许多生物过程发生在特定的时间和亚细胞位置。尽管存在高时空分辨率的活细胞分子变化描述性分析技术，但只有有限的方法允许实验控制活细胞中的蛋白质功能。我们建议用一种灵活的遗传编码系统来满足这一未满足的需求，该系统利用光来局部诱导两种蛋白质的结合。这种复杂的形成是生物学中蛋白质激活的普遍机制。我们提出的系统由两个具有高亲和力异二聚化能力的接头蛋白组成。融合到两个伙伴蛋白中的一个的光感应结构域将在暗状态下抑制异源二聚化。光的吸收将触发可逆的构象变化，这将减轻这种抑制，并允许二聚化。这些接头蛋白将融合到蛋白质中，当它们被共定位时，会触发生物反应。因此，接头蛋白的异源二聚化将导致融合伙伴相互结合并激活感兴趣的途径。因此，我们设想了一个通用系统，它将提供激活任意生物通路的能力，具有高时间和空间分辨率。重要的是，我们的策略是遗传编码的，适用于大多数细胞类型。这两个特点使绿色荧光蛋白（GFP）得到广泛应用，我们预计我们的策略也将具有广泛的实用性，并对生物学研究产生重大影响。公共卫生相关性：该项目提出了一种利用光在空间和时间上控制许多蛋白质激活和调节途径的通用策略。这种策略是遗传编码的，不需要外源辅助因子，因此它将适用于广泛的细胞和发育背景以及生化研究。

DESCRIPTION (provided by applicant): 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.