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

描述(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。虽然已经存在以高时空分辨率描述活细胞中分子变化的技术，但允许对活细胞中蛋白质功能进行实验控制的方法有限。我们建议通过一种灵活的、遗传编码的系统来满足这一未得到满足的需求，该系统使用光来局部诱导两种蛋白质的关联。这种复合体的形成是生物学中蛋白质激活的一种普遍机制。我们建议的系统由两个接头蛋白组成，这两个接头蛋白具有高亲和力的异二聚体能力。与两个配对蛋白中的一个融合的光敏结构域将在暗状态下抑制异源二聚作用。光的吸收将触发可逆的构象变化，从而解除这种抑制并允许二聚化。当共定位时，这些接头蛋白将与触发生物反应的蛋白融合。因此，接头蛋白的异源二聚化将导致融合伙伴相互关联并激活感兴趣的途径。因此，我们设想了一个通用系统，它将提供以高时间和空间分辨率激活任意生物路径的能力。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。这是使绿色荧光蛋白(GFP)得以广泛使用的两个特征，我们预计我们的策略将同样具有广泛的实用价值，并对生物研究产生重大影响。与公共健康相关：该项目提出了一种通用策略，利用光在空间和时间上控制许多蛋白质和调节通路的激活。这一策略是遗传编码的，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.