Control of Protein Activity Via Light Stimulated Dimerizers

这项工作旨在设计，优化和验证新的工具和策略来控制蛋白质活性， 光，其可以以空间和时间分辨率递送。在以前的工作中，我们率先使用了 拟南芥蓝光感受器隐花色素2作为光遗传学模块。我们 开发了一种基于与伴侣蛋白CIB 1相互作用的光诱导二聚化平台， 用于控制靶蛋白的活性和定位。在最近的工作中，我们开发了一种新的 光调节模块，p2 olig，其诱导蛋白质与光的寡聚化。其主要目的是 该项目旨在进一步开发可控制蛋白质的B12/CIB和B12 olig技术 二聚和低聚。具体而言，我们的目标是优化NH 2/CIB二聚体， 改进和扩展细胞生物学应用的性能（目标1）。在目标2中，我们将应用这些 二聚体来控制一组重要的和通用的调节酶的活性，允许操纵二聚体。 DNA重组、蛋白质切割和蛋白质光标记。重要的是，我们将进一步 优化和验证一种有前途的光激活Cre重组酶，我们将在 在啮齿动物脑中的体内。在目标3中，我们将开发和验证瞬时和局部破坏蛋白质的方法。 活性使用β 2和β 2 olig。这些工具将使研究人员能够快速，精确地破坏功能， 避免基因切除或敲除研究可能发生的补偿效应。

This work seeks to engineer, optimize, and validate new tools and strategies to control protein activity with light, which can be delivered with spatial and temporal resolution. In previous work, we pioneered use of the Arabidopsis thaliana blue light photoreceptor cryptochrome 2 (CRY2) as an optogenetic module. We developed a light-induced dimerization platform based on interaction with a partner protein, CIB1, which can be used to control activity and localization of target proteins. In recent work, we developed a new photoregulated module, CRY2olig, that induces protein oligomerization with light. The broad aims of this project are to further develop the CRY2/CIB and CRY2olig technologies allowing control of protein dimerization and oligomerization with light. Specifically, we aim to optimize CRY2/CIB dimerizers with improved and extended properties for cell biology applications (Aim 1). In Aim 2, we will apply these dimerizers to control activity of a set of important and versatile regulatory enzymes, allowing manipulation of DNA recombination, protein cleavage, and protein labeling with light. Importantly, we will carry out further optimization and validation of a promising light-activated Cre recombinase, activity of which we will test in vivo in rodent brain. In Aim 3, we will develop and validate methods to transiently and locally disrupt protein activity using CRY2 and CRY2olig. Such tools will enable researchers to quickly and precisely disrupt function, avoiding compensatory effects that can occur with genetic ablation or knockdown studies.