Control of Protein Activity Via Light Stimulated Dimerizers

这项工作旨在设计、优化和验证用于控制蛋白质活性的新工具和策略 光，可以以空间和时间分辨率传输。在以前的工作中，我们率先使用 拟南芥蓝光感受器隐花色素2(CRY2)作为光发生模块。我们 开发了一个基于与伙伴蛋白CIB1相互作用的光诱导二聚平台，CIB1可以 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CRY2olig，用光诱导蛋白质齐聚。这样做的主要目的是 项目是进一步开发CRY2/CIB和CRY2olig技术，使其能够控制蛋白质 光催化二聚和齐聚反应。具体地说，我们的目标是优化CRY2/CIB二聚体 改进和扩展用于细胞生物学应用的特性(目标1)。在目标2中，我们将应用这些 二聚体控制一组重要的和多功能的调节酶的活性，允许操纵 DNA重组、蛋白质裂解和蛋白质光标记。重要的是，我们将进一步开展 一种有前景的光激活Cre重组酶的优化和验证，我们将在 在啮齿类动物脑中的活体。在目标3中，我们将开发和验证瞬时和局部干扰蛋白质的方法 使用CRY2和CRY2olig的活动。这样的工具将使研究人员能够快速而准确地扰乱功能， 避免在基因消融或基因敲除研究中可能出现的补偿效应。

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.