Control of Protein Activity Via Light Stimulated Dimerizers

描述(申请人提供)：允许可诱导地控制蛋白质活动的系统，允许精确地操纵信号通路、蛋白质表达和其他过程，已经改变了实验生物学。虽然许多研究使用化学物质来扰乱这一过程，但近年来，科学家们已经开发出对光响应的“光遗传”工具，这种工具可以对活动进行精确的时空控制。在最近的工作中，我们开发了一个基于隐花色素(CRY2)及其相互作用伙伴CIB1的光诱导二聚平台，它可以控制蛋白质之间的相互作用和用光定位。我们的初步表征表明，该系统具有独特的特性，将有助于在模式生物和体内使用，因为它具有快速的动力学，是可逆的，并且完全由基因编码。该项目的广泛目标是进一步开发CRY2-CIB1二聚化技术，使其能够在广泛的生物应用中使用。具体地说，我们的目标是设计出更小、更单体、控制更严格、动态范围更大的改进型二聚体(目标1)。在目标2中，我们将生成一个优化的光激活蛋白的“工具包”，包括重组酶、转录激活剂和蛋白酶。总而言之，这些酶将使在基因、RNA和蛋白质水平上调节生物活性的不同方法成为可能。例如，一个强大的光激活的Cre重组酶将对神经科学、发育生物学等领域具有巨大的意义，并将在体内各种模型系统中广泛使用。在目标3中，我们将研究CRY2-CIB1系统的性质以及用于控制生物系统的光激活酶。

DESCRIPTION (provided by applicant): Systems allowing inducible control of protein activities, allowing precise manipulation of signaling pathways, protein expression and other processes, have transformed experimental biology. While a number of studies have used chemicals to perturb such processes, in recent years scientists have developed light-responsive 'optogenetic' tools, which allow precise spatio- temporal control of activity. In recent work, we have developed a light-induced dimerization platform based on cryptochrome (CRY2) and its interacting partner CIB1 that allows control of protein-protein interactions and localization with light. Our initial characterization indicates that this system has distinct properties that will facilitate use in model organisms and in vivo, as it has fast kinetics, is reversible, and is entirly genetically encoded. The broad aims of this project are to further develop the CRY2-CIB1 dimerization technology such that it can be used in a broad range of biological applications. Specifically, we aim to engineer improved dimerizers that are smaller, monomeric, more tightly controlled, and have improved dynamic range (Aim 1). In Aim 2, we will generate a 'toolkit' of optimized light-activated proteins, including a recombinase, a transcriptional activator, and a protease. Together, these enzymes will enable diverse approaches for regulation of biological activity at the levels of gene, RNA, and protein. A robust light-activated Cre recombinase, for example, would have enormous significance to neuroscience, developmental biology, and other fields, and would have widespread use in vivo in a variety of model systems. In Aim 3, we will examine the properties of the CRY2-CIB1 system and light-activated enzymes for control of biological systems.