Control of Protein Activity Via Light Stimulated Dimerizers

描述（由申请人提供）：允许蛋白质活性的诱导型控制、允许信号传导途径、蛋白质表达和其他过程的精确操纵的系统已经改变了实验生物学。虽然许多研究已经使用化学物质来干扰这些过程，但近年来科学家已经开发出光响应的“光遗传学”工具，可以精确地控制活动的时空。在最近的工作中，我们已经开发了一个基于隐花色素（cryptochrome，CIB 2）及其相互作用伴侣CIB 1的光诱导二聚化平台，该平台允许控制蛋白质-蛋白质相互作用和光定位。我们的初步表征表明，该系统具有独特的特性，这将有助于在模型生物和体内使用，因为它具有快速动力学，是可逆的，并且是完全遗传编码的。该项目的广泛目标是进一步开发B12-CIB 1二聚化技术，使其可用于广泛的生物学应用。具体而言，我们的目标是设计改进的二聚器，其更小，单体，更严格控制，并具有改进的动态范围（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，转录激活因子和蛋白酶。总之，这些酶将能够在基因、RNA和蛋白质水平上调节生物活性的多种方法。例如，一种强大的光激活Cre重组酶将对神经科学、发育生物学和其他领域具有巨大的意义，并将在各种模型系统中广泛使用。在目标3中，我们将研究用于控制生物系统的光活化酶和B12-CIB 1系统的性质。

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.