Design of Genetically Encoded Photoactivatable Proteins

描述（由申请人提供）：干扰细胞信号分子活性的诱导系统是探测活细胞和动物通路动力学和依赖性的有力工具。光激活或笼化是一种极好的诱导变化的方法，因为它几乎是瞬时的，激活可以在空间上定位。蛋白质的光激活通常需要在体外进行特定位点的化学修饰，产生的类似物通常难以添加到细胞中并且不可逆转地激活。我们的目标是创造光激活蛋白是遗传可编码的，因此，可以很容易地通过DNA转染引入活细胞。我们的设计策略利用了植物蛋白趋光素的天然光反应性LOV2结构域。当蓝光激活时，LOV2结构域中的黄素发色团与半胱氨酸450形成共价键，产生结构扰动，导致LOV2结构域C端螺旋（j1 -螺旋）展开。我们将测试光介导的LOV2 J1-螺旋展开是否可用于控制融合或嵌入J1-螺旋内的蛋白质或肽的活性。我们将重点关注在细胞迁移中激活关键信号通路的笼蛋白和肽。在目标1中，与LOV2结构域的融合将用于创建小gtpase Rac1， Cdc42和RhoA的光激活变体。初步研究表明，笼化需要GTPase表面残基与LOV2结构域之间良好的相互作用。LOV2-Rac1融合的晶体结构将被用作蛋白质设计模拟的模板，以确定稳定LOV2-GTPase融合的笼状状态的突变。在目标2中，多态蛋白质设计模拟将用于改变天然存在的肽激活剂和抑制剂的序列，以便它们可以在黑暗状态下嵌入折叠的j1 -螺旋中，但在明亮状态下仍然结合它们的靶蛋白。在目标3中，我们将测试光激活的LOV2变体及其结合伙伴是否可以用作诱导信号分子二聚化的模块。这些研究将揭示具有LOV2结构域的蛋白质光激活的一般策略，并为研究各种细胞过程提供强大的工具。

DESCRIPTION (provided by applicant): Inducible systems that perturb the activity of cell signaling molecules are powerful tools for probing pathway dynamics and dependencies in living cells and animals. Photoactivation, or caging, is an excellent method for inducing changes because it can be nearly instantaneous and activation can be spatially localized. Photoactivation of proteins has generally required site-specific chemical modification that is performed in vitro, generating analogs that are often difficult to add to cells and are irreversibly activated. Our goal is to create photoactivatable proteins that are genetically encodable, and therefore, can be readily introduced into living cells by DNA transfection. Our design strategy makes use of the naturally photoreactive LOV2 domain from the plant protein phototropin. When activated with blue light, the flavin chromophore in the LOV2 domain forms a covalent bond with cysteine 450, creating a structural perturbation that leads to the unfolding of the C- terminal helix of the LOV2 domain (the J1-helix). We will test if the light mediated unfolding of the LOV2 J1- helix can be used to control the activities of proteins or peptides that are either fused to or embedded within the J1-helix. We will focus on caging proteins and peptides that activate critical signaling pathways in cell migration. In aim 1, fusions with the LOV2 domain will be used to create photoactivatable variants of the small GTPases Rac1, Cdc42 and RhoA. Preliminary studies indicate that caging requires favorable interactions between surface residues on the GTPase and the LOV2 domain. A crystal structure of a LOV2-Rac1 fusion will be used as a template for protein design simulations to identify mutations that stabilize the caged state of LOV2-GTPase fusions. In aim 2, multi-state protein design simulations will be used to vary the sequences of naturally occurring peptide activators and inhibitors so that they can be embedded in the folded J1-helix in the dark state, but still bind their target proteins in the lit state. In aim 3, we will test if photoactivable LOV2 variants and their binding partners can be used as modules for inducing the dimerization of signaling molecules. These studies will reveal general strategies for the photoactivation of proteins with the LOV2 domain as well as provide powerful tools for studying a variety of cellular processes. PUBLIC HEALTH RELEVANCE: The correct timing and localization of signal transduction is critical to a variety of biological processes, including differentiation, growth and migration. We are developing new strategies for the rapid and reversible activation of signaling pathways in living cells and animals. These methods will allow biologists to gain a better understanding of pathways linked to a variety of diseases, including cancer, cardiovascular disease, and developmental disorders.