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中途径动力学和依赖性的有力工具。光活化，或笼，是一个很好的方法，诱导变化，因为它可以几乎是瞬时的，激活可以在空间上定位。蛋白质的光活化通常需要在体外进行位点特异性化学修饰，产生通常难以添加到细胞中并且被不可逆地活化的类似物。我们的目标是创造可遗传编码的光活化蛋白质，因此可以通过DNA转染容易地引入活细胞。我们的设计策略利用了来自植物蛋白趋光蛋白的天然光反应性LOV 2结构域。当用蓝光激活时，LOV 2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LOV 2结构域的C-末端螺旋（J1-螺旋）解折叠。我们将测试光介导的LOV 2 J1-螺旋的解折叠是否可用于控制与J1-螺旋融合或嵌入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LOV 2结构域的融合将用于产生小GTP酶Rac 1、Cdc 42和RhoA的可光活化变体。初步研究表明，笼需要GTdR和LOV 2结构域上的表面残基之间的有利的相互作用。LOV 2-Rac 1融合体的晶体结构将用作蛋白质设计模拟的模板，以鉴定稳定LOV 2-GTdR融合体的笼状状态的突变。在目标2中，多状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，使得它们可以在黑暗状态下嵌入折叠的J1-螺旋中，但仍然在发光状态下结合它们的靶蛋白。在目标3中，我们将测试可光活化的LOV 2变体及其结合配偶体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LOV 2结构域的蛋白质的光活化的一般策略，并为研究各种细胞过程提供强大的工具。 公共卫生相关性：信号转导的正确时机和定位对于各种生物过程（包括分化、生长和迁移）至关重要。我们正在开发新的策略，用于快速和可逆地激活活细胞和动物中的信号通路。这些方法将使生物学家能够更好地了解与各种疾病相关的途径，包括癌症，心血管疾病和发育障碍。

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.