Design of Genetically Encoded Photoactivatable Proteins

基因编码光活化蛋白质的设计

基本信息

批准号：
7865327
负责人：
BRIAN A KUHLMAN
金额：
$ 28.56万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-01 至 2014-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7865327
关键词：
Actins Adhesions Affinity Animals Binding Biological Process C-terminal Calpain Cardiovascular Diseases Cell physiology Cells Chemicals Chimeric Proteins Cysteine DNA Dependency Development Differentiation and Growth Dimerization Disease Family Filopodia Flavins Focal Adhesions Goals Guanosine Triphosphate Phosphohydrolases In Vitro Laboratories Libraries Life Light Link Malignant Neoplasms Mammalian Cell Mediating Methods Modeling Modification Molecular Models Monomeric GTP-Binding Proteins Mutation N-terminal PAK-1 kinase Pathway interactions Peptide Library Peptides Phage Display Plant Proteins Process Proteins Protocols documentation Reagent Signal Pathway Signal Transduction Signaling Molecule Signaling Protein Site Structure Surface System Tertiary Protein Structure Testing Time Transfection Two-Hybrid System Techniques Variant Vinculin Y protein Yeasts analog calpain inhibitor cell motility chromophore covalent bond design inhibitor/antagonist interest migration molecular modeling photoactivation phototropin programs public health relevance research study rho GTP-Binding Proteins simulation tool

项目摘要

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.

描述（由申请人提供）：扰乱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态和依赖性的强大工具。光激活或笼蔽是诱导变化的绝佳方法，因为它几乎是瞬时的，并且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激活的类似物。我们的目标是创造可基因编码的光活化蛋白质，因此可以通过 DNA 转染轻松引入活细胞中。我们的设计策略利用植物蛋白向光素的天然光反应性 LOV2 结构域。当用蓝光激活时，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 结构域的 C 末端螺旋（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。我们将重点关注激活细胞迁移中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型 GTPases Rac1、Cdc42 和 RhoA 的光激活变体。初步研究表明，笼化需要 GTP 酶和 LOV2 结构域上的表面残基之间存在有利的相互作用。 LOV2-Rac1 融合体的晶体结构将用作蛋白质设计模拟的模板，以识别稳定 LOV2-GTPase 融合体笼状态的突变。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，以便它们可以在暗态下嵌入折叠的 J1 螺旋中，但在亮态下仍然结合其目标蛋白状态。在目标 3 中，我们将测试可光激活的 LOV2 变体及其结合伴侣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。公共健康相关性：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。我们正在开发快速、可逆激活活细胞和动物信号通路的新策略。这些方法将使生物学家能够更好地了解与多种疾病相关的途径，包括癌症、心血管疾病和发育障碍。