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）：诱导系统扰动细胞信号分子的活性是探测活细胞和动物中途径动力学和依赖性的强大工具。光活化或笼子是诱导变化的极好方法，因为它几乎可以是瞬时的，并且可以在空间上进行激活。蛋白质的光活化通常需要在体外进行的位点特异性化学修饰，从而产生通常难以添加到细胞并不可逆地激活细胞的类似物。我们的目标是创建具有遗传编码的光活化蛋白，因此可以通过DNA转染很容易地引入活细胞。我们的设计策略利用了植物蛋白光蛋白的自然光反应性LOV2结构域。当用蓝光激活时，LOV2结构域中的黄素发色团形成与半胱氨酸450的共价键，形成了结构性扰动，从而导致LOV2域的C-末端螺旋的展开（J1-螺旋）（J1-螺旋）。我们将测试LOV2 J1-螺旋的光介导的展开，可用于控制与J1-螺旋中融合或嵌入J1-螺旋中的蛋白质或肽的活性。我们将重点放在激活细胞迁移中关键信号通路的蛋白质和肽上。在AIM 1中，与LOV2域的融合将用于创建小型GTPases Rac1，cdc42和Rhoa的光活化变体。初步研究表明，笼子需要GTPase和LOV2域的表面残基之间的有利相互作用。 LOV2-RAC1融合的晶体结构将用作蛋白质设计模拟的模板，以鉴定稳定LOV2-GTPase融合笼状状态的突变。在AIM 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，但仍会在点亮状态下结合其靶蛋白。在AIM 3中，我们将测试是否可以将可光活化的LOV2变体及其结合伙伴用作诱导信号分子二聚化的模块。这些研究将揭示具有LOV2结构域蛋白质光活化的一般策略，并为研究各种细胞过程提供了强大的工具。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，以快速，可逆地激活活细胞和动物的信号通路。这些方法将使生物学家能够更好地了解与各种疾病有关的途径，包括癌症，心血管疾病和发育障碍。