Design of Genetically Encoded Photoactivatable Proteins

基因编码光活化蛋白质的设计

• 批准号: 8258298
• 负责人: BRIAN A KUHLMAN
• 金额: $ 28.28万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 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/8258298
• 关键词: Actins; Adhesions; Affinity; Animals; Binding; Biological Process; C-terminal; Calpain; Cardiovascular Diseases; Cell physiology; Cells; Chemicals; Chimeric Proteins; Cysteine; DNA; Dependency; 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; developmental disease; inhibitor/antagonist; interest; migration; molecular modeling; photoactivation; phototropin; programs; public health relevance; research study; rho GTP-Binding Proteins; simulation; tool

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)：干扰细胞信号分子活性的可诱导系统是探测活细胞和动物中的通路动力学和依赖性的强大工具。光活化，或笼化，是一种很好的诱导变化的方法，因为它可以几乎是瞬时的，并且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的特定部位的化学修饰，产生的类似物通常很难添加到细胞中，并且是不可逆转的激活。我们的目标是创造可光激活的蛋白质，这些蛋白质在基因上是可编码的，因此，可以通过DNA转基因很容易地引入活细胞。我们的设计策略利用了植物蛋白趋光素的自然光反应LOV2结构域。当被蓝光激活时，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)的展开。我们将测试光介导的LOV2J1-螺旋的展开是否可以用来控制融合或嵌入J1-螺旋的蛋白质或多肽的活性。我们将专注于激活细胞迁移中关键信号通路的笼状蛋白和多肽。在目标1中，与LOV2结构域的融合将用于创建小GTP酶rac1、CDC42和RhoA的可光激活变体。初步研究表明，笼化需要GTPase表面残基和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融合的笼子状态的突变。在目标2中，多态蛋白质设计模拟将被用来改变自然产生的多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到折叠的J1-螺旋中，但仍然在发光状态下结合其目标蛋白质。在目标3中，我们将测试可光激活的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。 公共卫生相关性：信号转导的正确时机和定位对包括分化、生长和迁移在内的各种生物过程至关重要。我们正在开发新的策略，用于在活细胞和动物中快速和可逆地激活信号通路。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。