Mutually exclusive protein folding

互斥的蛋白质折叠

基本信息

批准号：
7256487
负责人：
STEWART N LOH
金额：
$ 25.94万
依托单位：
UPSTATE MEDICAL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-07-01 至 2008-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7256487
关键词：
Address Behavior Binding Binding Sites Biological Models Cancerous Catalytic Domain Cells Chimeric Proteins Class Complex Condition Consensus Coupled Coupling DNA DNA Binding DNA Binding Domain Databases Development Engineering Enzymes Equilibrium Escherichia Escherichia coli Free Energy Future Goals Human Ubiquitin In Vitro Life Ligand Binding Ligand Binding Domain Ligands Mediating Methodology Methods Modeling Molecular Mutation Oligonucleotides Plasmids Positioning Attribute Process Property Protein Subunits Proteins Purpose Reaction Research Personnel Ribonucleases Scheme Sequence Analysis Signal Pathway Signal Transduction Sorting - Cell Movement Structure Surface Temperature Tertiary Protein Structure Testing Thermodynamics Time Ubiquitin Variant War base cell killing cell type computerized data processing concept cytotoxic design in vivo killings molecular recognition novel numb protein programs protein folding protein protein interaction research study sensor synthetic construct therapeutic protein

项目摘要

DESCRIPTION (provided by applicant): Many proteins are built from structurally distinct subunits that communicate with each other by means of a conformational change in order to achieve overall function. The primary goal of this project is to create a new class of bi-functional, two-domain proteins that capture the properties of this conformationally-driven allosteric switch. This aim will be accomplished by implementing the novel concept of mutually exclusive folding, in which the free energy stored in the native structure of one subunit is used to drive unfolding of another subunit within the same molecule. A fusion protein is created by inserting one protein into a surface loop of another. A topological constraint causes the two domains to engage in a thermodynamic tug-of-war, from which only one can emerge in its folded state at any given-time. They cannot simultaneously exist in their native states. This conformational equilibrium cooperative, reversible, and controllable by ligand binding serves as a model for the coupled binding and folding mechanism widely used to mediate protein-protein interactions and cellular signaling processes. The unique properties afforded by mutually exclusive folding will be additionally exploited to develop two new applications. The first is an Escherichia coil based approach for rapidly selecting ultra-stable protein variants in vivo. The mutually exclusive folding design, combined with the use of a cytotoxic enzyme for one of the protein domains, results in a selection method of unprecedented versatility and throughput. The second is a class of cytotoxic enzymes that kills specific cell types. By virtue of the mutually exclusive folding design, activity of the catalytic domain is turned on or off by binding of a ligand to an engineered regulatory domain. Ligand binding domains from any one of a large number of proteins can perform this function. This switching mechanism forms the basis for developing cytotoxic proteins that are activated by a wide variety of cell-specific effector molecules, and can thus target cancerous or virally infected cells for destruction.

描述（由申请人提供）：许多蛋白质都是由结构上不同的亚基构建的，这些亚基通过构象变化相互通信以实现整体功能。该项目的主要目标是创建一类新的双功能，两域蛋白，以捕获这种构型驱动的变构开关的特性。这一目标将通过实现相互排斥的折叠的新颖概念来实现，其中一个亚基的天然结构中存储的自由能用于驱动同一分子中另一个亚基的展开。融合蛋白是通过将一种蛋白插入另一个蛋白的表面环中而产生的。拓扑约束会导致两个域进行热力学拔河，在任何给定时间中，只能从中出现一个折叠状态。他们不能同时存在于本国国家。这种构象平衡合作，可逆和可通过配体结合可控制的是耦合结合和折叠机制的模型，该模型广泛用于介导蛋白质 - 蛋白质相互作用和细胞信号传导过程。相互排斥的折叠提供的独特属性还将被利用以开发两个新的应用程序。首先是一种基于大胆管线圈的方法，用于在体内快速选择超稳的蛋白质变体。相互排斥的折叠设计，结合使用细胞毒性酶为蛋白质结构域之一，导致一种空前的多功能性和吞吐量的选择方法。第二个是一类杀死特定细胞类型的细胞毒性酶。凭借相互排斥的折叠设计，通过结合配体与工程调节域的结合，催化结构域的活性可以打开或关闭。来自大量蛋白质中任何一个的配体结合域都可以执行此功能。这种开关机制构成了开发由多种细胞特异性效应分子激活的细胞毒性蛋白的基础，因此可以靶向癌性或病毒感染的细胞来破坏。