Mutually exclusive protein folding

描述（由申请人提供）：许多蛋白质是由结构上不同的亚基构建而成的，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造一类新的双功能、双结构域蛋白，以捕获这种构象驱动的变构开关的特性。这一目标将通过实施互斥折叠的新概念来实现，其中存储在一个亚基的天然结构中的自由能被用来驱动同一分子内另一个亚基的展开。融合蛋白是通过将一种蛋白质插入另一种蛋白质的表面环而产生的。拓扑约束使这两个畴陷入一场热力学拉锯战，在任何给定的时间内，只有一个畴能以折叠态出现。他们不能同时存在于自己的国家。这种配体结合的协同、可逆和可控的构象平衡是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的耦合结合和折叠机制的模型。互斥折叠所提供的独特性质将被进一步利用来开发两个新的应用。第一种是基于埃希氏菌线圈的方法，用于快速选择体内超稳定的蛋白质变体。互斥折叠设计，结合使用细胞毒性酶的蛋白质结构域之一，导致前所未有的多功能性和吞吐量的选择方法。第二种是一类杀死特定细胞类型的细胞毒性酶。通过互斥折叠设计，催化结构域的活性通过配体与工程调节结构域的结合而打开或关闭。大量蛋白质中的任何一种的配体结合域都可以执行此功能。这种转换机制形成了产生细胞毒性蛋白的基础，这些蛋白被各种细胞特异性效应分子激活，从而可以靶向癌细胞或病毒感染的细胞进行破坏。

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.