Mutually exclusive protein folding

描述(申请人提供)：许多蛋白质是由结构不同的亚基组成的，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建一类新的双功能、两结构域蛋白质，以捕捉这种构象驱动的变构开关的特性。这一目标将通过实现互斥折叠的新概念来实现，在该概念中，存储在一个亚基的自然结构中的自由能被用来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质插入到另一种蛋白质的表面环中而产生的。拓扑约束导致这两个域进行热力学拉锯战，在任何给定的时间，只有一个域可能以折叠状态出现。它们不能同时存在于它们的原生状态。这种构象平衡的协作性、可逆性和配基结合的可控性为偶联结合和折叠机制提供了一个模型，该机制广泛用于介导蛋白质-蛋白质相互作用和细胞信号过程。互斥折叠所提供的独特特性将被额外利用来开发两种新的应用。第一种是基于大肠杆菌线圈的方法，用于在体内快速选择超稳定的蛋白质变体。相互排斥的折叠设计，结合对其中一个蛋白质结构域使用细胞毒酶，导致了一种前所未有的通用性和吞吐量的选择方法。第二种是一种细胞毒酶，可以杀死特定类型的细胞。由于相互排斥的折叠设计，催化结构域的活性通过将配体结合到工程的调节结构域来开启或关闭。来自大量蛋白质中任何一个的配基结合域都可以执行这一功能。这种转换机制形成了开发细胞毒蛋白的基础，这些蛋白被各种细胞特异性效应分子激活，因此可以针对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.