Radley Tracy L, Markowska Anna I, Bettinger Blaine T, Ha Jeung-Hoi, Loh Stewart N
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 E. Adams Street, Syracuse, NY 13210, USA.
J Mol Biol. 2003 Sep 19;332(3):529-36. doi: 10.1016/s0022-2836(03)00925-2.
Many proteins are built from structurally and functionally distinct domains. A major goal is to understand how conformational change transmits information between domains in order to achieve biological activity. A two-domain, bi-functional fusion protein has been designed so that the mechanical stress imposed by the folded structure of one subunit causes the other subunit to unfold, and vice versa. The construct consists of ubiquitin inserted into a surface loop of barnase. The distance between the amino and carboxyl ends of ubiquitin is much greater than the distance between the termini of the barnase loop. This topological constraint causes the two domains to engage in a thermodynamic tug-of-war in which only one can exist in its folded state at any given time. This conformational equilibrium, which is 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 position of the equilibrium can be adjusted by temperature or ligand binding and is monitored in vivo by cell death. This design forms the basis for a new class of cytotoxic proteins that can be activated by cell-specific effector molecules, and can thus target particular cell types for destruction.
许多蛋白质由结构和功能不同的结构域组成。一个主要目标是了解构象变化如何在结构域之间传递信息以实现生物活性。设计了一种双结构域、双功能融合蛋白，使得一个亚基的折叠结构施加的机械应力导致另一个亚基展开，反之亦然。该构建体由插入到核酸酶表面环中的泛素组成。泛素氨基和羧基末端之间的距离远大于核酸酶环末端之间的距离。这种拓扑限制导致两个结构域进行热力学拔河，在任何给定时间只有一个结构域能以折叠状态存在。这种构象平衡是协同的、可逆的且可通过配体结合控制的，可作为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偶联结合和折叠机制的模型。平衡位置可通过温度或配体结合来调节，并在体内通过细胞死亡进行监测。这种设计构成了一类新型细胞毒性蛋白的基础，这类蛋白可被细胞特异性效应分子激活，从而能够靶向特定细胞类型进行破坏。
