Gardino Alexandra K, Kern Dorothee
Department of Biochemistry, Brandeis University, Waltham, MA, USA.
Methods Enzymol. 2007;423:149-65. doi: 10.1016/S0076-6879(07)23006-X.
A fundamental concept of phosphorylation-mediated signaling is the precise switching between discrete functional conformations. According to the traditional view, phosphorylation induces a new, active conformation. In this chapter, a series of NMR experiments performed on a response regulator are described that challenge this traditional notion. The combination of NMR relaxation experiments with chemical shift data and the linkage to structure/function reveals a fundamentally different activation mechanism. The NMR data for the response regulator NtrC provide kinetic (rates of interconversion), thermodynamic (relative populations), and structural (chemical shift) information for the conformational exchange process. The results demonstrate that both the inactive and active states are present before phosphorylation, and activation occurs via a shift of this preexisting equilibrium. This concept is in accordance with the energy landscape view of proteins that embraces the existence of conformational substates. We conjecture that this population-shift mechanism is a general paradigm for response regulator activation and possibly more universal for phosphorylation-mediated signaling.
磷酸化介导信号传导的一个基本概念是在离散功能构象之间进行精确切换。按照传统观点，磷酸化会诱导产生一种新的活性构象。在本章中，描述了一系列针对一种应答调节蛋白进行的核磁共振实验，这些实验对这一传统观念提出了挑战。核磁共振弛豫实验与化学位移数据的结合以及与结构/功能的联系揭示了一种截然不同的激活机制。应答调节蛋白NtrC的核磁共振数据提供了构象交换过程的动力学（相互转换速率）、热力学（相对丰度）和结构（化学位移）信息。结果表明，在磷酸化之前，非活性状态和活性状态均已存在，激活是通过这种预先存在的平衡的移动而发生的。这一概念与包含构象亚态存在的蛋白质能量景观观点相一致。我们推测，这种丰度移动机制是应答调节蛋白激活的一般模式，可能对于磷酸化介导的信号传导更为普遍。
