Zhao Jason Ming, Lee Haeshin, Nome Rene A, Majid Sophia, Scherer Norbert F, Hoff Wouter D
Department of Physics, University of Chicago, IL 60637, USA.
Proc Natl Acad Sci U S A. 2006 Aug 1;103(31):11561-6. doi: 10.1073/pnas.0601567103. Epub 2006 Jul 19.
The Per-Arnt-Sim (PAS) domain is a ubiquitous protein module with a common three-dimensional fold involved in a wide range of regulatory and sensory functions in all domains of life. The activation of these functions is thought to involve partial unfolding of N- or C-terminal helices attached to the PAS domain. Here we use atomic force microscopy to probe receptor activation in single molecules of photoactive yellow protein (PYP), a prototype of the PAS domain family. Mechanical unfolding of Cys-linked PYP multimers in the presence and absence of illumination reveals that, in contrast to previous studies, the PAS domain itself is extended by approximately 3 nm (at the 10-pN detection limit of the measurement) and destabilized by approximately 30% in the light-activated state of PYP. Comparative measurements and steered molecular dynamics simulations of two double-Cys PYP mutants that probe different regions of the PAS domain quantify the anisotropy in stability and changes in local structure, thereby demonstrating the partial unfolding of their PAS domain upon activation. These results establish a generally applicable single-molecule approach for mapping functional conformational changes to selected regions of a protein. In addition, the results have profound implications for the molecular mechanism of PAS domain activation and indicate that stimulus-induced partial protein unfolding can be used as a signaling mechanism.
Per-Arnt-Sim（PAS）结构域是一种普遍存在的蛋白质模块，具有共同的三维折叠结构，参与生命各个领域的广泛调节和传感功能。这些功能的激活被认为涉及与PAS结构域相连的N端或C端螺旋的部分展开。在这里，我们使用原子力显微镜来探测光活性黄色蛋白（PYP）单分子中的受体激活，PYP是PAS结构域家族的一个原型。在有光照和无光照的情况下，对通过半胱氨酸连接的PYP多聚体进行机械展开实验，结果表明，与之前的研究不同，在PYP的光激活状态下，PAS结构域本身延长了约3纳米（在测量的10皮牛检测极限下），稳定性降低了约30%。对两个探测PAS结构域不同区域的双半胱氨酸PYP突变体进行的比较测量和引导分子动力学模拟，量化了稳定性的各向异性和局部结构的变化，从而证明了它们的PAS结构域在激活时会部分展开。这些结果建立了一种普遍适用的单分子方法，用于将功能构象变化映射到蛋白质的选定区域。此外，这些结果对PAS结构域激活的分子机制具有深远影响，并表明刺激诱导的蛋白质部分展开可作为一种信号传导机制。
