University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, USA.
Biophys J. 2012 Jan 18;102(2):325-32. doi: 10.1016/j.bpj.2011.11.4021.
Visualizing the three-dimensional structures of a protein during its biological activity is key to understanding its mechanism. In general, protein structure and function are pH-dependent. Changing the pH provides new insights into the mechanisms that are involved in protein activity. Photoactive yellow protein (PYP) is a signaling protein that serves as an ideal model for time-dependent studies on light-activated proteins. Its photocycle is studied extensively under different pH conditions. However, the structures of the intermediates remain unknown until time-resolved crystallography is employed. With the newest beamline developments, a comprehensive time series of Laue data can now be collected from a single protein crystal. This allows us to vary the pH. Here we present the first structure, to our knowledge, of a short-lived protein-inhibitor complex formed in the pB state of the PYP photocycle at pH 4. A water molecule that is transiently stabilized in the chromophore active site prevents the relaxation of the chromophore back to the trans configuration. As a result, the dark-state recovery is slowed down dramatically. At pH 9, PYP stops cycling through the pB state altogether. The electrostatic environment in the chromophore-binding site is the likely reason for this altered kinetics at different pH values.
在生物活性过程中可视化蛋白质的三维结构对于理解其机制至关重要。通常情况下，蛋白质的结构和功能都依赖于 pH 值。改变 pH 值可以深入了解参与蛋白质活性的机制。光致变色黄色蛋白（PYP）是一种信号蛋白，是研究光激活蛋白的时间依赖性的理想模型。已经在不同的 pH 条件下广泛研究了其光循环。然而，在采用时间分辨晶体学之前，中间产物的结构仍然未知。随着最新光束线的发展，现在可以从单个蛋白质晶体中收集全面的时间分辨劳埃数据序列。这使我们能够改变 pH 值。在这里，我们首次展示了在 pH 值为 4 的 PYP 光循环的 pB 状态下形成的短寿命蛋白质-抑制剂复合物的结构。在生色团活性位点中瞬时稳定的水分子阻止了生色团回至反式构型的松弛。因此，暗态恢复大大减慢。在 pH 值为 9 时，PYP 完全停止循环通过 pB 状态。在不同 pH 值下，这种动力学改变的可能原因是色团结合位点的静电环境。
