Chemical and Biological Switches

复杂的生命系统，如发育中的多细胞生物，仍然是我们对自然世界分子理解的一个几乎没有绘制的前沿。人们不能仅仅通过研究这些系统的孤立组成部分来实现对它们的分子理解;为了捕捉它们的本质，人们必须设计出完整地研究它们的方法。操纵分子结构和功能的光学方法提供了解开生命系统某些复杂性的可能性。它们可以用于在活体动物体内以非常精确的时间在特定位点靶向单个分子物种。这种方法的能力已经得到了广泛的认可（Nature Methods 2010中的“年度方法”），但是要使体内分子的光学控制普遍实用，还有很长的路要走。 我们建议创建广泛适用的工具来光控制生命系统中的蛋白质功能。我们将采取两种互补的方法：（一）我们将设计和合成偶氮苯衍生物，吸收可见光波长的光，并经历大的构象变化。同时，我们将开发一套规则，用于如何将这些分子连接到蛋白质上，以便偶氮苯异构化驱动蛋白质的功能变化。(II)我们将采用一种在细菌中发现的天然光敏蛋白（光敏黄蛋白）作为基因编码的光开关。使用结构引导的蛋白质工程方法，我们将使光活性黄蛋白的环状排列体可用于光控制各种靶蛋白。这些操纵蛋白质的光学工具的基本发展将提高我们理解复杂生命系统的能力。

Complex living systems, such as a developing multicellular organism, remain a barely charted frontier in our molecular understanding of the natural world. One cannot achieve a molecular understanding of such systems solely by studying their isolated components; to capture their essence one must devise methods to study them intact. Optical methods for manipulating molecular structure and function offer the potential to unravel some of the complexity of living systems. They can be used to target individual molecular species at specific sites with very precise timing inside living animals. The power of this approach has been recognized widely ("Method of the Year" in Nature Methods 2010) but there is still a long way to go to make optical control of molecules in vivo generally practical. We propose to create broadly applicable tools to photo-control protein function in living systems. We will take two complementary approaches: (I) We will design and synthesize derivatives of azobenzene that absorb light at visible wavelengths and undergo large conformational changes. Concurrently we will develop a set of rules for how to attach such molecules to proteins so that the azobenzene isomerization drives a functional change in the protein. (II) We will adapt a naturally light-sensitive protein found in bacteria (photoactive yellow protein) for use as a genetically encoded photoswitch. Using a structure-guided protein engineering approach we will make a circular permutant of photoactive yellow protein that can be used to photo-control a diverse array of target proteins. The fundamental development of these optical tools for manipulating proteins will enhance our ability to understand complex living systems.