Engineering Protein Structure and Interactions to Create Next Generation Optogenetic Tools

基于光的技术在最基本的层面上彻底改变了我们对生物系统的理解，并稳步引导研究人员走向新的诊断和治疗方法。作为能量的一种形式，光使我们能够感知形状和颜色，它是我们大多数人理解周围世界的主要手段。由于它的无所不在和无害，光是生物研究的一个强大而有吸引力的工具。如果利用得当，光可以让我们无创地观察和控制细胞和模式生物中的生物。在过去的二十年里，研究人员一直在寻找越来越多的方法来利用光能来探测模式生物中的细胞和组织。在这个方向上的第一个重大突破可以追溯到1994年，当时研究人员首次报告说，水母的一种特殊蛋白质可以被引入其他动物的细胞中，并使它们在蓝光照射下发出绿光。十年后，另一个重大突破出现了，研究人员报告了一种来自藻类的蛋白质，当它被引入哺乳动物的神经元时，会使神经元在蓝光照射下放电。这两项突破作为“光遗传学”工具蛋白质与光相互作用的典型例子，使研究人员能够可视化或操纵细胞。在本提案中，我们的目标是为光遗传学工具工具箱添加全新的功能。由于光遗传工具必须是蛋白质，我们将使用蛋白质工程的策略来改变光遗传工具的特性，并赋予它们在自然界中不存在的特性。作为一个研究主题，我们将努力创造单一的“光遗传信标”结构，存在于感兴趣的细胞内，并提供细胞活动的彩色读数。第二个研究主题建立在我们最近的一份工程蛋白质的报告上，该蛋白质可以利用光分成两部分。我们现在建议用这种蛋白质在细胞内建造可以用光打开或关闭的笼子。这些笼可用于非侵入性捕获或释放蛋白质，并将为生物学家提供一种破译细胞内信号通路的强大新工具。作为第三个主题，我们将致力于开发一种改进的系统，将蛋白质相互作用或功能的变化转化为可以通过显微镜轻松可视化的颜色变化。总之，我们确信我们的工作将代表光遗传学领域的下一步，并为我们对细胞生物学，健康和疾病的理解的未来突破奠定基础。

Light-based technologies are revolutionizing our understanding of biological systems at the most fundamental level and steadily guiding researchers towards new diagnostic and therapeutic approaches. As a form of energy, light enables us to perceive shape and colour, and it is the primary means by which most of us make sense of the world around us. By virtue of its very ubiquity and innocuousness, light is a powerful and attractive tool for biological research. If properly harnessed, light can allow us to non-invasively visualize and control biology as it happens in cells and model organisms. Over the past two decades, researchers have been figuring out an ever-increasing number of ways to harness light energy to probe cells and tissues in model organisms. One of the first major breakthroughs in this direction dates to 1994, when researchers first reported that a particular protein from jellyfish could be introduced into the cells of other animals, and cause them to glow green when illuminated by blue light. Another major breakthrough came a decade later, when researchers reported a protein from algae that, when introduced into mammalian neurons, would make the neurons fire upon illumination with blue light. These two breakthroughs stand tall as the prototypical examples of “optogenetic” tools proteins that interact with light and enable researchers to either visualize or manipulate cells. In this proposal we aim to add entirely new capabilities to the toolbox of optogenetic tools. As optogenetic tools are necessarily proteins, we will use the strategy of protein engineering to change the properties of optogenetic tools and bestow them with properties that do not occur in nature. As one research Theme, we will work to create singular “optogenetic beacon” structures that exist inside the cell of interest and provide a colourful readout of the cell's activity. A second research Theme builds upon our recent report of an engineered protein that can be split into two pieces using light. We now propose to use this protein to build cages inside the cell that can be opened or closed with light. These cages can be used to non-invasively trap or release proteins and will give biologists a powerful new tool for deciphering intracellular signalling pathways. As a third Theme, we will work to develop an improved system that converts changes in protein interactions or function into colour changes that can be easily visualized through a microscope. Altogether, we are certain that our work will represent the next step forward for the field of optogenetics and lay the foundation for future breakthroughs in our understanding of cell biology, in health and disease.