International Collaboration in Chemistry: BLUF Domain blue light photosensors - a paradigm for optogenetics

各种各样的生物体都能感知光并对光作出反应。最明显的例子是植物的光合作用，它将阳光转化为化学能，以及视觉色素的反应，它将落在视网膜上的光子转化为大脑中的视觉信号。负责这些过程的蛋白质复合体-光系统I和视紫红质-已经被研究了很多年。虽然我们对它们的了解仍在发展，但在确定光响应的机制方面已经取得了很大的进展。近年来，在植物和细菌中广泛发现了一类新的光敏蛋白家族--光活性黄素蛋白。这些还没有被很好地描述，但已经被证明是各种光敏反应中的冥想因素。例如，使细菌远离有害光源的恐光反应，植物的趋光性，叶片优先吸收阳光的方向，以及细菌在强光下关闭不必要的生物合成的光遗传控制。后一种过程的一个例子是AppA蛋白，它在黑暗中与抑制蛋白PpsR结合，但在光中经历结构变化来释放抑制蛋白。这就是我们将要研究的蛋白质复合体。在这里提出的工作中，我们将结合两种类型的先进技术。首先，先进的光谱方法将被用来探测黄素蛋白对光吸收的结构动力学。特别是，我们将结合光谱可见区的光谱学，告诉我们黄素蛋白在蓝光吸收后发生的过程，以及产生结构动力学的红外测量。由于初级过程的快速性和蛋白质的低浓度，需要极敏感的方法，如我们实验室和哈威尔研究中心的激光科学设施中开发的那些方法。对于红外光谱中的结构研究，也有必要利用LSF Harwell正在开发的新的飞秒到毫秒的方法。这些将使我们能够第一次研究导致生物事件的完整结构动力学。第二个关键工具是先进的化学生物学方法。现在有可能用非天然氨基酸来定位标记蛋白质。我们计划通过两种方式利用这一最新发展。首先，我们将放置包含特定IR标记的残基，这些标记在被认为参与结构变化的途径沿线的已知位置以特征频率吸收。通过计时黄素激发和特定残基开始变化之间的延迟，我们将能够绘制出结构变化的详细机制。接下来，我们将修改黄素附近的残基，以改变触发结构变化的主要事件。通过这种方式，我们的目标是优化和控制黄素的光响应。正是这最后一个方面，即控制光响应的潜力，为在更广泛的背景下应用这一新知识提供了一个令人兴奋的机会。自2009年以来，光学控制细胞内反应的想法--光遗传学--一直引起极大的兴奋。这个想法起源于GFP技术的成功，在GFP技术中，一种荧光蛋白(GFP)被编码来标记活细胞中的特定蛋白质。在光遗传学中，具有特定光学寻址功能的蛋白质是以类似的方式进行遗传编码的。一旦就位，这一功能就可以被光刺激。APPA是一个很好的候选者，特别是如果可以招募和控制APPA的光诱导复杂解离机制来结合和释放任意伙伴(例如药物分子)的话。这种光学可寻址功能将是向前迈出的一大步。

A wide variety of organisms sense and respond to light. The most obvious examples are photosynthesis in plants, which converts sunlight into chemical energy, and the response of the vision pigments, which translate the photons falling onto the retina into vision signals in the brain. The protein complexes responsible for these processes - photosystem I and rhodopsin respectively - have been studied for many years. Although our understanding of them is still evolving great progress has been made in determining the mechanism of the photoresponse. In recent years a new family of light sensitive proteins, the photoactive flavoproteins, have been found widely in plants and bacterial. These are as yet much less well characterised but have been shown to be the meditating factor in a variety of photosensitive responses. For example the photophobic response which causes bacteria to swim away from a damaging light source, phototaxis in plants, the orientation of leaves to preferentially absorb the sun and photogenetic control, by which bacteria turn off unnecessary biosynthesis in strong light. An example of the latter process is the protein AppA, which in the dark binds a repressor protein PpsR, but in light undergoes a structure change to release the repressor. This is the protein complex we will study.In the work proposed here we will combine two types of advanced technology. First advanced spectroscopic methods will be used to probe structural dynamics of flavoproteins in response to light absorption. In particular we will combine spectroscopy in the visible region of the spectrum, to tell us about the processes in the flavoprotein occurring after blue light absorption, with infra-red measurements which yield structural dynamics. Because of the fast nature of the primary processes and the low concentration of the protein extremely sensitive methods, such as those developed in our laboratories and in the Laser for Science Facility at the Harwell Research Complex, are required. For the structural studies in the IR it will also be necessary to exploit the new femto- to millisecond methods under development at the LSF Harwell. These will allow us to study for the first time the complete structural dynamics responsible for the biological event.The second critical tool is advanced methods of chemical biology. It is now possible to site specifically label a protein with unnatural amino acids. We plan to exploit this recent development in two ways. First we will place residues containing specific IR labels, which absorb at characteristic frequencies at known sites along the pathway thought to be involved in the structure change. By timing the delay between flavin excitation and the onset of change in the specific residue we will be able to map out the detailed mechanism of the structure change. Next we will modify the residues in the vicinity of the flavin to alter the primary events which trigger structural change. In this way we aim to optimize and control the flavin photoresponse.It is this last aspect, the potential to control the photoresponse, which provides an exciting opportunity to apply this new knowledge in a much wider context. Since 2009 the idea of optically controlling intracellular responses - optogenetics - has been generating great excitement. This idea has its origins in the success of GFP technology, where a fluorescent protein (GFP) was encoded to label a specific protein in a living cell. In opto-genetics a protein with a specific optically addressable function is genetically encoded in a similar way. Once in place the function can be stimulated by light. AppA is an excellent candidate, particularly if the light induced complex dissociation mechanism of AppA can be recruited and controlled to bind and release an arbitrary partner (for example a drug molecule). Such an optically addressable function would be an immense step forward.