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

各种各样的有机体都能感知并对光做出反应。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物--分别是光系统I和视紫红质--已经被研究了很多年。虽然我们对它们的理解仍在不断发展，但在确定光响应机制方面已经取得了很大进展。近年来，在植物和细菌中发现了一类新的光敏蛋白，即光敏黄素蛋白。这些还没有得到很好的表征，但已被证明是各种光敏反应中的冥想因素。例如，导致细菌游离有害光源的嫌光反应，植物中的趋光性，叶子优先吸收太阳的方向和光生控制，细菌通过这种控制关闭强光中不必要的生物合成。后一过程的一个例子是蛋白AppA，其在黑暗中结合阻遏蛋白PpsR，但在光照下经历结构变化以释放阻遏物。这就是我们要研究的蛋白质复合体。在这里提出的工作中，我们将结合联合收割机两种类型的先进技术。首先，先进的光谱方法将用于探测响应于光吸收的黄素蛋白的结构动力学。特别是，我们将联合收割机光谱在可见光区的光谱，告诉我们的过程中发生的黄素蛋白后，蓝光吸收，红外测量产生的结构动力学。由于初级过程的快速性质和蛋白质的低浓度，需要极其灵敏的方法，例如在我们的实验室和Harwell研究中心的激光科学设施中开发的方法。对于IR中的结构研究，还需要利用LSF Harwell正在开发的新的毫微微到毫秒方法。这将使我们能够第一次研究负责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。现在可以用非天然氨基酸对蛋白质进行位点特异性标记。我们计划从两个方面利用这一最新发展。首先，我们将放置含有特定IR标记的残基，其在被认为参与结构变化的途径的沿着已知位点以特征频率吸收。通过对黄素激发和特定残基发生变化之间的延迟进行计时，我们将能够绘制出结构变化的详细机制。接下来，我们将修饰黄素附近的残基，以改变引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，控制光响应的潜力，这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-已经产生了巨大的兴奋。这个想法起源于GFP技术的成功，其中荧光蛋白（GFP）被编码以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以类似的方式进行遗传编码。一旦到位，该功能可以被光刺激。AppA是一个很好的候选者，特别是如果AppA的光诱导复合物解离机制可以被募集和控制以结合和释放任意配偶体（例如药物分子）。这种光学寻址功能将是一个巨大的进步。

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.