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

各种各样的生物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。虽然我们对它们的认识还在不断发展，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——光活性黄蛋白。这些尚未被很好地表征，但已被证明是各种光敏反应中的冥想因素。例如，导致细菌远离有害光源的避光反应，植物的趋光性，叶子优先吸收阳光的方向和光发生控制，细菌在强光下关闭不必要的生物合成。后一过程的一个例子是蛋白质AppA，它在黑暗中结合抑制蛋白PpsR，但在光线下经历结构变化以释放抑制蛋白。这就是我们要研究的蛋白质复合物。在这里提出的工作中，我们将结合两种先进技术。首先，将采用先进的光谱方法来探测黄酮类蛋白对光吸收的结构动力学响应。特别是，我们将结合光谱可见区域的光谱学，告诉我们在蓝光吸收后发生的黄蛋白过程，以及产生结构动力学的红外测量。由于初级过程的快速性质和蛋白质的低浓度，需要非常敏感的方法，例如在我们的实验室和哈维尔研究中心的激光科学设施中开发的方法。对于红外光谱中的结构研究，还需要利用哈维尔LSF正在开发的新的飞至毫秒方法。这将使我们第一次能够研究导致生物事件的完整结构动力学。第二个关键工具是化学生物学的先进方法。现在有可能用非天然氨基酸对蛋白质进行定位特异性标记。我们计划以两种方式利用这一最新发展。首先，我们将放置含有特定IR标签的残基，这些残基以特征频率吸收被认为与结构变化有关的已知位点。通过计时黄素激发和特定残基变化开始之间的延迟，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。目的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，提供了一个令人兴奋的机会，将这一新知识应用于更广泛的背景下。自2009年以来，光控制细胞内反应的想法——光遗传学——已经引起了极大的兴奋。这个想法源于绿色荧光蛋白技术的成功，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，具有特定光学可寻址功能的蛋白质以类似的方式被遗传编码。一旦到位，这个功能就可以用光来刺激。AppA是一个很好的候选者，特别是如果AppA的光诱导复解离机制可以被招募和控制以结合和释放任意伙伴（例如药物分子）。这样一个光学可寻址的函数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.