Structural Dynamics in LOV Domain Photosensor Proteins

我们呼吸的氧气和我们吃的食物最终来自光合作用，即植物和细菌将太阳光线转化为有用的化学能。然而，我们可以有太多的阳光。就像人类会因为阳光中有害的紫外线而患上皮肤癌一样，植物和细菌也会因为过多的阳光而受损。由于这些相互冲突的需求，对于广泛的生物体来说，具有某种感知光水平的手段是必不可少的。植物有这样的工具是显而易见的，任何人谁曾经在窗台上种植水芹，并看到它转向光。我们在这个项目中主要关注的是植物和细菌如何感知光线，以及这个过程是否可以在人类应用中加以利用。在本论文中，我们重点研究了一个特别有用的光传感器蛋白家族--LOV（Light-Oxygen-Voltage，光氧电压）结构域。LOV结构域蛋白是称为黄素蛋白的更大组的一部分。“Flavo-”意味着黄色，表明这些蛋白质是有色的，因此具有吸收光能的能力。在包括LOV结构域的光敏黄素蛋白中，这种能量被转化为蛋白质中的一些有用的结构变化。然后刺激相关蛋白质的进一步变化，最终引起特定的生物反应。已知这一复杂的事件链在以下方面很重要：确定花何时开放;使叶子转向太阳;使细菌游泳远离有害的阳光;控制昼夜节律等。在少数情况下，这些LOV结构域蛋白的结构已经确定，其他实验已经显示了它们与什么二级蛋白（或DNA）复合，这告诉我们它们的功能。然而，人们对光敏黄素蛋白的作用机制知之甚少，除了蛋白质结合吸收蓝光的黄素分子之外。我们研究的核心问题是光吸收事件如何转化为特定的结构变化，作为启动活细胞其他过程的信号。在这项工作中，我们将使用一些最复杂的激光光谱学方法来记录蛋白质吸收光后发生的变化。正是通过将这种先进的物理方法应用于生命系统，我们才能开始理解（甚至控制）生命的化学性质。在这种情况下，我们将用一个短脉冲的蓝光（不到1亿分之一秒）刺激蛋白质反应，并使用另一个短脉冲的光来拍摄结构变化的超快“快照”。我们将跟踪这些结构的变化，从激发的时间一直到最终信号状态的形成。通过这样在真实的时间里观察蛋白质的功能，我们将对植物如何"看到"光的机制有新的认识。然后，我们将使用蛋白质化学的一些技巧来测试，探测和操纵这些结构变化。我们对这些蛋白质的兴趣不仅仅是好奇它们是如何工作的。最近，科学家们人工地将光激活蛋白质掺入各种细胞中，然后利用光来触发特定的反应。最著名的例子是使用光激活小鼠大脑中神经元的放电，但随着对其他光激活蛋白（如LOV结构域）的更好理解，刺激各种新现象将成为可能。在具有时间和空间分辨率的活细胞中刺激特定过程的能力将为试图了解细胞功能的科学家提供强大的新工具，并将为健康科学的各种研究提供信息。

The oxygen we breathe and the food we eat ultimately derive from photosynthesis, the conversion of the sun's rays into useful chemical energy by plants and bacteria. However, we can have too much sunshine. Just as humans can suffer from skin cancer due to harmful UV rays in the sun, so plants and bacteria can be damaged by too much sunlight. As a result of these conflicting demands it is essential for a wide range of living organisms to have some means of sensing light levels. That plants have such tools is obvious to anyone who has ever grown cress on a windowsill and seen it turn towards the light. What we are principally concerned with in this project is precisely how plants and bacteria sense light, and whether this process can be exploited in human applications. In this proposal we focus on one particularly useful family of photosensor proteins, the LOV (Light-Oxygen-Voltage) domains.Over the past twenty years many proteins have been discovered which detect light. The LOV domain proteins are part of a much larger group called the flavoproteins. 'Flavo-' means yellow indicating that these proteins are colored and thus have the ability to absorb light energy. In the photoactive flavoproteins, which includes the LOV domains, this energy is converted it into some useful structure change in the protein. This then stimulates further changes in associated proteins which ultimately gives rise to a specific biological response. This complex chain of events in known to be important in: determining when flowers open; making leaves turn towards the sun; causing bacteria to swim away from harmful sunlight; controlling circadian rhythms, etc. In a few cases the structures of these LOV domain proteins have been determined, and other experiments have shown what secondary proteins (or DNA) they are complexed with, which informs us about their function. However, very little is known about the mechanism of operation of photoactive flavoproteins, beyond the fact that the proteins binds a flavin molecule which absorbs blue light. The question at the heart of our research is how is the event of light absorption can be converted into a specific structure change which acts as a signal to initiate other processes in living cells.In this work we will use some of the most sophisticated methods of laser spectroscopy to record what happens to the proteins after they have absorbed light. It is through the application of such advanced physical methods to living systems that we can begin to understand (and even control) the chemistry of life. In this case we will stimulate the protein response with a short pulse of blue light (less than 100 million billionths of a second long) and use another short pulse of light to take ultrafast 'snapshots' of the structural changes as they happen. We will follow these structure changes right from the time of excitation all the way through to formation of the final signalling state. By thus observing protein function in real time we will obtain new insights into the mechanism of how plants 'see' light. We will then use some tricks of protein chemistry to test, probe and manipulate these structure changes. Our interest in these proteins is not simply curiosity as to how they work. Recently scientists have artificially incorporated light-activated proteins into various cells and then used light to trigger a particular response. The most famous example is the use of light to activate the firing of neurons in the brains of mice, but as other light-activated proteins (such as LOV domains) become better understood it will become possible to stimulate a variety of new phenomena. The ability to stimulate a specific process in a living cell with both time and space resolution will represent a powerful new tool for scientists trying to understand cellular functions, and will inform a variety of research in health sciences.