Structural Dynamics in LOV Domain Photosensor Proteins

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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.