Structural Dynamics in LOV Domain Photosensor Proteins

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.

Our proposal aims to extend the range of ultrafast spectroscopy and to couple it with chemical biology as an aid to probing biological function. The project is therefore largely fundamental science, but a number of areas for impact in the non-academic sphere have been identified.

Life science research has major potential benefits to society and the economy through the development of new healthcare technologies, which will then be beneficial to patients and to the companies who supply the technology. We see in particular the application of optobiology to probe cell function as having potential applications in health science. A key component in delivering this impact in health is the development of advanced instrumentation. This is a major area of economic activity in which UK based companies are active. Our research will lead to the development of novel tools of optobiology. These will be applied in instruments designed for high resolution 3D imaging, especially microscopy, and will require for their application a variety of optoelectronic devices.

This impact can only be realized if influential people in the field are aware of our research. The primary route for dissemination is through high quality research publications. Most major technology and biotech companies have access to literature. In addition all of our published work will appear immediately on acceptance in a publicly searchable database hosted by UEA. Further, we will present our ongoing and preliminary work at key laser and photonics instrumentation conferences such as the Munich Laser Photonics Conference and CLEO, as well as at academic conferences attended by instrument suppliers and manufacturers (e.g. Time Resolved Vibrational Spectroscopy and European Photobiology Congress). In addition we will remain alert to the possibilities of exploiting some of our own technical innovations in instrument development. The possibility of licencing such developments is actively discussed with the Research and Enterprise services group at UEA.

A further beneficiary of this research will be the people trained in the project, which will include the PDRA appointed, PhD students and Masters students. We anticipate high quality outputs in an important and dynamic field, which will directly benefit the PDRA. Career development is an important part of the 'The Concordat to Support the Career Development of Researchers' to which UEA subscribes. In this case we will provide detailed advice and assistance to the PDRAS in the development and authoring of research fellowships, typically an important next step for PDRAs. In this way the research supports the development of next generation physical scientists, specifically those with skills applicable in life sciences, which is a priority area.

Chemistry research students will benefit from training on one hand in laser methods and instrumentation and on the other they will learn about sample preparation and handling techniques in life science. These are both skills which would make them valuable assets in environments (academic or industrial) aiming to apply this research.