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

Lead Research Organisation: University of East Anglia
Department Name: Chemistry

Abstract

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.

Planned Impact

The major impact for this research outside the academic sphere is likely to come through its important contribution to the method of optogenetics. GFP can be thought of as the first example of a genetically encoded optical response, where the 'output' in response to electronic excitation is fluorescence. The dramatic success of GFP has lead to a search for other genetically encoded optically induced outputs. The two which have been considered so far are rhodopsins and flavoproteins. Rhodopsins have been succesful in controlling nerve response in vivo as optically gated ion channels. To deliver optically more complex targets requires a different mechanism and the flavoproteins are the most promising target. Under light excitation the fast excited state dynamics lead to a local structural change in the flavin binding pocket, which extends on a slower timescale into a macroscopic structure change releasing a protein co-factor. The target of optogenetics is to engineer a genetically encoded protein which binds and then photochemically releases a specific target.

The advantages of this technology in physiology and cell biology are obvious. Once the optical delivery method has been understood and optimised in the manner described in the main proposal it may be possible to design the complex partner to deliver specific molecules. The delivery of the molecule to a specific site by the genetically encoded light-activated carrier (e.g. one based on AppA) will allow a host of intracellular processes to be studied for the first time. The knowledge gained will have important implications for health and medicine.

The objective of our research is to understand the photoinduced structure change and therefore develop AppA as a robust and flexible delivery vehicle for optogenetics. That AppA operates in vivo is already an encouraging result. Optimization of the ability to control delivery of the binding partner will be a critical step in realizing the full potential of this technology.

A secondary impact of the work described is in the training of new researchers at the important but challenging spectroscopy/life sciences interface. In this International Collaboration the researchers will benefit from exposure to both advanced biology and cutting edge spectroscopy. Moreover they will do this in the context of international collaboration, and while working with a central research facility (Rutherford Laboratory in this case). The researchers will thus be ideally qualified to manage complex interdisciplinary research programmes, an increasingly important skill.
 
Description New information on the mechanism of light sensing actions over 10 to the power of 9 decades in time.

New information on the primary mechanism has been obtained showing that different BLUF domains operate differently, and that formation of metastable electron transfer intermediates is not correlated with photoactivity.

new data on the light to dark relaxation has been obtained through unnatural amino acid substitution. The data show that a proton transfer is rate determining
Exploitation Route The mechanistic data will challenge the basic understanding of BLUF function and stimulate new quantum chemical calculations.

The microsecond data add fundamental new information to the understanding of allostery and should inform the debate on protein function.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The work is in progress. We have already published work on the femtosecond to millisecond dynamics of the blue light sensing mechanism. The mechanistic questions raised will influence the debate on optogenetics We have extended experiments to new classes of proteins of importance in both optogenetics and bioimaging
First Year Of Impact 2014
Sector Education,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Cultural,Societal

 
Description Photactive proteins 
Organisation Stony Brook University
Department Department of Chemistry
Country United States 
Sector Academic/University 
PI Contribution Ultrafast measurements and analysis
Collaborator Contribution protein preparation, measurements and analysis
Impact dozens of papers, proceedings and two grants
Start Year 2006
 
Description photo switching proteins 
Organisation RIKEN
Country Japan 
Sector Private 
PI Contribution measurement and analysis, isotope labelling
Collaborator Contribution protein preparation and characterization
Impact one publication more to come
Start Year 2011