Controllable model membranes and new quantitative analyses to interrogate light harvesting proteins

This proposal will develop new model systems and analysis methods to quantify the biophysical properties of important Light Harvesting (LH) proteins with high spatiotemporal resolution. This will provide insight into the physical basis of photosynthetic processes which are crucial for life on Earth but poorly understood. It will also have wider relevance for the potential use of LH proteins in nanotechnology, photonics and plasmonics. We need to develop a suite of "model membranes" (idealized experimental models of biomembranes) which incorporate LH proteins and offer a balance between complexity, controllability and amenability to tools that can quantify structure and optical properties. We need the ability to manipulate the density of LH proteins within these model membranes. We need to quantify how the organization and optical properties of these LH proteins change in response to important physicochemical stimuli. The proposed project will develop three types of model membranes for studying LH proteins, which progress from mostly synthetic to close-to-natural membranes. These "model membranes" will be manipulated by applying external electric fields to direct the migration of LH proteins within membranes, for the first time. The nanoscale organization and photophysical properties of these LH proteins will be compared in each type of membrane by an advanced microscopy and spectroscopy combined system.

My previous research has demonstrated that the clustering of LH proteins is correlated to energy dissipation (fluorescence quenching) and my expertise with correlative microscopy and spectroscopy tools make me uniquely well-placed for performing quantitative studies of energy transfer processes. In Leeds, we have a new Fluorescence Lifetime Imaging Microscope combined with Atomic Force Microscopy (FLIM+AFM) instrument which will allow unprecedented direct correlation and quantification of optical properties and nanoscale organization (I was co-investigator on the grant acquiring this instrument). This combined microscope is ideal to study LH proteins and gives us a competitive edge. In summary, this project will provide a comparative analysis of the advantages and limitations of new membrane models and, in doing so, quantify the crucial relationship between organization and optical properties of light harvesting proteins. These new models could be applied to bacterial and mammalian membranes of relevance for health and agriculture. Furthermore, knowledge of the important LHCII protein could be relevant for the development of alternative solar nanotechnologies or the production of enhanced crops.

Who might benefit? Potential economic and societal beneficiaries:
1. New industrial and academic users of our membrane formation methodology and analyses.
2. The scientific microscope technology industry.
3. The alternative solar technology industry.
4. The general public (from increased engagement and understanding and industry).
Beneficiaries #1, #2 and #4 are quite reachable during the 3 year project, whereas #3 is an exciting long-term possibility which will be better defined as we engage with this community.

How might they benefit?:
1. Researchers in academia or industry may use our methodology and benefit from having a more effective method to form and manipulate models of biomembranes and improved analysis routines to gain more quantitative information. For example, once success is demonstrated on naturally-fluorescent LHCII, where its function is easily probed throughout, these approaches could be used to study other membrane systems, i.e., alternative proteins and lipids. The three types of model membrane developed by this research grant (Objectives 1-3) would allow researchers to study the mobility and interactions of other fluorescently-tagged membrane proteins within membranes, manipulating them via electrophoresis, if desired. Use of our analysis routines applying FLIM would allow researchers to quantify protein-protein interactions (e.g. FLIM-based FRET to measure average distances between two proteins). This could lead to future industrial and economic benefit as medically-important membrane proteins are some of the most actively researched.
2. Microscope technology developers and their customers would benefit from the new microscopy sample holder designs (i.e. flow-chambers) which will be developed during this grant which allow combined electrophoresis and microscopy (see section 1.2 in the Case for Support). This could be a new commercialization opportunity. These sample holders would be straightforward to manufacture based on the 3-D CAD specification which would be produced. This would allow other researchers who already have access to AFM or fluorescence microscopes (10,000s globally) the ability to visualize the effect of electric field on their own samples with the use of a simple accessory. For example, this would allow other researchers the ability to easily perform electrophoresis as a separation technique or method to cluster their membrane proteins of interest.
3. The alternative solar technology industry would benefit from the potential that our research could lead to increases in the efficiency of their products. Understanding the biophysics of light harvesting proteins could guide the future exploitation of LH proteins in nanotechnologies, for example, understanding which protein geometries, densities and linkage chemistry promote the effective transfer of energy and/or electrons to conducting solid surfaces. This could assist the field of biophotovoltaics and future development of improved photoeletrochemical devices where LH proteins may be attached to electrodes. Alternatively, an improved understanding of light-harvesting proteins may lead to "bio-inspired" solutions, whereby biological components are not used directly, but instead lead to the construction of new inorganic materials following the design motifs and strategies of the highly efficient biological systems.
4. Adults with an interest in science in the UK and children in local schools will benefit culturally and educationally from our planned public engagement events. We will aim to inspire, increase curiosity and improve understanding of those who attend. The public discourse would be more informed if more members of the public have an increased recognition of the scientific challenges and related environmental issues in solar energy research. This could feed into important debates over sustainability and the economics of alternative energy and challenge conventional wisdom.