The role of the light antenna protein LHC-II in the structure and dynamic reorganization of photosynthetic membranes

Lead Research Organisation: University of Leeds
Department Name: Astbury Centre

Abstract

Background
Lipopolysaccharide (LPS) is a complex lipoglycan, which acts as a major structural component of the outer membrane of Gram-negative bacteria and potent toxin when released from cells ("endotoxin"). It is important that we understand LPS-lipid molecular interactions which may affect toxicity. We previously found that LPS inserts into model membranes and causes major perturbations: (i) lipid tubule formation, (ii) membrane perforation and (iii) multilayer formation. These changes were proposed to be governed by local electrostatic interactions, however, a detailed mechanism is required.

Objectives
(1) Elucidate the molecular basis of LPS-lipid interaction.
(2) Qualify any correlation between pathogenicity of LPS source and membrane disruptiveness.
(3) Determine physical property changes induced by LPS on membranes.
(4) Test biologically-relevant inhibitors of LPS action.

Novelty/Timeliness
We were the first group to observe perturbation of supported lipid bilayers by LPS and with our expertise in this system and required experimental techniques, we are uniquely-placed to reveal the underlying molecular mechanism. Whilst many have investigated either the pathogenicity (endotoxic shock) or isolated LPS (physical characterization), the dynamic changes caused by LPS on a combined lipid/LPS model system have not been fully investigated.

Experimental approach
State-of-the-art microscopy will be used to visualize dynamic membrane organizational changes at the nanoscale (atomic force microscopy (AFM), 1-nm-resolution at 8 fps) and track membrane fluidity (fluorescence microscopy). This will allow qualitative assessment of the effect of LPS from different bacteria (pathogenic/ non-pathogenic) on different lipid systems (charged, "lipid raft"-like). A quantitative analysis of physical changes induced by LPS on membranes will be undertaken using differential scanning calorimetry (melting transition temperatures), quartz crystal microbalance (mass/viscosity) and Langmuir trough measurements (membrane lateral pressure). Multilayers will be characterized with neutron reflectivity (layer thickness). Finally, various components of the immune system (e.g. LPS Binding Protein) will be screened for ability to inhibit LPS-induced damage.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011151/1 01/10/2015 30/09/2023
1940236 Studentship BB/M011151/1 01/10/2017 30/09/2021 Sophie Meredith
 
Description The structure and light-harvesting function of photosynthetic membranes is heavily dependant on the arrangement of the proteins that are contained within the membrane. The native membrane is extremely complicated, and the mechanisms that control the membrane structure and energy transfer within photosynthetisis are intensely debated.

So far, we have been able to develop model systems, that are vastly simplified compared to the native system and offer a greater degree of modularity and access from surface specific techniques (fluorescence lifetime measurements and atomic force microscopy). These model systems have allowed use to probe the relationship between the structural arrangements of proteins and fluorescenct molecules, and the resultant energy transfer within the system. These systems often contain a limited number of photosynthetic components, allowing us to test individual parameters outside of the native system, in order to build up a comprehensive understanding of the specifc roles of certain proteins.

In an example model system, we were able to demonstrate the artifical enhancement of photosynthetic absorption by introducing a complementary chorophore. Unlike previous approaches, our method didn't rely on the covalent / chemical alteration of the native protein, and maintained a native-like membrane environment that has been shown to be crucial for protein stability and function. We have since developed this approach to artifically enhance the absorption of native thylakoid extracts as part of a patterned hybrid-thylakoid membrane.

We are now using these models to develop an experimental system specifically to test the effects of protein concentration on the membrane structure, and on the protein-protein energy transfer. We will be using time-resolved fluorescence lifetime microscopy to capture the dynamic rearrangment of proteins within our model system, to observe these effects in real-time with extremely high resolution temporal data, and complementary topological information from Atomic Force Microscopy.
Exploitation Route As part of this funding, we have begun to set up the Standard Operating Procedures and dedicated Lab space for the creation of similar patterned model membranes at the University of Leeds. These model systems, could theoretically be applied to any membrane based system containing lipids or membrane proteins and as a result could have far-reaching applications in biophysical research and biotechnology.

The spatial control that has been demonstrated over these systems, makes this approach particularly appealling for the development of bio-hybrid devices or future experiments, where rapid quantitative results could be acheived through pattern recognition anaylsis. In addition to this, the succesful demonstration of photosynthetic enhancement could be used to produce photonic devices with a higher efficicency, and better light-harvesting for alternative energy sources.
Sectors Energy,Environment,Pharmaceuticals and Medical Biotechnology

URL https://doi.org/10.1039/C9NR04653D
 
Description JSPS-Royal Society International Exchanges Scheme - Model Membrane Platforms for Light Harvesting 
Organisation Kobe University
Country Japan 
Sector Academic/University 
PI Contribution The team in Leeds have extensive experience with microscopy and spectroscopy of photosynthetic proteins and novel techniques for synthetic lipid membrane manipulation. Their newly-acquired combined Atomic Force Microscopy (AFM) + Fluorescence Lifetime Imaging Microscopy (FLIM) instrument is ideal for accurate analysis of these photo-active membranes: mapping of membrane structure via AFM correlated to optical properties via FLIM.
Collaborator Contribution The Kobe team are world-leaders in constructing microscale patterns of membranes and their use for quantitative evaluation of membrane protein function. Using their established techniques, we will be undertaking multiple visits between institutions to facilitate the knowledge transfer of their methods to Leeds.
Impact Outcomes from this collaboration are still pending, though we have been able to make rapid progress on the early stages of this collaboration. We anticipate multiple high-impact publications as a result of this collaboration, and are in the process of reveiwing and preparing these for submission.
Start Year 2019