The aims of the collaboration project, of which this project plays a key part, are to establish a new field of “low-dimensional chemistry” in order to synthesize manipulate and characterise the components of the photosynthetic system of Rhodobacter Sphaeroides. At a fundamental level, the physical processes involved in membrane biochemistry are to be investigated. Specifically, the environment in which the light harvesting components are found, their structure and function, including proton gradient formation, diffusion mechanisms, electron transport and the molecular association between them were to be studied.
“The ultimate goal of the collaboration project was the reconstruction, on a chip in a synthetic low-dimensional system, of the complete photosynthetic pathway of the bacterium Rhodobacter Sphaeroides.”
The creation of a homogeneous, fluid, polymer supported lipid bilayer to contain and maintain the integrity of these proteins, whilst under investigation, was a key part of the project.
To create this successful system many polymer brush supports were examined, which were anionic, cationic and zwitterionic polyelectrolytes. Some were pH responsive and some were fully charged and did not change with pH. The properties of a range of lipid vesicles were studied using DLS, so that the zeta potential of the liposomes could be measured and then tuned specifically to be attracted to the oppositely charged polymer brush surfaces. It was found that at very low charge (a zwitterionic lipid vesicle or surface) no vesicles were attracted. At very high charge (a highly cationic or anionic polymer or lipid vesicle) vesicles were attracted to the surface, but remained intact and did not fuse to form a bilayer. These highly charged combinations of surfaces and vesicles created an intriguing “de-quenching halo” effect, which is worthy of further investigation. At a specific point of low to mid-charge interaction a fluid bilayer was formed on a brush which had a surface of low charge and was interacting with a charged vesicle. The successful polymer brush support was created using a short novel amino acid polycysteine methacrylate brush, which is pH responsive and can be micro-patterned. The vesicles used were composed of 25 mol % DOTAP, a cationic lipid, in combination with a zwitterionic POPC lipid. The diffusion coefficient of the bilayer deposited on the brush was measured using FRAP and found to match the rates measured for lipid bilayers on glass, which is considered to be the “standard” for comparison. The mobile fractions also compared very well to this standard. AFM scans showed a homogeneous surface and break-through force measurement confirmed a bilayer of 5 nm in thickness, as expected for a single bilayer.
Attempts were subsequently made to incorporate proteins into this system by a number of methods, including creating proteoliposomes containing light harvesting components from the Rhodobacter Sphaeroides. The most promising of these was the development of a “one-step detergent depletion method” for creating a solubilised bilayer, adding protein and rinsing away the detergent in a single experiment. The characterisation of the system by TIRF, dark field and AFM microscopy was indicating success and paves the way for future work.
In testing the polymer brushes as candidates for supporting a lipid bilayer, one with the potential to be used to create compartments in a corral for segregating the individual protein components of a light harvesting bacterium was also confirmed. This protein and lipid resistant Poly(2-(methacryloyloxy)ethyl phosphorylcholine), will facilitate the study of their function, by micro-patterning. In conjunction with the PCysMA this system has the potential be used to create corrals between these compartments which allow the free movement of ions between the proteins. The long term goal is to generate energy following light absorption by the proteins.
The project is truly multidisciplinary and has presented the opportunity for collaboration between researchers in the disciplines of physics, chemistry and biology. The work presented here combines knowledge of membrane biophysics with polymer chemistry, and protein biochemistry.
