Investigating non-photochemical quenching in model membranes

Non-photochemical quenching of chlorophyll fluorescence (NPQ) is a photoprotective process that harmlessly dissipates excess excitation energy as heat. qE is the major component of NPQ in plants, starting with a proton gradient (ΔpH) that triggers a conformational change in light harvesting complex II (LHCII), which leads to the creation of a quencher. The protein PsbS and the carotenoid zeaxanthin are also needed for energy dissipation in a natural environment. A major issue for studying qE is that the thylakoid membrane is extremely crowded with protein, making mechanistic analysis a challenge, while studies on isolated LHCII remove the native lipid: protein and protein: protein interactions. In this thesis, a model-membrane approach was taken to study the qE mechanism, as model membranes such as liposomes and nanodiscs bridge the gap between studies of intact thylakoid membranes and isolated complexes. LHCII bound to either violaxanthin or zeaxanthin were separately incorporated into liposomes at various protein concentrations. Increasing the concentration of LHCII in the membrane increased quenching, however the presence of zeaxanthin had no effect on quenching. To probe the effect of the membrane environment, a single LHCII was incorporated into different liposome and nanodisc membranes. Each membrane environment caused some quenching in LHCII, and smaller membrane areas increased both LHCII photodamage and switching between quenched and unquenched conformations. Finally, a fluorescently tagged PsbS construct was incorporated into liposomes, allowing controlled orientation of PsbS in themembrane. The addition of PsbS in its correct orientation to liposomes containing zeaxanthin enriched LHCII significantly increased quenching, even in the absence of ΔpH. Overall, the work presented in this thesis has characterised the effect that PsbS, zeaxanthin, LHCII: LHCII interactions and the membrane itself have on quenching. The advantages and limitations of model membranes are also discussed throughout this thesis.