An investigation of the PsbS protein isolated from spinach chloroplast membranes
Dissipation of excess light energy in plant photosynthetic membranes plays an important role in the response of plants to the environment, providing short-term balancing between the intensity of sunlight and photosynthetic capacity. The carotenoid zeaxanthin and the photosystem Il subunit PsbS play vital roles in this process, but the mechanism of their action is largely unexplained. This thesis reports a novel procedure for the extraction of the PsbS protein from spinach thylakoids, including a detailed account of the developmental process and characterisation of the isolated protein. The ability of the PsbS protein to bind xanthophyll cycle carotenoids in vitro was assessed, leading to the observation that the isolated protein was able to bind exogenous zeaxanthin, the binding resulting in a strong red shift in the absorption spectrum, and the appearance of characteristic features in the resonance Raman spectrum and a distinct circular dichroism spectrum, indicating pigment-protein, as well as specific pigmentpigment, interaction. A strong shift in the absorption spectrum of PsbS phenylalanine residues after zeaxanthin binding was observed. It is concluded that zeaxanthin binding to PsbS is the origin of the well known energy dissipation-related 535-nm absorption change. The ability of this PsbS-zeaxanthin complex to affect the rate of chlorophyll fluorescence quenching of the major LHcn antenna protein is detailed, revealing an increase in the rate of quenching, whilst the magnitude of quenching remained constant. The altered properties of zeaxanthin and PsbS after in vitro reconstitution and their subsequent effect on LHCnb provide the first direct indication about how they regulate energy dissipation.