Fluorescence correlation spectroscopy of the main plant light-harvresting complex

Abstract

Light-harvesting complex II, LHCII, is vital for the light-dependent reaction of photosynthesis in higher plants. It acts as the starting point for photosynthesis in Photosystem II (PS II) by increasing the absorption of sunlight and efficiently transferring excitation energy to reaction centres, where water-splitting takes place. On top of this, LHCII acts as a molecular switch for a fast photo-protective mechanism that protects PS II from over-absorption of solar energy. This protective non-photochemical quenching (NPQ) is linked to a pH gradient over the thylakoid lipid membranes in which LHCII resides. Aggregation of LHCII is also caused by a drop in pH accompanying an increased pH gradient and has been proposed to be a useful model with which NPQ can be studied. This dissertation describes an experimental study on LHCII aggregation using Bio-Beads adsorbent to remove detergent from solutions containing isolated LHCII at two pH levels, namely pH 7.5 and pH 5.5. Two experimental techniques were combined to investigate the sizes of LHCII aggregates and fluorescence lifetimes simultaneously: fluorescence correlation spectroscopy (FCS) and time-correlated single-photon counting (TCSPC). FCS calibration was done by measuring the effective detection volume of a microscope using two different methods. NPQ measurements of LHCII fluorescence during detergent removal showed that LHCII aggregation was not ultrasensitive to the adsorption of detergent to Bio-Beads. Results from applying FCS showed an increase in hydrodynamic radii of LHCII over detergent adsorption time, from single trimeric radius to aggregate level radii, with the increase being more dramatic for pH 5.5. A sudden increase in radii after 45 minutes of detergent removal for all samples was found. Two-component fluorescence decays were analysed, showing a decrease in average fluorescence lifetimes from 3.6 – 4.0 ns to < 2.1 ns at pH 7.5 and to < 1.3 ns at pH 5.5. A comparison between fluorescence intensities and particle sizes as well as between fluorescence lifetimes and particle sizes was obtained for two separate repetitions of the study.