Impact of the intensity threshold on binary switching analysis in single molecule spectroscopy of phycobilisomes

Abstract

The functional dynamics of photosynthetic light-harvesting pigment-protein complexes play an essential physiological role. The dynamic switching between different states – associated with different spectroscopic signatures – allows these complexes to regulate the excitation energy flow in the photosynthetic apparatus as a direct response to changing environmental conditions. To a large extent, the robustness of the photosynthetic processes depends on this functional flexibility of the light-harvesting complexes. Single molecule spectroscopy (SMS) is a method of choice to investigate the spectroscopic properties of individual molecules or complexes and allows the direct observation of switches between emissive states. The fluorescence intensity dynamics of individual light-harvesting complexes are commonly analyzed using a two-state model, corresponding to an ON and OFF state, respectively. In this model, each switch across the intensity threshold that separates the two states is counted as an intensity switch. However, multichromophoric systems in general, and protein complexes binding multiple pigments in particular, are known to switch among various intensity levels, suggesting that the choice of the intensity threshold may significantly influence the two-state statistics. Here, we investigated the fluorescence data of a very large complex – the main light harvesting complex of cyanobacteria, the phycobilisome – and determined that the intensity threshold has only a limited impact on the binary switching analysis of the complex for an extended range of threshold positions.