Antenna proteins play a major role in the regulation of light-harvesting in photosynthesis. However, less is known about a possible link between their sizes (oligomerization state) and fluorescence intensity (number of photons emitted). Here, we used a microscopy-based method, Fluorescence Correlation Spectroscopy (FCS), to analyze different antenna proteins at the particle level. The direct comparison indicated that Chromera Light Harvesting (CLH) antenna particles (isolated from Chromera velia) behaved as the monomeric Light Harvesting Complex II (LHCII) (from higher plants), in terms of their radius (based on the diffusion time) and fluorescence yields. FCS data thus indicated a monomeric oligomerization state of algal CLH antenna (at our experimental conditions) that was later confirmed also by biochemical experiments. Additionally, our data provide a proof of concept that the FCS method is well suited to measure proteins sizes (oligomerization state) and fluorescence intensities (photon counts) of antenna proteins per single particle (monomers and oligomers). We proved that antenna monomers (CLH and LHCIIm) are more "quenched" than the corresponding trimers. The FCS measurement thus represents a useful experimental approach that allows studying the role of antenna oligomerization in the mechanism of photoprotection.

• Klíčová slova
• Chromera velia, antenna proteins, fluorescence correlation spectroscopy, light-harvesting, microscopy, photosynthesis, protein diffusion, protein oligomerization,
• MeSH
• bílkoviny řas chemie   metabolismus   MeSH
• fluorescence * MeSH
• fluorescenční spektrometrie MeSH
• fotosyntéza * MeSH
• kinetika MeSH
• multimerizace proteinu MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bílkoviny řas MeSH

Antenna protein aggregation is one of the principal mechanisms considered effective in protecting phototrophs against high light damage. Commonly, it is induced, in vitro, by decreasing detergent concentration and pH of a solution of purified antennas; the resulting reduction in fluorescence emission is considered to be representative of non-photochemical quenching in vivo. However, little is known about the actual size and organization of antenna particles formed by this means, and hence the physiological relevance of this experimental approach is questionable. Here, a quasi-single molecule method, fluorescence correlation spectroscopy (FCS), was applied during in vitro quenching of LHCII trimers from higher plants for a parallel estimation of particle size, fluorescence, and antenna cluster homogeneity in a single measurement. FCS revealed that, below detergent critical micelle concentration, low pH promoted the formation of large protein oligomers of sizes up to micrometers, and therefore is apparently incompatible with thylakoid membranes. In contrast, LHCII clusters formed at high pH were smaller and homogenous, and yet still capable of efficient quenching. The results altogether set the physiological validity limits of in vitro quenching experiments. Our data also support the idea that the small, moderately quenching LHCII oligomers found at high pH could be relevant with respect to non-photochemical quenching in vivo.

• Klíčová slova
• antenna proteins, detergent critical micelle concentration, fluorescence correlation spectroscopy, non-photochemical quenching, photoprotection, photosynthesis, protein oligomerization,
• MeSH
• chlorofyl chemie   genetika   účinky záření   MeSH
• fluorescence MeSH
• fluorescenční spektrometrie MeSH
• fotosyntéza genetika   MeSH
• fotosystém II (proteinový komplex) genetika   účinky záření   MeSH
• fototrofní procesy genetika   MeSH
• homeodoménový protein Antennapedia chemie   genetika   MeSH
• koncentrace vodíkových iontů MeSH
• proteinové agregáty genetika   MeSH
• shluková analýza MeSH
• světlo škodlivé účinky   MeSH
• světlosběrné proteinové komplexy chemie   genetika   MeSH
• tylakoidy chemie   genetika   účinky záření   MeSH
• zeaxanthiny genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chlorofyl MeSH
• fotosystém II (proteinový komplex) MeSH
• homeodoménový protein Antennapedia MeSH
• proteinové agregáty MeSH
• světlosběrné proteinové komplexy MeSH
• zeaxanthiny MeSH