Centre Algatech, Institute of Microbiology of the Czech Academy of Sciences, Opatovický Mlýn, 379 81 Třeboň, Czech Republic.
Faculty of Science, University of South Bohemia in České Budějovice, Branišovská 31a, 370 05 České Budějovice, Czech Republic.
Int J Mol Sci. 2021 Mar 15;22(6):2969. doi: 10.3390/ijms22062969.
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.
天线蛋白聚集是被认为有效保护光养生物免受高光破坏的主要机制之一。通常，在体外通过降低纯化天线溶液中的去污剂浓度和 pH 值来诱导这种聚集；由此产生的荧光发射减少被认为是体内非光化学猝灭的代表。然而，对于通过这种方法形成的天线颗粒的实际大小和组织知之甚少，因此这种实验方法的生理相关性值得怀疑。在这里，荧光相关光谱 (FCS) 这种准单分子方法在体外猝灭高等植物的 LHCII 三聚体时被应用，以便在单次测量中平行估计颗粒大小、荧光和天线簇的均一性。FCS 表明，在去污剂临界胶束浓度以下，低 pH 值促进了高达微米级的大蛋白质寡聚物的形成，因此显然与类囊体膜不兼容。相比之下，在高 pH 值下形成的 LHCII 簇更小且更均匀，但仍然能够有效地猝灭。这些结果共同设定了体外猝灭实验的生理有效性限制。我们的数据还支持这样一种观点，即在高 pH 值下发现的小的、适度猝灭的 LHCII 低聚物可能与体内非光化学猝灭有关。
