Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA.
J Chem Phys. 2022 Jul 21;157(3):035102. doi: 10.1063/5.0095763.
Photosynthetic organisms use pigment-protein complexes to capture the sunlight that powers most life on earth. Within these complexes, the position of the embedded pigments is all optimized for light harvesting. At the same time, the protein scaffold undergoes thermal fluctuations that vary the structure, and, thus, photophysics, of the complexes. While these variations are averaged out in ensemble measurements, single-molecule spectroscopy provides the ability to probe these conformational changes. We used single-molecule fluorescence spectroscopy to identify the photophysical substates reflective of distinct conformations and the associated conformational dynamics in phycoerythrin 545 (PE545), a pigment-protein complex from cryptophyte algae. Rapid switching between photophysical states was observed, indicating that ensemble measurements average over a conformational equilibrium. A highly quenched conformation was also identified, and its population increased under high light. This discovery establishes that PE545 has the characteristics to serve as a photoprotective site. Finally, unlike homologous proteins from the evolutionarily related cyanobacteria and red algae, quenching was not observed upon photobleaching, which may allow for robust photophysics without the need for rapid repair or replacement machinery. Collectively, these observations establish the presence of a rich and robust set of conformational states of PE545. Cryptophytes exhibit particularly diverse energetics owing to the variety of microenvironments in which they survive, and the conformational states and dynamics reported here may provide photophysical flexibility that contributes to their remarkable ability to flourish under diverse conditions.
光合作用生物利用色素-蛋白复合物来捕获阳光，而阳光为地球上大多数生命提供能量。在这些复合物中，嵌入色素的位置都经过优化以进行光捕获。与此同时，蛋白质支架会经历热波动，从而改变复合物的结构和光物理性质。虽然这些变化在整体测量中被平均掉了，但单分子光谱学提供了探测这些构象变化的能力。我们使用单分子荧光光谱学来识别反映不同构象的光物理亚态以及与之相关的构象动力学，这些研究对象是来自 cryptophyte 藻类的藻红蛋白 545（PE545）色素-蛋白复合物。观察到光物理状态的快速切换，表明整体测量平均值是在构象平衡上。还确定了一个高度猝灭的构象，并且在高光下其种群增加。这一发现表明 PE545 具有作为光保护位点的特征。最后，与进化上相关的蓝细菌和红藻中的同源蛋白不同，在光漂白时没有观察到猝灭，这可能允许稳健的光物理特性而不需要快速修复或替换机制。总之，这些观察结果确立了 PE545 存在丰富而稳健的构象状态。cryptophytes 由于其生存的各种微环境而表现出特别多样化的能量学，而这里报告的构象状态和动力学可能提供光物理灵活性，这有助于它们在各种条件下茁壮成长的非凡能力。
