Department of Chemistry, Stanford University, Stanford, CA 94305.
Department of Chemistry, Stanford University, Stanford, CA 94305
Proc Natl Acad Sci U S A. 2017 Sep 12;114(37):9779-9784. doi: 10.1073/pnas.1705435114. Epub 2017 Aug 28.
Phycobilisomes are highly organized pigment-protein antenna complexes found in the photosynthetic apparatus of cyanobacteria and rhodophyta that harvest solar energy and transport it to the reaction center. A detailed bottom-up model of pigment organization and energy transfer in phycobilisomes is essential to understanding photosynthesis in these organisms and informing rational design of artificial light-harvesting systems. In particular, heterogeneous photophysical behaviors of these proteins, which cannot be predicted de novo, may play an essential role in rapid light adaptation and photoprotection. Furthermore, the delicate architecture of these pigment-protein scaffolds sensitizes them to external perturbations, for example, surface attachment, which can be avoided by study in free solution or in vivo. Here, we present single-molecule characterization of C-phycocyanin (C-PC), a three-pigment biliprotein that self-assembles to form the midantenna rods of cyanobacterial phycobilisomes. Using the Anti-Brownian Electrokinetic (ABEL) trap to counteract Brownian motion of single particles in real time, we directly monitor the changing photophysical states of individual C-PC monomers from in free solution by simultaneous readout of their brightness, fluorescence anisotropy, fluorescence lifetime, and emission spectra. These include single-chromophore emission states for each of the three covalently bound phycocyanobilins, providing direct measurements of the spectra and photophysics of these chemically identical molecules in their native protein environment. We further show that a simple Förster resonant energy transfer (FRET) network model accurately predicts the observed photophysical states of C-PC and suggests highly variable quenching behavior of one of the chromophores, which should inform future studies of higher-order complexes.
藻胆体是高度组织化的色素-蛋白天线复合物，存在于蓝藻和红藻的光合作用器官中，可收集太阳能并将其传递到反应中心。藻胆体中色素组织和能量转移的详细自下而上模型对于理解这些生物的光合作用以及为人工光捕获系统的合理设计提供信息至关重要。特别是，这些蛋白质的异质光物理行为（不能从头预测）可能在快速光适应和光保护中发挥重要作用。此外，这些色素-蛋白支架的精细结构使其对外界干扰敏感，例如表面附着，通过在游离溶液或体内进行研究可以避免这种情况。在这里，我们展示了 C-藻蓝蛋白（C-PC）的单分子表征，C-PC 是一种三色素的胆红素蛋白，可自组装形成蓝藻藻胆体的中天线棒。使用反布朗运动电动（ABEL）陷阱实时抵消单个粒子的布朗运动，我们通过同时读取它们的亮度、荧光各向异性、荧光寿命和发射光谱，直接监测游离溶液中单个 C-PC 单体不断变化的光物理状态。这些包括三个共价结合的藻蓝胆素的每个色素的单色素发射状态，提供了这些化学上相同的分子在其天然蛋白环境中的光谱和光物理性质的直接测量。我们进一步表明，简单的Förster 共振能量转移（FRET）网络模型准确预测了 C-PC 的观察到的光物理状态，并表明一个发色团的猝灭行为具有高度可变性，这应该为进一步研究更高阶复合物提供信息。
