Voltage-controlled fluorescence switching of a single redox protein
Voltage-controlled fluorescence switching of a single redox protein
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DOI：
10.1016/j.bios.2014.07.051
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发表时间：
2015-05-15
影响因子：
12.6
通讯作者：
Sanghamitra, Nusrat J. M.
中科院分区：
文献类型：
--
作者：
Akkilic, Namik;Kamran, Muhammad;Sanghamitra, Nusrat J. M.
通过荧光共振能量转移（FRET）在单分子（SM）水平上监测了铜绿假单胞菌氧化还原蛋白野生型天青蛋白（wt-Az）的异质电子转移（ET），每次一个电子。用有机荧光团（Cy 5）标记天青蛋白分子，并使用Cy 5与氧化还原中心（铜）之间的FRET耦合来研究在光学配置中半透明的10 nm薄金电极的ET。通过使用共聚焦显微镜和用于控制电极电位的双恒电位仪，监测单个Az-Cy 5分子的氧化和还原过程。在天青蛋白分子的氧化还原中心的氧化状态下，共价连接的Cy 5的荧光发射在很大程度上被FRET淬灭（“关"状态），而发射在还原后恢复（”开"状态）。这里提出的工作，显示直接控制单个氧化还原蛋白质的氧化还原开关事件及其热力学分散。我们表明，分布的中点电位（Eo）的个别天青分子的峰值在45.7 +/- 0.5 mV与饱和甘汞电极（SCE）的半高宽为15 mV。(C)2014爱思唯尔有限公司版权所有。
Heterogeneous electron transfer (ET) of the redox protein, wild-type azurin (wt-Az) from Pseudomonas aeruginosa, was monitored at the single-molecule (SM) level by fluorescence resonance energy transfer (FRET), one electron at a time. Azurin molecules were labeled with an organic fluorophore (Cy5), and the FRET-coupling between Cy5 and the redox center (copper) was used to study ET to a semi-transparent, 10 nm thin gold electrode in an optical configuration. By using a confocal microscope and a bipotentiostat for control of the electrode potential, the oxidation and reduction processes of individual Az-Cy5 molecules were monitored. In the oxidized state of the redox center of the azurin molecule, the fluorescence emission of the covalently attached Cy5 was largely quenched by FRET ('off'-state), whereas the emission was recovered upon reduction ('on'-state). The work presented here, shows directly controlled single redox switching events of an individual redox protein and its thermodynamic dispersion. We show that the distribution of midpoint potentials (Eo) of individual azurin molecules peaks at 45.7 +/- 0.5 mV with a full width at half maximum of 15 mV vs saturated calomel electrode (SCE). (C) 2014 Elsevier B.V. All rights reserved.