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首页> 外文会议>Conference on Single Molecule Spectroscopy and Imaging; 20080119-21; San Jose,CA(US) >Monitoring the conformational dynamics of a single potassium transporter by ALEX-FRET
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Monitoring the conformational dynamics of a single potassium transporter by ALEX-FRET

机译：通过ALEX-FRET监测单个钾转运蛋白的构象动力学

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摘要

Conformational changes of single proteins are monitored in real time by Forster-type resonance energy transfer, FRET. Two different fluorophores have to be attached to those protein domains, which move during function. The distance between the fluorophores is measured by relative fluorescence intensity changes of FRET donor and acceptor fluorophore, or by fluorescence lifetime changes of the FRET donor. The fluorescence spectrum of a single FRET donor fluorophore is influenced by local protein environment dynamics causing apparent fluorescence intensity changes on the FRET donor and acceptor detector channels. To discriminate between those spectral fluctuations and distance-dependent FRET, alternating pulsed excitation schemes (ALEX) have recently been introduced which simultaneously probe the existence of a FRET acceptor fluorophore. Here we employ single-molecule FRET measurements to a membrane protein. The membrane-embedded KdpFABC complex transports potassium ions across a lipid bilayer using ATP hydrolysis. Our study aims at the observation of conformational fluctuations within a single P-type ATPase functionally reconstituted into liposomes by single-molecule FRET and analysis by Hidden-Markov-Models.
机译：通过Forster型共振能量转移FRET实时监测单个蛋白质的构象变化。必须将两个不同的荧光团连接至在功能过程中移动的那些蛋白质结构域。荧光团之间的距离是通过FRET供体和受体荧光团的相对荧光强度变化或FRET供体的荧光寿命变化来测量的。单个FRET供体荧光团的荧光光谱受局部蛋白质环境动力学的影响，导致FRET供体和受体检测器通道上的荧光强度发生明显变化。为了区分那些频谱波动和与距离相关的FRET，最近引入了交替脉冲激发方案（ALEX），该方案同时探测FRET受体荧光团的存在。在这里，我们采用单分子FRET测量膜蛋白。嵌入膜的KdpFABC复合物使用ATP水解将钾离子转运通过脂质双层。我们的研究旨在观察通过单分子FRET功能重构为脂质体的单个P型ATPase的构象波动，并通过Hidden-Markov模型进行分析。

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