利用ALEX-FRET监测单个钾转运蛋白的构象动力学
Monitoring the conformational dynamics of a single potassium transporter by ALEX-FRET
摘要：
通过福斯特型共振能量转移（FRET）实时监测单个蛋白质的构象变化。两种不同的荧光团必须附着在这些蛋白质结构域上，它们在功能过程中移动。通过FRET供体和受体荧光团的相对荧光强度变化，或通过FRET供体的荧光寿命变化来测量荧光团之间的距离。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明显荧光强度变化。为了区分这些光谱波动和距离依赖性FRET，交替脉冲激发方案（ALEX）最近已被引入，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。膜包埋的KdpFABC复合物利用ATP水解将钾离子转运穿过脂质双层。我们的研究旨在观察内的一个单一的P-型ATP酶功能重组成脂质体的单分子FRET和分析的隐藏马尔可夫模型的构象波动。
英文摘要：
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.
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