Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers.
Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers.
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DOI：
10.1038/srep33257
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发表时间：
2016-09-19
影响因子：
4.6
通讯作者：
Cordes T
中科院分区：
文献类型：
--
作者：
Ploetz E;Lerner E;Husada F;Roelfs M;Chung S;Hohlbein J;Weiss S;Cordes T
先进的显微镜方法允许通过测量结构中的单个分子距离来获得关于生物分子中的（动态）构象变化的信息。然而，这是非常具有挑战性的捕获的三维生化状态，结合相关的结构变化或构象串扰的多蛋白质复合物中使用一维测定的全部深度。在本文中，我们解决了这个基本问题，通过扩展标准的分子标尺的基础上Förster共振能量转移（FRET）到一个二维的测定，通过结合蛋白诱导荧光增强（PIFE）。我们表明，供体亮度（通过PIFE）和能量转移效率（通过FRET）可以同时报告，例如，双链DNA（dsDNA）与未标记蛋白（BamHI、EcoRV和T7 DNA聚合酶gp 5/trx）相互作用后的构象状态。PIFE-FRET检测使用已建立的标记方案和单分子荧光检测方案（交替激光激发，ALEX）。除了定量研究PIFE和FRET统治者的特点，我们概述了可能的应用ALEX为基础的PIFE-FRET扩散和固定化分子的单分子研究。最后，我们研究了E.大肠杆菌RNA聚合酶与PIFE-FRET和提供直接证据的物理存在和附近的聚合酶，导致结构变化和scrunching的转录DNA泡。
Advanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-protein complexes using one-dimensional assays. In this paper we address this fundamental problem by extending the standard molecular ruler based on Förster resonance energy transfer (FRET) into a two-dimensional assay via its combination with protein-induced fluorescence enhancement (PIFE). We show that donor brightness (via PIFE) and energy transfer efficiency (via FRET) can simultaneously report on e.g., the conformational state of double stranded DNA (dsDNA) following its interaction with unlabelled proteins (BamHI, EcoRV, and T7 DNA polymerase gp5/trx). The PIFE-FRET assay uses established labelling protocols and single molecule fluorescence detection schemes (alternating-laser excitation, ALEX). Besides quantitative studies of PIFE and FRET ruler characteristics, we outline possible applications of ALEX-based PIFE-FRET for single-molecule studies with diffusing and immobilized molecules. Finally, we study transcription initiation and scrunching of E. coli RNA-polymerase with PIFE-FRET and provide direct evidence for the physical presence and vicinity of the polymerase that causes structural changes and scrunching of the transcriptional DNA bubble.
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影响因子：
14.9
作者：
Craggs TD;Hutton RD;Brenlla A;White MF;Penedo JC
通讯作者：
Penedo JC
影响因子：
16.8
作者：
Gouridis, Giorgos;Schuurman-Wolters, Gea K.;Poolman, Bert
通讯作者：
Poolman, Bert
影响因子：
48
作者：
Campos, Luis A.;Liu, Jianwei;Munoz, Victor
通讯作者：
Munoz, Victor
DOI：
10.1073/pnas.0912664107
发表时间：
2010-02-02
影响因子：
11.1
作者：
Etson, Candice M.;Hamdan, Samir M.;van Oijen, Antoine M.
通讯作者：
van Oijen, Antoine M.
影响因子：
--
作者：
DIPAOLO, RE;SCAFFARDI, LB;BILMES, GM
通讯作者：
BILMES, GM