Single-pair fluorescence resonance energy transfer of nucleosomes in free diffusion:: Optimizing stability and resolution of subpopulations

被引：34

作者：

Gansen, Alexander ^{[1
]}

Hauger, Florian ^{[1
]}

Toth, Katalin ^{[1
]}

Langowski, Joerg ^{[1
]}

机构：

[1] German Canc Res Ctr, Div Biophys Macromol B040, D-69120 Heidelberg, Germany

来源：

ANALYTICAL BIOCHEMISTRY | 2007年 / 368卷 / 02期

关键词：

single-pair FRET; nucleosome reconstitution; nucleosome remodeling; fluorophore stability; chromatin structure;

D O I：

10.1016/j.ab.2007.04.047

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

We applied fluorescence detection methods on the single-molecule level to study structural variations and dynamic processes occurring within nucleosomes. Four fluorescent nucleosome constructs were made by attaching donor and acceptor fluorophores to different positions of two nucleosome positioning sequences and reconstituting nucleosornes by salt dialysis. The photochemical and biochemical stability of nucleosomes under single-molecule conditions was optimized by adding inert protein and free radical capturing additives, allowing us to define the best experimental conditions for single-molecule spectroscopy on highly diluted solutions of nucleosome complexes. We could demonstrate for the first time the resolution of conformational subpopulations of nUcleosornes by single-pair fluorescence resonance energy transfer in a freely diffusing system and could show the effect of thermally induced nucleosome repositioning. (c) 2007 Elsevier Inc. All rights reserved.

引用

页码：193 / 204

页数：12

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