Imaging of conformational changes of proteins with a new environment-sensitive fluorescent probe designed for site-specific labeling of recombinant proteins in live cells

J. Nakanishi, T. Nakajima, M. Sato, T. Ozawa, K. Tohda, Y. Umezawa*

*この研究の対応する著者

研究成果: Article査読

74 被引用数 (Scopus)

抄録

We demonstrate herein a new method for imaging conformational changes of proteins in live cells using a new synthetic environment-sensitive fluorescent probe, 9-amino-6,8-bis(1,3,2-dithioarsolan-2-yl)-5H-benzo[α]phenoxazin5-one, This fluorescent probe can be attached to binant proteins containing four cysteine residues at the i, i + 1, i + 4, and i + 5 positions of an α-helix. The specific binding of the fluorescent probe to this 4Cys motif enables fluorescent labeling inside cells by its extracellular administration. The high sensitivity of the fluorophore to its environment enables monitoring of the conformational changes of the proteins in live cells as changes in its fluorescence intensity. The present method was applied to calmodulin (CAM), a Ca2+-binding protein that was wellknown to expose hydrophobic domains, depending on the Ca2+ concentration. A recombinant CaM fused at its C-terminal with a helical peptide containing a 4Cys motif was labeled with the fluorescent probe inside live cells. The fluorescence intensity changed reversibly depending on the intracellular Ca2+ concentration, which reflected the conformational change of the recombinant CAM in the live cells.

本文言語English
ページ(範囲)2920-2928
ページ数9
ジャーナルAnalytical chemistry
73
13
DOI
出版ステータスPublished - 2001 7月 1
外部発表はい

ASJC Scopus subject areas

  • 分析化学

フィンガープリント

「Imaging of conformational changes of proteins with a new environment-sensitive fluorescent probe designed for site-specific labeling of recombinant proteins in live cells」の研究トピックを掘り下げます。これらがまとまってユニークなフィンガープリントを構成します。

引用スタイル