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

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

Research output: Contribution to journalArticle

69 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)2920-2928
Number of pages9
JournalAnalytical chemistry
Volume73
Issue number13
DOIs
Publication statusPublished - 2001 Jul 1
Externally publishedYes

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Fluorescent Dyes
Recombinant Proteins
Labeling
Imaging techniques
Calmodulin
Proteins
Fluorescence
Fluorophores
Cysteine
Carrier Proteins
Peptides
Monitoring

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Imaging of conformational changes of proteins with a new environment-sensitive fluorescent probe designed for site-specific labeling of recombinant proteins in live cells. / Nakanishi, Jun; Nakajima, T.; Sato, M.; Ozawa, T.; Tohda, K.; Umezawa, Y.

In: Analytical chemistry, Vol. 73, No. 13, 01.07.2001, p. 2920-2928.

Research output: Contribution to journalArticle

Nakanishi, Jun ; Nakajima, T. ; Sato, M. ; Ozawa, T. ; Tohda, K. ; Umezawa, Y. / Imaging of conformational changes of proteins with a new environment-sensitive fluorescent probe designed for site-specific labeling of recombinant proteins in live cells. In: Analytical chemistry. 2001 ; Vol. 73, No. 13. pp. 2920-2928.
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