Imaging of Conformational Changes of Proteins with a New Environment-Sensitive Fluorescent Probe Designed for Site-Specific Labeling of Recombinant Proteins in Live Cells

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[a]phenoxazin-5-one. This fluorescent probe can be attached to recombinant proteins contain...

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Veröffentlicht in:Analytical chemistry (Washington) 2001-07, Vol.73 (13), p.2920-2928
Hauptverfasser: Nakanishi, Jun, Nakajima, Takahiro, Sato, Moritoshi, Ozawa, Takeaki, Tohda, Kohji, Umezawa, Yoshio
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container_end_page 2928
container_issue 13
container_start_page 2920
container_title Analytical chemistry (Washington)
container_volume 73
creator Nakanishi, Jun
Nakajima, Takahiro
Sato, Moritoshi
Ozawa, Takeaki
Tohda, Kohji
Umezawa, Yoshio
description 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[a]phenoxazin-5-one. This fluorescent probe can be attached to recombinant 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 well-known 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.
doi_str_mv 10.1021/ac001528p
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Chem</addtitle><date>2001-07-01</date><risdate>2001</risdate><volume>73</volume><issue>13</issue><spage>2920</spage><epage>2928</epage><pages>2920-2928</pages><issn>0003-2700</issn><eissn>1520-6882</eissn><coden>ANCHAM</coden><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[a]phenoxazin-5-one. This fluorescent probe can be attached to recombinant 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 well-known 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.</abstract><cop>Washington, DC</cop><pub>American Chemical Society</pub><pmid>11467536</pmid><doi>10.1021/ac001528p</doi><tpages>9</tpages></addata></record>
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source MEDLINE; American Chemical Society Journals
subjects Amino Acid Sequence
Biological and medical sciences
Cell Line
Chromatography, High Pressure Liquid
Diverse techniques
Fluorescence
Fluorescent Dyes - chemistry
Fundamental and applied biological sciences. Psychology
Humans
Molecular and cellular biology
Molecular Sequence Data
Molecules
Nuclear Magnetic Resonance, Biomolecular
Protein Conformation
Proteins
Recombinant Proteins - chemistry
Scientific imaging
title Imaging of Conformational Changes of Proteins with a New Environment-Sensitive Fluorescent Probe Designed for Site-Specific Labeling of Recombinant Proteins in Live Cells
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