Solvent-Sensitive Dyes to Report Protein Conformational Changes in Living Cells
Covalent attachment of solvent-sensitive fluorescent dyes to proteins is a powerful tool for studying protein conformational changes, ligand binding, or posttranslational modifications. We report here new merocyanine dyes that make possible the quantitation of such protein activities in individual l...
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Published in | Journal of the American Chemical Society Vol. 125; no. 14; pp. 4132 - 4145 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
WASHINGTON
American Chemical Society
09.04.2003
Amer Chemical Soc |
Subjects | |
Online Access | Get full text |
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Summary: | Covalent attachment of solvent-sensitive fluorescent dyes to proteins is a powerful tool for studying protein conformational changes, ligand binding, or posttranslational modifications. We report here new merocyanine dyes that make possible the quantitation of such protein activities in individual living cells. The quantum yield of the new dyes is sharply dependent on solvent polarity or viscosity, enabling them to report changes in their protein environment. This is combined with other stringent requirements needed in a live cell imaging dye, including appropriate photophysical properties (excitation >590 nm, high fluorescence quantum yield, high extinction coefficient), good photostability, minimal aggregation in water, and excellent water solubility. The dyes were derivatized with iodoacetamide and succinimidyl ester side chains for site-selective covalent attachment to proteins. A novel biosensor of Cdc42 activation made with one of the new dyes showed a 3-fold increase in fluorescence intensity in response to GTP-binding by Cdc42. The dyes reported here should be useful in the preparation of live cell biosensors for a diverse range of protein activities. |
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Bibliography: | istex:840D7BC100E6246CA65300BA3D349C8645C4C83E ark:/67375/TPS-NM72NM0L-V Medline NIH RePORTER ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0002-7863 1520-5126 |
DOI: | 10.1021/ja0290882 |