Solvent-sensitive dyes to report protein conformational changes in living cells
Alexei Toutchkine1, Vadim Kraynov, Klaus Hahn
1Department of Cell Biology, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Journal of the American Chemical Society
|April 3, 2003
Summary
New merocyanine dyes enable live cell imaging of protein activity. These solvent-sensitive fluorescent probes covalently attach to proteins, reporting environmental changes for studying cellular processes.
Area of Science:
- Biochemistry
- Cell Biology
- Chemical Biology
Background:
- Fluorescent dyes are crucial for monitoring protein dynamics.
- Existing dyes often lack the necessary properties for live-cell imaging and quantitative analysis.
Purpose of the Study:
- To develop novel merocyanine dyes for live-cell imaging of protein activity.
- To create dyes with solvent sensitivity for reporting protein environment changes.
- To enable site-selective covalent attachment to proteins.
Main Methods:
- Synthesis of new merocyanine dyes with specific photophysical properties.
- Derivatization with iodoacetamide and succinimidyl ester for covalent protein labeling.
- Development of a biosensor for Cdc42 activation using the new dyes.
Main Results:
- The new dyes exhibit sharp dependence of quantum yield on solvent polarity/viscosity.
- Dyes possess excellent photophysical properties, photostability, and water solubility.
- A Cdc42 activation biosensor showed a 3-fold fluorescence increase upon GTP-binding.
Conclusions:
- The developed merocyanine dyes are suitable for live-cell imaging of protein activities.
- These dyes can be used to create versatile biosensors for diverse cellular processes.
- The findings advance the study of protein conformational changes and interactions in living cells.
Related Concept Videos
03:31Labeling DNA Probes
DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
01:19Protein Dynamics in Living Cells
Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...