Related Experiment Video
Updated: May 30, 2026
10:57
Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules
Published on: November 2, 2009
Fluorescent nucleoside triphosphates for single-molecule enzymology.
Christopher P Toseland1, Martin R Webb
1MRC National Institute for Medical Research, London, UK. chris.toseland@med.uni-muenchen.de
Methods in Molecular Biology (Clifton, N.J.)
|August 3, 2011
Summary
Researchers developed fluorescent ATP analogs to study enzyme kinetics. These tools track nucleotide binding and conformational changes in real-time, aiding the understanding of crucial cellular energy transfer processes.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Nucleoside triphosphate (NTP) and diphosphate interconversion is vital for cellular energy.
- Enzymes like triphosphatases, kinases, and ATP synthases catalyze these reactions.
- Understanding enzyme kinetics and conformational changes is key to cellular processes.
Purpose of the Study:
- To explore the role of fluorescent nucleotides in single-molecule studies.
- To develop methods for preparing ATP analogs with specific fluorophores.
- To investigate nucleotide binding, release, and conformational changes.
Main Methods:
- Selection of appropriate fluorophores and nucleotide modifications.
- Preparation of Adenosine Triphosphate (ATP) analogs.
- Utilizing fluorescent nucleotides to monitor single-molecule events.
Main Results:
- Demonstrated the utility of fluorescent nucleotides for studying enzyme mechanisms.
- Provided methods for synthesizing ATP analogs with diethylaminocoumarin and Cy3 fluorophores.
- Enabled real-time tracking of nucleotide-dependent conformational changes.
Conclusions:
- Fluorescent nucleotides are powerful tools for dissecting enzyme mechanisms at the single-molecule level.
- The developed ATP analogs facilitate the study of energy-transducing enzymes.
- This approach enhances the understanding of fundamental cellular reactions involving NTPs.
Related Concept Videos
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...
01:14Photoluminescence: Applications
Photoluminescence offers a wide range of applications due to its inherent sensitivity and selectivity. This technique allows for both direct and indirect analyses of the analyte. Direct quantitative analysis is possible when the analyte exhibits a favorable quantum yield for fluorescence or phosphorescence. However, an indirect analysis may be feasible if the analyte is not fluorescent or phosphorescent, or if the quantum yield is unfavorable. Indirect methods include reacting the analyte with...