Related Experiment Video
Updated: Oct 19, 2025
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Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques
Published on: November 2, 2018
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Synchronized Real-time Measurement of Sec-mediated Protein Translocation.
Riti Gupta1, Dmitri Toptygin2, Christian M Kaiser2,3
1CMDB Graduate Program, Johns Hopkins University, 3400 N Charles St., Baltimore, MD 21218, USA.
Bio-Protocol
|September 20, 2021
Summary
This study introduces a real-time assay to measure protein translocation via the Sec translocon (SecYEG/SecA) in bacteria. The new method, combined with kinetic modeling, provides high-resolution mechanistic insights into protein export.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Biology
Background:
- The Sec translocon (SecYEG/SecA) is crucial for bacterial protein export.
- Existing translocation assays lack high time resolution.
- Understanding translocation kinetics is vital for mechanistic insights.
Purpose of the Study:
- To develop and implement a real-time assay for measuring protein translocation kinetics.
- To enable high-resolution mechanistic analysis of the Sec translocon.
- To analyze translocation kinetics using a detailed kinetic model.
Main Methods:
- Implementation of a real-time translocation assay using reconstituted Sec translocon.
- Engineering of substrate proteins for assay priming.
- Development of a detailed kinetic model including on- and off-pathway processes.
Main Results:
- Successful development of a real-time assay for measuring protein translocation.
- High-resolution kinetic data of the Sec translocon's function.
- Kinetic model effectively analyzes translocation dynamics.
Conclusions:
- The developed assay and model enable detailed mechanistic studies of Sec-dependent protein translocation.
- Real-time measurements provide unprecedented resolution of protein export dynamics.
- This approach advances our understanding of fundamental protein transport mechanisms.
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