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High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay
Published on: June 1, 2014
Design and introduction of a disulfide bridge in firefly luciferase: increase of thermostability and decrease of pH
Mehdi Imani1, Saman Hosseinkhani, Shahin Ahmadian
1Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Summary
Engineered firefly luciferase with a disulfide bridge exhibits enhanced thermal stability and pH insensitivity. One mutant, A296C/A326C, significantly increased specific activity and maintained bioluminescence at higher temperatures.
Area of Science:
- Biochemistry
- Protein Engineering
- Enzymology
Background:
- Firefly luciferase exhibits thermal sensitivity and pH-dependent spectral properties, limiting its applications.
- Protein stability can be enhanced by introducing disulfide bridges to reduce unfolding entropy.
- Disulfide bridges can be engineered into enzymes to improve their stability and functionality.
Purpose of the Study:
- To engineer disulfide bridges into Photinus pyralis firefly luciferase to enhance its thermal and pH stability.
- To investigate the impact of disulfide bridges on enzyme activity, spectral properties, and conformational stability.
- To compare the effects of disulfide bridges at different locations within the luciferase structure.
Main Methods:
- Site-directed mutagenesis was used to introduce single disulfide bridges into firefly luciferase, creating A103C/S121C and A296C/A326C mutants.
- Enzyme activity assays were performed to measure specific activity and pH sensitivity.
- Spectroscopic methods, including bioluminescence emission spectrum analysis and circular dichroism (CD) spectroscopy, were used to assess spectral properties and secondary structure changes.
- Thermal denaturation studies were conducted to evaluate conformational stability.
Main Results:
- The A103C/S121C mutant showed increased thermal stability but decreased specific activity.
- The A296C/A326C mutant displayed significant thermal stability, relative pH insensitivity, and a 7.3-fold increase in specific activity.
- The bioluminescence emission spectrum of the A296C/A326C mutant remained stable at elevated temperatures (37°C).
- Far-UV CD analysis indicated only minor secondary structure alterations in both mutants.
- Thermal denaturation analysis confirmed increased conformational stability for both mutants compared to the native enzyme.
Conclusions:
- Disulfide bridge engineering can significantly enhance the stability and functionality of firefly luciferase.
- The A296C/A326C mutation provides a highly stable and active firefly luciferase variant with improved pH insensitivity.
- The location of the disulfide bridge, particularly near the enzyme's active site, critically influences its kinetic characteristics and stability.
