Engineering of mCherry variants with long Stokes shift, red-shifted fluorescence, and low cytotoxicity
Yi Shen1, Yingche Chen1, Jiahui Wu1
1Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
Abstract:
MCherry, the Discosoma sp. mushroom coral-derived monomeric red fluorescent protein (RFP), is a commonly used genetically encoded fluorophore for live cell fluorescence imaging. We have used a combination of protein design and directed evolution to develop mCherry variants with low cytotoxicity to Escherichia coli and altered excitation and emission profiles. These efforts ultimately led to a long Stokes shift (LSS)-mCherry variant (λex = 460 nm and λem = 610 nm) and a red-shifted (RDS)-mCherry variant (λex = 600 nm and λem = 630 nm). These new RFPs provide insight into the influence of the chromophore environment on mCherry's fluorescence properties, and may serve as templates for the future development of fluorescent probes for live cell imaging.
Insights
Researchers engineered new versions of the mCherry fluorescent protein for improved live cell imaging. These red fluorescent protein (RFP) variants offer altered spectral properties and reduced toxicity, aiding in advanced microscopy applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Microscopy
Background:
- mCherry, a red fluorescent protein (RFP) from Discosoma sp. mushroom coral, is vital for live cell fluorescence imaging.
- Existing mCherry variants can exhibit cytotoxicity and have limitations in excitation/emission profiles.
Purpose of the Study:
- To develop mCherry variants with reduced cytotoxicity in Escherichia coli.
- To engineer mCherry with modified excitation and emission spectra for enhanced fluorescence imaging.
Main Methods:
- Utilized a combination of protein design and directed evolution techniques.
- Focused on altering the chromophore environment within the mCherry protein structure.
Main Results:
- Successfully created a long Stokes shift (LSS)-mCherry variant (excitation: 460 nm, emission: 610 nm).
- Developed a red-shifted (RDS)-mCherry variant (excitation: 600 nm, emission: 630 nm).
- Demonstrated that chromophore environment significantly influences mCherry fluorescence properties.
Conclusions:
- New mCherry variants offer improved spectral characteristics and lower cytotoxicity for live cell imaging.
- These engineered fluorescent proteins can serve as foundational tools for developing novel probes in microscopy.
- Provides valuable insights into structure-function relationships governing RFP fluorescence.
