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Influence of Fluorescence Lifetime Selections and Conformational Flexibility on Brightness of FusionRed Variants
Open accessPublished version of a preprint
Abstract
Fluorescent proteins (FPs) for bioimaging are typically developed by screening mutant libraries for clones with improved photophysical properties. This approach has resulted in FPs with high brightness, but the mechanistic origins of the improvements are often unclear. We focused on improving the molecular brightness in the FusionRed family of FPs with fluorescence lifetime selections on targeted libraries, with the aim of reducing nonradiative decay rates. Our new variants show fluorescence quantum yields of up to 75% and lifetimes >3.5 ns. We present a comprehensive analysis of these new FPs, including trends in spectral shifts, photophysical data, photostability, and cellular brightness resulting from codon optimization. We also performed all-atom molecular dynamics simulations to investigate the impact of side chain mutations. The trajectories reveal that individual mutations reduce the flexibility of the chromophore and side chains, leading to an overall reduction in nonradiative rates.
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Highly accurate protein structure prediction with AlphaFold
Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Žídek, A., Potapenko, A., et al. (2021) · Nature · doi:10.1038/s41586-021-03819-2 · Cited by 43,764
Accurate prediction of protein structures and interactions using a 3-track network
Baek, M., DiMaio, F., Anishchenko, I., Dauparas, J., Ovchinnikov, S., Lee, G. R., Wang, J., Cong, Q., Kinch, L. N., Schaeffer, R. D., et al. (2021) · doi:10.1101/2021.06.14.448402 · Cited by 5,549
Non-canonical amino acids in protein engineering
Link, A. J., Mock, M. L., & Tirrell, D. A. (2003) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2003.10.011 · Cited by 393
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Metadata
- Authors
- Mukherjee, S., Douglas, N., & Jimenez, R.
- Affiliations
- National Institute of Standards and Technology (US)
- University of Colorado Boulder (US)
- Corresponding author
- Jimenez, Ralph
- Venue
- The Journal of Physical Chemistry Letters
- Publisher
- American Chemical Society (ACS)
- Published
- 2024-02-05
- Volume
- 15
- Issue
- 6
- Pages
- 1644-1651
- ISSN
- 1948-7185 (ISSN-L)
- OpenAlex
- W4391542369
- Cited by
- 6
- References
- 3
- Citations by year
- 2026: 3 · 2025: 2 · 2024: 1
- FWCI
- 1.16
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- Locations
- The Journal of Physical Chemistry Letters (publishedVersion)
- ChemRxiv (acceptedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Funders
- Division of Physics (PHY 2317149)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Physical Sciences › Materials Science › Materials Chemistry › Luminescence and Fluorescent Materials
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › bioluminescence and chemiluminescence research
- Keywords
- fluorescent proteins, fluorescence lifetime, molecular brightness, nonradiative decay, conformational flexibility, codon optimization, photostability, quantum yield, wavelength shift, molecular dynamics simulation
- MeSH terms
- Coloring Agents, Fluorescence, Mutation, Protein Conformation, Spectrometry, Fluorescence, Green Fluorescent Proteins, Molecular Dynamics Simulation
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