StayRose: A photostable StayGold derivative redshifted by genetic code expansion

Area of Science:

• Biochemistry
• Molecular Biology
• Microscopy

Background:

• Photobleaching of fluorescent proteins hinders high-quality microscopy.
• Monomeric StayGold offers improved photostability.
• Development of multicolor fluorescent proteins is crucial for simultaneous target labeling.

Purpose of the Study:

• To create redshifted variants of the photostable StayGold fluorescent protein.
• To investigate the use of unnatural amino acids for spectral tuning.
• To develop new tools for multicolor fluorescence microscopy.

Main Methods:

• Sequence engineering of StayGold.
• Genetic code expansion to incorporate 3-aminotyrosine.
• Spectroscopic analysis of the resulting protein (StayRose).
• In vivo photostability testing in E. coli and zebrafish embryos.
• High-resolution crystal structure determination of StayRose.

Main Results:

• A novel red fluorescent protein, StayRose, was engineered from StayGold.
• StayRose exhibits excitation/emission maxima at 530 nm/588 nm.
• Monomeric mStayRose demonstrated high photostability in vivo.
• Crystal structure confirmed the amino chromophore modification within a stable fold.

Conclusions:

• StayRose provides a redshifted, photostable fluorescent protein option.
• This work advances the development of multicolor StayGold derivatives.
• Genetic code expansion is a viable strategy for tuning fluorescent protein properties.