Combined Structural and Computational Study of the mRubyFT Fluorescent Timer Locked in Its Blue Form

Area of Science:

• Biophysics
• Structural Biology
• Fluorescent Proteins

Background:

• mRubyFT is a monomeric fluorescent timer protein with high brightness and photostability.
• The red form structure of mRubyFT is known, but the blue form remains structurally uncharacterized.
• Understanding the blue form is key to optimizing fluorescent timer applications.

Purpose of the Study:

• To determine the high-resolution structure of the blue form of mRubyFT.
• To elucidate the chromophore conformation and interactions within the blue form.
• To investigate the protonation states and spectral properties of the blue form.

Main Methods:

• X-ray crystallography of the mRubyFT S148I variant at 1.8 Å resolution.
• Molecular dynamic simulations (MD) with quantum mechanic/molecular mechanic (QM/MM) potentials.
• Excited state MD simulations.

Main Results:

• A detailed model of the blue chromophore conformation and its interactions with neighboring residues was proposed.
• The imidazolidinone moiety is a conjugated π-system, and the methine bridge is not oxidized.
• Hydrogen bonding between the phenolic moiety and T163 was identified.
• Two protonation states (anion and zwitterion) of the blue form were characterized, both in enolate tautomeric forms.
• Excitation leads to the same anionic fluorescent state for both protonation states.

Conclusions:

• The study provides the first structural insights into the blue form of the mRubyFT fluorescent timer.
• Structural and computational data explain the spectral properties of the blue form.
• This work facilitates the rational design of improved fluorescent timer proteins.