Intrinsic dynamics in ECFP and Cerulean control fluorescence quantum yield

Area of Science:

• Biophysics
• Molecular Biology
• Biochemistry

Background:

• Enhanced cyan fluorescent protein (ECFP) and Cerulean are crucial for FRET-based cell imaging.
• Their distinct spectroscopic properties are key to their function as FRET donors.

Purpose of the Study:

• To elucidate the structural basis for the improved fluorescence of Cerulean over ECFP.
• To understand the origin of the double-peak spectroscopic signature in these proteins.

Main Methods:

• Denaturation experiments to identify the source of spectroscopic signatures.
• High-resolution crystal structure determination at physiological pH.
• Molecular dynamics simulations to analyze protein dynamics.

Main Results:

• The indole ring of the chromophore is responsible for the double-peak spectroscopic signature.
• Two conformations in the N-terminal half of the seventh strand interact with the chromophore.
• Mutations in Cerulean (Y145A, H148D) stabilize chromophore interactions, enhancing planarity and reducing quenching.

Conclusions:

• The enhanced fluorescence of Cerulean results from stabilized chromophore interactions and reduced nonradiative decay.
• Significant dynamical flexibility near the chromophore influences optical properties.
• Findings provide a basis for designing improved fluorescent protein mutants.