Yellow fluorescent protein phiYFPv (Phialidium): structure and structure-based mutagenesis

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• The marine jellyfish Phialidium harbors wild-type phiYFP, a rare naturally occurring yellow-orange fluorescent protein.
• Yellow fluorescent proteins are valuable tools in biological imaging and research.
• Understanding the structural basis of fluorescence is crucial for protein engineering.

Purpose of the Study:

• To determine the crystal structure of the improved yellow fluorescent protein phiYFPv.
• To elucidate the molecular mechanisms underlying its yellow emission spectrum.
• To guide further engineering of phiYFPv and related fluorescent proteins.

Main Methods:

• X-ray crystallography to determine the 3D structure of phiYFPv at 2.05 Å resolution.
• Structure-based site-directed mutagenesis to identify key functional residues.
• Spectroscopic analysis to characterize fluorescence properties.

Main Results:

• The crystal structure of phiYFPv revealed a chromophore with a bicyclic structure typical of green-emitting fluorophores.
• Antiparallel π-stacking between chromophore Tyr66 and proximal Tyr203, along with Val205, was identified as critical for the observed yellow emission at 537 nm.
• Mutagenesis studies confirmed the roles of specific residues in the chromophore environment.

Conclusions:

• The unique yellow fluorescence of phiYFPv originates from specific π-stacking interactions within the chromophore environment.
• These structure-function insights enable the rational design of improved yellow fluorescent proteins.
• The findings facilitate the enhancement of phiYFPv and the development of the biomarker tagYFP.