Structural evidence for a dehydrated intermediate in green fluorescent protein chromophore biosynthesis

Area of Science:

• Biochemistry
• Structural Biology
• Fluorescent Proteins

Background:

• Green fluorescent proteins (GFPs) are vital tools in biological research.
• The acGFPL protein represents a novel, non-fluorescent GFP-like protein family.
• Understanding GFP maturation mechanisms is crucial for developing new fluorescent probes.

Purpose of the Study:

• To elucidate the high-resolution crystal structures of aceGFP and its mutant aceGFP-G222E.
• To investigate the immature and UV-photoconverted states of aceGFP-G222E.
• To determine the structural basis for the novel maturation pathway of aceGFP.

Main Methods:

• High-resolution crystallography
• Site-directed mutagenesis
• UV irradiation studies

Main Results:

• The colorless aceGFP-G222E structure reveals a trapped intermediate state with backbone cyclization and a non-oxidized Tyr-66.
• UV irradiation induces photoconversion to a green fluorescent state with a conventional coplanar chromophore.
• Mutagenesis identified Tyr-220 as critical for an alternative maturation pathway.

Conclusions:

• The structure of immature aceGFP-G222E provides insights into GFP chromophore biosynthesis intermediates.
• A novel maturation pathway for aceGFP involving Tyr-220, distinct from the canonical Glu-222, has been proposed.
• This work deepens the understanding of fluorescent protein structure-function relationships and engineering potential.