Chromophore formation in green fluorescent protein

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Chemistry

Background:

• Green fluorescent protein (GFP) from Aequorea Victoria possesses an intrinsic chromophore.
• The chromophore arises from cyclization and oxidation of an internal tripeptide motif.
• S65T-GFP mutant recovered from inclusion bodies lacks a mature chromophore, indicating aggregation before folding.

Purpose of the Study:

• To monitor and elucidate the in vitro chromophore formation process in S65T-GFP.
• To identify the kinetic steps and order of events in GFP chromophore maturation.
• To determine if GFP chromophore formation is an autocatalytic process.

Main Methods:

• Utilized the S65T-GFP chromophore mutant recovered from Escherichia coli inclusion bodies.
• Monitored chromophore formation in vitro using kinetic analysis.
• Determined the rate constants for individual steps in the reaction pathway.

Main Results:

• GFP chromophore formation proceeds through three distinct kinetic steps.
• Protein folding is the initial slow step (k(f) = 2.44 x 10(-3) s(-1)).
• An intermediate cyclization step (k(c) = 3.8 x 10(-3) s(-1)) is followed by a slow oxidation step (k(ox) = 1.51 x 10(-4) s(-1)).

Conclusions:

• GFP chromophore formation is an ordered, three-step kinetic process.
• The process occurs de novo from purified denatured protein.
• GFP chromophore formation is concluded to be an autocatalytic process.