Light-activated reassembly of split green fluorescent protein

Area of Science:

• Biochemistry
• Biophysics
• Molecular Biology

Background:

• Truncated green fluorescent protein (GFP) lacking the 11th β-strand is a target for bioconjugation and semisynthetic protein applications.
• Standard refolding of truncated GFP does not restore its ability to bind the synthetic 11th strand peptide.

Purpose of the Study:

• To investigate the mechanism behind the light-activated reassembly of truncated GFP with its synthetic 11th strand peptide.
• To explore the potential of this light-driven reassembly for controlling protein-protein interactions.

Main Methods:

• Absorption spectroscopy
• Fluorescence spectroscopy
• Raman spectroscopy
• Kinetic modeling

Main Results:

• Refolded truncated GFP contains a chromophore in the trans configuration.
• Light exposure induces a photostationary state involving trans and cis chromophore conformations.
• Only truncated GFP with the cis chromophore configuration binds the synthetic 11th strand peptide.

Conclusions:

• The reassembly of split GFP is a light-activated process dependent on chromophore isomerization.
• This light-controlled mechanism provides a novel method for regulating protein-protein interactions.
• The findings have implications for bioconjugation, imaging, and the development of functional semisynthetic proteins.