Trigger factor assisted folding of green fluorescent protein

Area of Science:

• Biochemistry
• Structural Biology
• Protein Folding Dynamics

Background:

• Green fluorescent protein (GFP) variants are crucial tools in molecular biology.
• Understanding protein folding pathways is essential for protein engineering and drug development.
• The role of enzymes like TF (thioredoxin fold) in protein folding is complex and multifaceted.

Purpose of the Study:

• To elucidate the equilibrium and kinetic folding pathways of a specific GFP variant (GFPuv).
• To investigate the dual role of TF as both a catalyst and a chaperone in GFPuv folding.
• To characterize the thermodynamic and kinetic parameters governing GFPuv folding.

Main Methods:

• Utilized manual mixing and stopped-flow techniques for kinetic studies.
• Employed multiple probes including tryptophan fluorescence, chromophore fluorescence, and DTNB reactivity.
• Analyzed guanidine-induced unfolding and refolding kinetics of GFPuv.

Main Results:

• GFPuv folding and unfolding proceed in a stepwise manner, populating a stable, compact intermediate.
• This intermediate retains significant green fluorescence compared to the denatured state.
• Wild-type TF accelerates folding at low concentrations by catalyzing proline isomerization but retards it at high concentrations.

Conclusions:

• GFPuv folding involves a stable, fluorescent intermediate.
• TF exhibits dual activity, acting as an enzyme and a chaperone, with concentration-dependent effects on GFPuv folding.
• A general mechanism for TF-assisted protein folding is proposed, highlighting its complex regulatory role.