Snapshots of a protein folding intermediate

Area of Science:

• Biochemistry
• Protein dynamics
• Thermostable proteins

Background:

• Cytochrome c folding pathways are well-studied but exhibit rapid kinetics.
• Thermostable proteins offer unique insights into protein folding stability.
• Understanding folding intermediates is crucial for protein stability and function.

Purpose of the Study:

• To investigate the folding dynamics of Thermus thermophilus cytochrome c(552).
• To identify and characterize folding intermediates in a thermostable protein.
• To compare the folding kinetics of T. thermophilus cytochrome c(552) with cytochrome c.

Main Methods:

• Time-resolved fluorescence energy transfer (TR-FRET) using site-specific fluorescent probes.
• Kinetics studies to analyze unfolding and refolding pathways.
• Continuous flow mixing for millisecond timescale kinetic measurements.

Main Results:

• Observed both equilibrium unfolding and distinct refolding intermediates.
• Identified two-state or three-state denaturation transitions depending on the monitored region.
• Characterized an unfolding intermediate with native contacts in beta-sheet and C-terminal helix regions.
• Detected a millisecond-timescale refolding intermediate ensemble (heterogeneous mixture).
• Found no microsecond burst phase, contrasting with cytochrome c.

Conclusions:

• Thermus thermophilus cytochrome c(552) folding kinetics differ significantly from cytochrome c.
• An observable, albeit slower, refolding intermediate exists in this thermostable protein.
• The absence of a microsecond burst phase suggests a distinct folding pathway for T. thermophilus cytochrome c(552).