Two-state expansion and collapse of a polypeptide

Area of Science:

• Biophysics
• Protein Folding Dynamics
• Biochemistry

Background:

• Protein folding is initiated by polypeptide chain collapse from expanded to compact, yet denatured, states.
• Theoretical models and simulations propose this collapse as a two-state transition with barrier-crossing kinetics.
• Homopolymer and random heteropolymer collapse are generally considered continuous and multi-phasic processes.

Purpose of the Study:

• To investigate the kinetic mechanism of polypeptide collapse.
• To resolve the complete time-course of protein collapse at high temporal resolution.
• To compare experimental findings with theoretical predictions for homopolymer collapse.

Main Methods:

• Utilized a novel rapid-mixing flow technique to study late stages of collapse (>45 microseconds).
• Employed laser temperature-jump coupled with fluorescence spectroscopy for nanosecond time resolution.
• Analyzed the time-course of denatured cytochrome c collapse.

Main Results:

• The polypeptide collapse process was found to be exponential over time.
• The collapse is thermally activated, exhibiting an apparent activation energy of approximately 9 k(B)T (adjusted for solvent viscosity).
• Experimental data indicates a significant free energy barrier governing the collapse process.

Conclusions:

• Polypeptide collapse is kinetically a two-state transition, contrary to continuous models.
• The observed free energy barrier results in a collapse time scale significantly slower than predicted by Langevin models for homopolymer collapse.
• This study provides critical insights into the fundamental kinetics of protein folding initiation.