Slow refolding kinetics in yeast iso-2 cytochrome c

Area of Science:

• Biochemistry
• Protein Folding Dynamics
• Enzyme Kinetics

Background:

• Saccharomyces cerevisiae iso-2 cytochrome c exhibits complex folding pathways.
• Two distinct slow folding reactions, tau 1a and tau 1b, have been identified.

Purpose of the Study:

• To investigate the kinetics and thermodynamics of slow folding reactions in iso-2 cytochrome c.
• To elucidate the nature of the slow refolding species and their formation mechanisms.

Main Methods:

• Kinetic analysis of refolding using absorbance and fluorescence spectroscopy.
• Temperature dependence studies to determine activation enthalpies.
• Double-jump experiments to probe refolding intermediates.
• Amplitude measurements under varying unfolding conditions.

Main Results:

• Two slow folding reactions, tau 1a (absorbance-detected) and tau 1b (fluorescence-detected), were characterized with distinct rates.
• Activation enthalpies for tau 1a and tau 1b were determined to be 27 kcal/mol and 21 kcal/mol, respectively.
• Slow folding species are generated at different rates under unfolding conditions.
• The amplitudes of slow refolding were independent of initial guanidine hydrochloride concentration and pH.

Conclusions:

• The observed properties of the slow refolding species are consistent with proline imide bond isomerization.
• These findings contribute to understanding the intricate mechanisms of protein folding.