Cytochrome c folds through a smooth funnel

Area of Science:

• Biochemistry
• Protein Folding Dynamics
• Biophysical Chemistry

Background:

• Cytochrome c folding is characterized by nonnative His-heme kinetic traps that impede the native state formation.
• These kinetic traps can represent the majority of folding species, with their dissociation rate limiting overall folding.
• Understanding these traps is crucial for elucidating protein folding mechanisms.

Purpose of the Study:

• To compare the unfolding and folding kinetics of wild-type yeast iso-2 cytochrome c with a double mutant lacking His-heme kinetic traps (H33N,H39K iso-2).
• To investigate the role of His-heme kinetic traps in the folding pathway and their impact on folding rates.
• To determine the thermodynamic properties of the transition states for both proteins.

Main Methods:

• Utilized a temperature jump (T-jump) relaxation technique to study folding and unfolding kinetics.
• Monitored absorbance changes at 287 nm to measure solvent exclusion from aromatic residues.
• Applied a two-state model to calculate folding and unfolding rate constants and thermodynamic activation parameters.

Main Results:

• The double mutant H33N,H39K iso-2 exhibited a four to eight times faster observed relaxation time (tau(obs)) compared to wild-type iso-2 cytochrome c.
• Thermodynamic properties of the transition states were found to be very similar between the wild-type and mutant proteins.
• A small activation enthalpy for folding was observed, suggesting a predominantly entropic kinetic barrier.

Conclusions:

• Elimination of His-heme kinetic traps significantly accelerates cytochrome c folding.
• The folding barrier for cytochrome c is largely entropic, consistent with a smooth funnel folding landscape model.
• Kinetic traps play a critical role in modulating protein folding rates.