Multiple kinetic intermediates accumulate during the unfolding of horse cytochrome c in the oxidized state.

The research article investigates the process of unfolding kinetics for the horse cytochrome c protein in an oxidized state. Different temperature and chemical conditions were tested, resulting in the discovery of two distinct kinetic unfolding intermediates.

Research Methodology

• The research team studied the unfolding kinetics of horse cytochrome c in the oxidized state at three different temperatures: 10, 22, and 34 degrees Celsius. The process was further analyzed based on guanidine hydrochloride (GdnHCl) concentration levels.
• Two methods were used for monitoring the unfolding process: rapid measurements of far-ultraviolet circular dichroism (far-UV CD) and fluorescence quenching due to energy transfer from tryptophan to the heme part of the protein.

Results & Observations

• At 10 degrees Celsius, the decrease in far-UV CD signal unique to protein unfolding occurred in two phases, defined as the ‘burst phase’ and a slower phase happening over tens to hundreds of milliseconds.
• The researchers observed that the burst phase unfolding amplitude increased cooperatively with an increase in GdnHCl concentration, exhibiting a transition midpoint of 3.2 M at 10 degrees Celsius.
• In contrast, no changes were recorded in fluorescence during the burst phase at this particular temperature.
• Unfolding kinetics measured at both 22 and 34 degrees Celsius, either through fluorescence or far-UV CD, were biphasic. Several discrepancies were noted between the transition midpoints and burst phase amplitudes dependencies on GdnHCl concentration in the two methods.

Key Conclusions

• At least two kinetic unfolding intermediates accumulate during the protein unfolding process. The researchers defined these intermediates as IU1 and IU2.
• Intermediate IU1 loses all the native-state secondary structure during the burst phase with GdnHCl, while IU2 loses both secondary structure and native-like compactness.
• The existence of these kinetic unfolding intermediates is suggested by the nonlinear relationship between GdnHCl concentration and the apparent unfolding rate constant, especially pronounced at 10 and 22 degrees Celsius.
• The stabilities of IU1 and IU2 were found to decrease steadily with the increase in temperature from 10 to 34 degrees Celsius, suggesting that hydrogen bonding interactions primarily stabilize the structures within these intermediates.