Effect of urea and alkylureas on the stability and structural fluctuation of the M80-containing Ω-loop of horse cytochrome c.

This research investigates how various denaturants affect the structural instability of a subcomponent of ferrocytochrome c, a protein complex. Specifically, they determined how this structural fluctuation was influenced by varying concentrations of urea and alkylureas under certain experimental conditions.

Objective and Approach

• The study focuses on understanding how different denaturants, such as urea and alkylureas, influence the structural fluctuation of the M80-containing Ω-loop, a part of the ferrocytochrome c protein complex.
• The research was conducted at a particular pH and temperature, and the reaction rate coefficient of carbon monoxide (CO) association with ferrocytochrome c was measured under different concentrations of denaturants.

Findings

• Researchers observed that when the concentration of denaturants was increased within the subdenaturing limit, the CO-association reaction slowed down. This indicates that these concentrations decreased the structural fluctuation of the Ω-loop.
• It was found that urea reduced the structural fluctuation of the Ω-loop more compared to tetramethylurea (TMU).
• Upon performing intermolecular docking between horse cytochrome c and the denaturant molecules, it was discovered that as the alkyl groups on the urea molecule increased, polyfunctional interactions between the denaturant and the Ω-loop decreased. This suggested that the reduced structural dynamics of the Ω-loop with increasing alkyl groups on the urea molecule was due to a decrease in denaturant-mediated cross-linking interactions.

Implications

• These denaturant-mediated interactions are expected to decrease the conformational entropy of the protein, which means they would reduce the number and variety of possible conformations the protein can take.
• An analysis of the reaction rate-temperature data indicated a consistent decrease in conformational entropy of the protein from the native to subdenaturing region.
• Thermodynamic analysis provided insight that the thermodynamic stability of the protein decreases with an increase in denaturant concentration or hydrophobicity of urea derivatives.
• The research also highlights the essential role of water activity in the stabilization of ferrocytochrome c and that the destabilization of ferrocytochrome c by denaturant occurs primarily due to disturbance of hydrophobic interactions and hydrogen bonding.