Temperature and pressure effects on C112S azurin: volume, expansivity, and flexibility changes

Area of Science:

• Biochemistry
• Protein dynamics
• Biophysical chemistry

Background:

• Proteins undergo conformational changes crucial for their function.
• Understanding protein unfolding mechanisms provides insights into protein stability and disease.
• Azurin is a small blue copper protein involved in bacterial electron transfer.

Purpose of the Study:

• To investigate the pressure-induced unfolding of the C112S mutant azurin from Pseudomonas aeruginosa.
• To characterize the thermodynamic and kinetic parameters governing protein unfolding under pressure.
• To elucidate the distinct pathways and transition states involved in protein folding and unfolding.

Main Methods:

• Monitoring unfolding via fluorescence emission spectral shifts and phosphorescence intensity.
• Evaluating free energy changes (ΔG) as a function of pressure and temperature.
• Utilizing pressure-jump experiments to determine kinetic activation parameters (ΔV*, ΔH*, ΔS*).

Main Results:

• The dependence of ΔG on temperature exhibited a concave profile across all studied pressures.
• A positive heat capacity change (ΔCp) of approximately 4.3 kJ mol⁻¹ deg⁻¹ was observed.
• Pressure-induced unfolding proceeded in two distinct steps, indicating initial flexibility increase followed by significant structural changes.

Conclusions:

• The folding and unfolding pathways of C112S azurin likely involve different transition states.
• Pressure-induced unfolding is a multi-step process initiated by increased protein flexibility, possibly due to water penetration.
• Subsequent higher pressures lead to major alterations in the tryptophan environment, signifying complete unfolding.