To Boil an Egg: Substrate Binding Affects Critical Stability in Thermal Unfolding of Proteins

Area of Science:

• Biophysics
• Structural Biology
• Biochemistry

Background:

• Protein thermal unfolding is vital for drug candidate screening.
• Ligand and substrate interactions significantly influence protein stability, transition temperature, and cooperativity.
• Understanding these thermal behaviors is key to characterizing protein dynamics.

Purpose of the Study:

• To monitor the thermal evolution of protein secondary structure using synchrotron radiation circular dichroism.
• To investigate the impact of substrates on protein thermal behavior.
• To quantify protein transition strength and proximity to a critical point using Landau free energy expansion.

Main Methods:

• Synchrotron radiation circular dichroism (SRCD) to monitor secondary structure changes during thermal unfolding.
• Landau free energy expansion to model protein stability and phase transitions.
• Calculation of the relative separation (τ₊) between transition temperature (T_m) and spinodal (T₊) from the equation of state.

Main Results:

• Lysozyme exhibited the weakest thermal transition (τ₊ = -0.0167).
• Holo-albumin showed a weaker transition (τ₊ = -0.0208) compared to monomeric apo-albumin (τ₊ = -0.0242).
• Apo-albumin displayed a noncooperative melt (τ₊ = -0.00532) at 45 °C, with an amyloidogenic increase in beta content.

Conclusions:

• Protein thermal stability and transition cooperativity vary significantly between different proteins and their states (e.g., apo vs. holo albumin).
• The Landau free energy expansion provides a quantitative measure of transition strength and proximity to critical points.
• Specific structural transitions, like that in apo-albumin, can lead to noncooperative melting and altered secondary structures with potential implications for protein aggregation.