Resolving the enthalpy of protein stabilization by macromolecular crowding

Area of Science:

• Biophysics
• Structural Biology
• Biochemistry

Background:

• Cellular environments are crowded, impacting protein stability.
• Most protein stability studies occur in dilute solutions, not reflecting in vivo conditions.
• Understanding crowding effects is crucial for protein folding and function.

Purpose of the Study:

• To investigate the thermodynamic effects of polyethylene glycols (PEGs) on protein stability.
• To elucidate the molecular mechanisms behind PEG-induced protein stabilization.
• To challenge and refine existing theories of macromolecular crowding.

Main Methods:

• Utilized 19F nuclear magnetic resonance (NMR) spectroscopy.
• Studied the reversible, two-state unfolding thermodynamics of the drk protein's N-terminal Src homology 3 domain.
• Employed varying concentrations and molecular weights of polyethylene glycols (PEGs).

Main Results:

• PEG-induced protein stabilization was accompanied by heat release, with entropy disfavoring folding.
• Smaller PEGs and ethylene glycol stabilized the protein through favorable binding to the folded state.
• Larger PEGs stabilized via excluded volume interactions and exothermic nonideal mixing, not chemical or soft interactions.

Conclusions:

• Macromolecular crowding influences protein stability through subtle molecular interactions.
• Both folded and unfolded protein states interact with crowding agents.
• Current crowding theories need refinement to account for diverse interaction mechanisms and thermodynamic contributions.