Cosolutes, Crowding, and Protein Folding Kinetics

Area of Science:

• Biochemistry
• Biophysics
• Chemical Physics

Background:

• Protein stability is traditionally attributed to steric repulsions from macromolecular cosolutes.
• Recent evidence suggests chemical interactions significantly influence or dominate these crowding effects.
• Understanding these interactions is crucial for comprehending protein folding in cellular environments.

Purpose of the Study:

• To investigate the impact of small and large cosolutes on the folding and unfolding kinetics of a model protein (drk SH3 domain).
• To differentiate between steric and chemical contributions to protein stability under crowding conditions.
• To evaluate the suitability of synthetic polymers as models for physiological crowding.

Main Methods:

• Utilized 19F Nuclear Magnetic Resonance (NMR) spectroscopy to monitor protein folding/unfolding kinetics.
• Employed various small (trimethylamine N-oxide, sucrose, urea) and large (Ficoll, lysozyme) cosolutes.
• Analyzed temperature dependence to determine changes in activation enthalpy and entropy.

Main Results:

• Enthalpic effects on protein folding kinetics are more complex than equilibrium measurements indicate.
• Even seemingly inert polymers like Ficoll exhibit enthalpic effects due to their macromolecular nature.
• Activation entropies reveal significant contributions from solvent and cosolute, beyond protein configurational entropy.

Conclusions:

• Steric repulsions alone are insufficient to explain macromolecular cosolute effects on protein stability and kinetics.
• Chemical interactions play a dominant role in modulating crowding effects on protein folding.
• Synthetic polymers like Ficoll are inadequate models for studying protein folding kinetics in cellular environments, unlike globular proteins such as lysozyme.