Resolving the enthalpy of protein stabilization by macromolecular crowding

Abstract

Proteins in the cellular milieu reside in environments crowded by macromolecules and other solutes. Although crowding can significantly impact the protein folded state stability, most experiments are conducted in dilute buffered solutions. To resolve the effect of crowding on protein stability, we use 19 F nuclear magnetic resonance spectroscopy to follow the reversible, two-state unfolding thermodynamics of the N-terminal Src homology 3 domain of the Drosophila signal transduction protein drk in the presence of polyethylene glycols (PEGs) of various molecular weights and concentrations. Contrary to most current theories of crowding that emphasize steric protein-crowder interactions as the main driving force for entropically favored stabilization, our experiments show that PEG stabilization is accompanied by significant heat release, and entropy disfavors folding. Using our newly developed model, we find that stabilization by ethylene glycol and small PEGs is driven by favorable binding to the folded state. In contrast, for larger PEGs, chemical or soft PEG-protein interactions do not play a significant role. Instead, folding is favored by excluded volume PEG-protein interactions and an exothermic nonideal mixing contribution from release of confined PEG and water upon folding. Our results indicate that crowding acts through molecular interactions subtler than previously assumed and that interactions between solution components with both the folded and unfolded states must be carefully considered.

Molecular Crowders Can Induce Collapse in Hydrophilic Polymers via Soft Attractive Interactions

“…For the large-sized PEG molecules, soft interactions did not play a significant role. 30 This is similar to what we observe for the hydrophilic polymers where we find that the preferential binding coefficients of the C state are higher than those of the E state. In another study, the RNA structure was found to be stabilized in crowded solutions of PEG via enthalpic stabilization.…”

“…It was shown that ethylene glycol and small oligomers of polyethylene glycol (PEG) bind to both folded and unfolded states via attractive interactions; however, stronger attraction to the folded state is observed that stabilizes the folded state. For the large-sized PEG molecules, soft interactions did not play a significant role . This is similar to what we observe for the hydrophilic polymers where we find that the preferential binding coefficients of the C state are higher than those of the E state.…”

“…The role of soft interactions or attractive energy of the crowders due to the chemical nature of the crowder and the test macromolecule remains unexplored while examining such systems. A recent view has emerged where experimental − as well as simulation ,− studies have pointed out that the attractive interactions between crowders and the test macromolecule can play a crucial role in determining protein folding equilibria and protein–protein interactions. Enthalpic, rather than entropic, stabilization of the folded states of proteins has been observed in the macromolecular crowded environment. ,, A recent simulation study found that moderately attractive crowder–protein interactions destabilized the protein association, whereas for a critical attraction strength the destabilization effect was canceled by the volume exclusion effects.…”

“…Enthalpic, rather than entropic, stabilization of the folded states of proteins has been observed in the macromolecular crowded environment. 30,32,33 A recent simulation study found that moderately attractive crowder−protein interactions destabilized the protein association, whereas for a critical attraction strength the destabilization effect was canceled by the volume exclusion effects. A higher attraction strength resulted in nonspecific complex formation.…”

Excluded Volume and Weak Interactions in Crowded Solutions Modulate Conformations and RNA Binding of an Intrinsically Disordered Tail

“…Indeed, weak interactions complicate the interpretation of ensemble experiments, where different protein (or nucleic acid) conformations cannot be resolved. However, single-molecule measurements ,,, and carefully designed ensemble experiments , can leverage such interactions to explore the interplay between purely entropic effects and enthalpic contributions. In this respect, the use of cytomimetic solutions based on polymers and other solutes can provide physical insights into the properties of the cellular medium .…”