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A supercooled protein folds unexpectedly

21 November 2019

At low enough temperatures, a small simulated peptide regains its ordered structure.

Human bodies have a narrow range of temperatures at which they function properly. Unsurprisingly, proteins behave similarly: At ambient temperatures they fold as needed for their biological purposes, but if they get too hot or too cold, their structures begin to come apart. The details of what happens to proteins away from their conformational sweet spot, and how or why it happens, could provide insight into how they manage to find their functional forms at physiological conditions.

Graph of amount of folding at different temperatures

Daniel Kozuch, Frank Stillinger, and Pablo Debenedetti at Princeton University found an unexpected result while using molecular dynamics simulations to probe the structure of a small protein at low temperatures. Earlier work also led by Debenedetti looked at the denaturation of Trp-cage, a 20-amino-acid model protein, in liquid water supercooled to 210 K. Those simulations showed a peak in the fraction of folded proteins at room temperature, followed by a steep drop-off as the temperature decreased, as shown in the first figure. But when the researchers lowered the temperature even further, they found a surprising result: At 194 K, the proteins refolded.

The supercooled folded structure was remarkably similar, though not identical, to that at ambient temperature. Denaturation of Trp-cage by cooling is characterized by the unfolding of a small helix (see second figure), and that feature returned at sufficiently low temperatures. Additionally, the helix formation was accompanied by the development of a more compact hydrophobic core.

Comparison of structure at ambient temperature and cold denatured

To seek an explanation for the structural difference, the researchers turned to the surrounding water molecules. The temperature at which the protein refolded was the same temperature at which the water finished transitioning from a high- to a low-density state. The water remained liquid, so the solvent had no long-range order, but on short length scales the molecules became tetrahedrally coordinated (see the article by Pablo Debenedetti and Gene Stanley, Physics Today, June 2003, page 40). Because of such locally ordered configurations, it was no longer energetically favorable for the water molecules to solvate the protein’s core; hence the collapse.

Although proteins don’t run the risk of becoming supercooled in vivo, their behavior at below-freezing temperatures may have implications for cryo-microscopy and cryo-preservation techniques. (D. J. Kozuch, F. H. Stillinger, P. G. Debenedetti, J. Chem. Phys. 151, 185101, 2019.)

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