The Cold-Unfolded State Is Expanded but Contains Long-and Medium-Range Contacts and Is Poorly Described by Homopolymer Models

Natalie E. Stenzoski, Junjie Zou, Andrea Piserchio, Ranajeet Ghose, Alex S. Holehouse, Daniel P. Raleigh

Research output: Contribution to journalArticlepeer-review

4 Scopus citations


Cold unfolding of proteins is predicted by the Gibbs-Helmholtz equation and is thought to be driven by a strongly temperature-dependent interaction of protein nonpolar groups with water. Studies of the cold-unfolded state provide insight into protein energetics, partially structured states, and folding cooperativity and are of practical interest in biotechnology. However, structural characterization of the cold-unfolded state is much less extensive than studies of thermally or chemically denatured unfolded states, in large part because the midpoint of the cold unfolding transition is usually below freezing. We exploit a rationally designed point mutation (I98A) in the hydrophobic core of the C-terminal domain of the ribosomal protein L9 that allows the cold denatured state ensemble to be observed above 0 °C at near neutral pH and ambient pressure in the absence of added denaturants. A combined approach consisting of paramagnetic relaxation enhancement measurements, analysis of small-angle X-ray scattering data, all-atom simulations, and polymer theory provides a detailed description of the cold-unfolded state. Despite a globally expanded ensemble, as determined by small-angle X-ray scattering, sequence-specific medium-and long-range interactions in the cold-unfolded state give rise to deviations from homopolymer-like behavior. Our results reveal that the cold-denatured state is heterogeneous with local and long-range intramolecular interactions that may prime the folded state and also demonstrate that significant long-range interactions are compatible with expanded unfolded ensembles. The work also highlights the limitations of homopolymer-based descriptions of unfolded states of proteins.

Original languageEnglish
Pages (from-to)3290-3299
Number of pages10
Issue number36
StatePublished - Sep 15 2020


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