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Understanding the forces governing protein folding

27 October 2010

NIMR scientists have described the first structural characterization of a cold denatured protein. The research is published in the Journal of the American Chemical Society.

When heated above room temperature proteins lose their ordered structure, becoming what is generally called a random coil. This denatured state often leads to the formation of insoluble aggregates, a distinctive feature of severe diseases such as Alzheimer’s and Parkinson’s. These diseases are often called misfolding diseases, because they are related to protein misfolding, the onset of a random coil state even without heating.

It is now widely recognised that proteins also undergo “cold denaturation”, that is a transition from folded to unfolded state at temperatures below room temperature. It is a property of globular proteins believed to be driven by the hydration of polar and non-polar groups as well as the decrease of hydrophobic interactions. To date still little is known, at a detailed structural level, of cold-denatured proteins. The main reason why cold denaturation has not been explored more thoroughly is that this process is difficult to study because, for most proteins, it occurs at temperatures well below the freezing temperature of water.

Yfh1 is a protein identified by Annalisa Pastore’s group in NIMR’s Division of Molecular Structure. It is the yeast ortholog of frataxin, a mitochondrial human protein responsible for the neurodegenerative disease Friedreich’s ataxia. It is interesting in this context as its cold denaturation occurs at temperatures above 0°C and at physiological conditions.

Piero Temussi, working both at NIMR and at the Universita di Napoli Federico II, has described the first structural characterization of Yfh1. Yfh1 is thought to be the first example of a full-length natural protein that can undergo cold denaturation at temperatures accessible to NMR analysis, without the need of addition of destabilizing cosolvents and/or introduction of ad hoc mutations.

By achieving nearly full assignment of the NMR spectrum, they have shown that at -1 ºC, Yfh1 has all the features of an unfolded protein, although retaining some local, residual secondary structure. The effect is not uniform along the sequence and does not merely reflect the secondary structural features of the folded species. The N-terminus seems to be dynamically more flexible, although retaining some nascent helix character. Interestingly, this region is the one containing functionally important hot-spots. The β-sheet region and the C-terminal helix are completely unfolded, although experiencing some conformational exchange, partly due to the presence of several prolines.

While many decades of biophysical studies have led to the identification of the main forces which lead to protein unfolding caused by thermal denaturation, many phenomena that are connected with the action of intra-molecular forces at low temperature remain poorly understood. Our study opens the way to a deeper understanding of the forces governing protein folding, stability and denaturation and hence of some important aspects of misfolding diseases.

Piero Temussi

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Ribbon representation of the tertiary structure of Yfh1 (PDB code 2ga5) at 25 C. The fold contains an N-terminal import signal (residues 1-12) followed by two helices (helix 1 and helix 2), which sandwich a six stranded β-sheet. The side chains of proline residues are indicated.