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The anatomy of unfolding of Yfh1 is revealed by site-specific fold stability analysis measured by 2D NMR spectroscopy

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Rita Puglisi, Gogulan Karunanithy, D. Flemming Hansen, Annalisa Pastore, Piero Andrea Temussi

Original languageEnglish
Article number127
Journalcommunication chemistry - Nature
Issue number1
Early online date6 Sep 2021
Accepted/In press11 Aug 2021
E-pub ahead of print6 Sep 2021
Published6 Sep 2021

Bibliographical note

Funding Information: This manuscript is meant in celebration of the 80th birthday of Prof. Robert Kaptein. We thank Geoff Kelly and Tom Frenkiel of the MRC Biomedical NMR Centre for helpful discussions and technical support, Neri Niccolai and Franca Fraternali for help with their software SADIC and PopS, respectively. We are also thankful to Rolf Boelens for his detailed comments and to Jochen Balbach and Dmitry Korzhnev for their constructive criticisms. We acknowledge access to the NMR spectrometers at the Randall unit of King’s College London and at the MRC Biomedical NMR Centre in the Francis Crick Institute. The Crick Institute receives its core funding from Cancer Research UK (FC001029), the UK Medical Research Council (FC001029) and the Wellcome Trust (FC001029). The research was supported by UK Dementia Research Institute (RE1 3556) that is funded by the Medical Research Council, Alzheimer’s Society and Alzheimer’s Research UK. Publisher Copyright: © 2021, The Author(s). Copyright: Copyright 2021 Elsevier B.V., All rights reserved.

King's Authors


Most techniques allow detection of protein unfolding either by following the behaviour of single reporters or as an averaged all-or-none process. We recently added 2D NMR spectroscopy to the well-established techniques able to obtain information on the process of unfolding using resonances of residues in the hydrophobic core of a protein. Here, we questioned whether an analysis of the individual stability curves from each resonance could provide additional site-specific information. We used the Yfh1 protein that has the unique feature to undergo both cold and heat denaturation at temperatures above water freezing at low ionic strength. We show that stability curves inconsistent with the average NMR curve from hydrophobic core residues mainly comprise exposed outliers that do nevertheless provide precious information. By monitoring both cold and heat denaturation of individual residues we gain knowledge on the process of cold denaturation and convincingly demonstrate that the two unfolding processes are intrinsically different.

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