Cold Denaturation Unveiled: Molecular Mechanism of the Asymmetric Unfolding of Yeast Frataxin

Sanfelice, Domenico; Morandi, Edoardo; Pastore, Annalisa; Niccolai, Neri; Temussi, Piero Andrea

doi:10.1002/cphc.201500765

Cited by 39 publications

(47 citation statements)

References 30 publications

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“…We arbitrarily chose to set a cut-off at values of the unfolding temperatures (Tm and Tc) that differed, on average, less than 1.5 K from those corresponding to the average (RAD_0.1). This difference is smaller than the variability that we had observed among different preparations and measurements of the same protein (Pastore et al, 2007;Sanfelice et al, 2014;Sanfelice et al, 2015;Alfano et al, 2017;Puglisi et al, 2020). The residues selected according to this criterion are E71, E75, E89, L91, D101, L104, S105, M109, T110, F116, Y119, I130, L132, A133, F142, D143, L152, L158, T159, D160, T163, and K168 (Figure 1c).…”

Section: Residues Consistent With or Outliers From The Global Behaviourcontrasting

confidence: 57%

Anatomy of unfolding: The site-specific fold stability of Yfh1 measured by 2D NMR

Puglisi

Pastore

Temussi

2020

Preprint

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Abstract. 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 a detailed 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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Section: Residues Consistent With or Outliers From The Global Behaviourcontrasting

confidence: 57%

Anatomy of unfolding: The site-specific fold stability of Yfh1 measured by 2D NMR

Puglisi

Pastore

Temussi

2020

Preprint

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“…Although similar parabolic free-energy curves have been reported for other globular proteins (3), direct observation of cold denaturation in experiment is often frustrated by freezing of the solvent because the low-temperature unfolding conditions usually lie below water's freezing point (4,5). Experimental investigations have therefore involved proteins that cold-denature above the freezing point of water (5,12,13), or systems whose thermophysical properties are altered by chemical denaturants (14,15), freezing-point depressants (16), or pressurization (4). Alternatively, cold denaturation has been studied in deeply supercooled samples, stabilized by confining the protein in emulsified water droplets (17,18) or by encapsulation in reverse micelles (19).…”

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confidence: 77%

Computational investigation of cold denaturation in the Trp-cage miniprotein

Kim

Palmer

Debenedetti

2016

Proc. Natl. Acad. Sci. U.S.A.

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The functional native states of globular proteins become unstable at low temperatures, resulting in cold unfolding and impairment of normal biological function. Fundamental understanding of this phenomenon is essential to rationalizing the evolution of freeze-tolerant organisms and developing improved strategies for long-term preservation of biological materials. We present fully atomistic simulations of cold denaturation of an α-helical protein, the widely studied Trp-cage miniprotein. In contrast to the significant destabilization of the folded structure at high temperatures, Trp-cage cold denatures at 210 K into a compact, partially folded state; major elements of the secondary structure, including the α-helix, are conserved, but the salt bridge between aspartic acid and arginine is lost. The stability of Trp-cage's α-helix at low temperatures suggests a possible evolutionary explanation for the prevalence of such structures in antifreeze peptides produced by coldweather species, such as Arctic char. Although the 3 10 -helix is observed at cold conditions, its position is shifted toward Trp-cage's C-terminus. This shift is accompanied by intrusion of water into Trp-cage's interior and the hydration of buried hydrophobic residues. However, our calculations also show that the dominant contribution to the favorable energetics of low-temperature unfolding of Trp-cage comes from the hydration of hydrophilic residues.cold denaturation | Trp-cage miniprotein | protein folding T he functional native states of globular proteins that are stable near physiological conditions become labile when changes in temperature, pressure, and solvent composition alter their environment. The partial or complete unfolding of secondary and tertiary structure associated with this loss of stability can strongly affect protein behavior, leading to significantly impaired biological function (1, 2). Denaturation upon heating is a ubiquitous and well-studied phenomenon in which proteins gain configurational entropy and unfold as a result of increased kinetic energy. By contrast, the mechanisms responsible for pressure-induced unfolding, and for denaturation of globular proteins at low temperatures, remain incompletely understood (3-5).Fundamental understanding of cold denaturation is important due to its ecological implications and relevance to industrial processing of proteins and biological materials. Freeze-tolerant organisms such as the Arctic char, for example, thrive in subfreezing habitats where cold denaturation can occur (6). Biopharmaceuticals are also exposed to cold conditions that can result in denaturation when they are lyophilized into freeze-dried solids to prolong their shelf life (7,8). Natural cryoprotectants such as sugars and polyols stabilize proteins against denaturation in cold-weather species (6), and similar compounds have been used to mitigate the damaging effects of freeze-drying in pharmaceutical formulations (7, 9).Cold denaturation was first reported by Hopkins in 1930 (10). Brandts (11) subsequently observed ...

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“…Frataxin, which is a protein involved in the assembly of iron-sulfur clusters, is also related to Friedreich’s ataxia, a fatal neurodegenerative condition15. We considered yeast frataxin because this protein represents one of the few examples for which both cold and hot denatured states have been observed at neutral pH and without addition of destabilizing agents (at 272 K and 323 K, respectively), and characterized by nuclear magnetic resonance (NMR) chemical shifts and circular dichroism (CD)15161718. As shown by the pioneering work of Privalov, the thermodynamic analysis of cold denaturation, compared to that of the thermal denaturation, offers unique advantages for understanding the molecular determinants of hydrophobicity1920.…”

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confidence: 99%

Towards a structural biology of the hydrophobic effect in protein folding

Camilloni

Bonetti

Morrone

et al. 2016

Sci Rep

117

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The hydrophobic effect is a major driving force in protein folding. A complete understanding of this effect requires the description of the conformational states of water and protein molecules at different temperatures. Towards this goal, we characterise the cold and hot denatured states of a protein by modelling NMR chemical shifts using restrained molecular dynamics simulations. A detailed analysis of the resulting structures reveals that water molecules in the bulk and at the protein interface form on average the same number of hydrogen bonds. Thus, even if proteins are ‘large’ particles (in terms of the hydrophobic effect, i.e. larger than 1 nm), because of the presence of complex surface patterns of polar and non-polar residues their behaviour can be compared to that of ‘small’ particles (i.e. smaller than 1 nm). We thus find that the hot denatured state is more compact and richer in secondary structure than the cold denatured state, since water at lower temperatures can form more hydrogen bonds than at high temperatures. Then, using Φ-value analysis we show that the structural differences between the hot and cold denatured states result in two alternative folding mechanisms. These findings thus illustrate how the analysis of water-protein hydrogen bonds can reveal the molecular origins of protein behaviours associated with the hydrophobic effect.

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Cold Denaturation Unveiled: Molecular Mechanism of the Asymmetric Unfolding of Yeast Frataxin

Cited by 39 publications

References 30 publications

Anatomy of unfolding: The site-specific fold stability of Yfh1 measured by 2D NMR

Anatomy of unfolding: The site-specific fold stability of Yfh1 measured by 2D NMR

Computational investigation of cold denaturation in the Trp-cage miniprotein

Towards a structural biology of the hydrophobic effect in protein folding

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