Unbiased Cold Denaturation:  Low- and High-Temperature Unfolding of Yeast Frataxin under Physiological Conditions

Abstract

Protein unfolding can be induced both by heating and by cooling from ambient temperatures. 1 Accurate analysis of heat and cold denaturation processes has the potential to unveil hitherto obscure aspects of protein stability and dynamics. 2 For instance, while heat denaturation is generally highly cooperative, cold denaturation has been suggested to occur in a noncooperative fashion. 3,4 This view has been recently supported by an NMR study of ubiquitin in reverse micelles at very low temperatures, 5 but this is still controversial since Van Horn et al., 6 on the basis of similar NMR data, and Kitahara et al., 7 by an NMR study at 2 kbar, found a simple two-state behavior for the low-temperature unfolding of ubiquitin.To reach a consensus on this debate and other general issues, it is necessary to investigate cold denaturation further. However, since the cold denaturation of most proteins occurs well below the freezing point of water, full access to the cold denatured state is normally limited for the obvious reason that water freezes at 0 °C. The most common approach to circumvent this difficulty has been to try to raise the temperature of cold denaturation using destabilizing agents such as extreme pH values, chemical denaturants, cryosolvents, or very high pressure. 7-10 Alternatively, some laboratories used proteins destabilized by a combination of point mutations and denaturing agents. 9 The main drawback of these approaches is that it is not generally easy to extrapolate results to physiological conditions. On the other hand, there are methods aimed at keeping water in a supercooled condition, but these studies have also invariably used destabilized proteins. 11,12Following a different approach, we looked for a protein whose cold denaturation could be studied without the need for destabilization in a normal buffer at physiological pH within a temperature range accessible to several techniques. Here we describe the cold and heat denaturation of yeast frataxin (Yfh1) measured both by NMR and CD spectroscopies. In a systematic study of the factors that influence the thermal stability of the frataxin fold, we had previously shown that although they share the same fold, three orthologues from E. coli (CyaY), S. cerevisiae (Yfh1) and H. sapiens (hfra), are characterized, under the same conditions, by a remarkable variation of melting temperatures. 13 Yfh1, the one with lowest heat denaturation temperature, seemed a promising candidate for cold denaturation above 0 °C. Yfh1 and 15 Nlabeled Yfh1 were expressed in E. coli as described by He et al. 14 Since variations of ionic NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript strength lead to significant increases in the melting temperature, we restricted the present investigation to solutions of Yfh1 in salt-free buffers.We recorded 1D and 2D NMR spectra of Yfh1 either in TRIS at pH 7.0 or in HEPES at pH 7.0 in the temperature range −5 to 45 °C. Typically, 0.3-0.5 mM unlabeled or 15 N uniformly labeled protein samples were used. Thanks to t...

The Role of Hydration in Protein Stability: Comparison of the Cold and Heat Unfolded States of Yfh1

“…The thermal unfolding process recorded on different protein batches is also highly reproducible and can reliably be interpreted as a two-state transition with transition points around 7 and 30°C and the point of maximal stability around 19°C. 4 We estimated elsewhere that, at the point of maximal stability, ca 20 ÷ 30% of the protein is unfolded in rough agreement with the presence of 15 ÷ 25 (depending on the structure used as reference 9,10 ) unstructured residues that belong to the N-terminal tail that contains the processed part of the mitochondrial import signal and the presence of unstructured loops. 11 We first tried to select equivalent states devoid of residual folded populations at low and high temperatures by back calculations based on the published thermodynamic parameters.…”

“…11 We first tried to select equivalent states devoid of residual folded populations at low and high temperatures by back calculations based on the published thermodynamic parameters. 4,7 CD suggests − 5°C and 45°C as equivalent points along the unfolding curve, being roughly equidistant from the point of maximal stability and from the midpoints of unfolding. At these temperatures, the residual population of folded Yfh1 is estimated to be ca 7%, which should be hardly detectable by most of the standard commercial NMR instruments.…”

“…Best fit of the data at this temperature is obtained by assuming the first 30 residues unfolded and flexible, in agreement with the estimate from the ΔG curve. 4 The ensemble distributions selected by the EOM analysis at 0 and 50°C are shifted to larger R g values but with important differences. The R g distribution at 50°C is closer to the one of a completely random pool, whereas that at 0°C has a significant fraction of compact and folded conformations (corresponding to the peak around 2 nm).…”

“…The samples were all in a 20 mM Hepes buffer at pH 7.0 and 2 mM DTT and carefully desalted by dialysis since even small differences in desalting and/or buffer composition are known to influence the Yfh1 stability. 4,30 Fused silica cuvettes of 1 mm path length (Hellma) were used. CD spectra were typically recorded with 0.2 nm resolution and base-line corrected by subtraction of the appropriate buffer spectrum.…”

“…A noninvasive and facile way to observe cold denaturation was found with the identification of a protein, Yfh1, whose cold denaturation occurs at neutral pH and without addition of destabilizing agents at temperature sufficiently high to be easily attainable experimentally. 4 Yfh1 is the yeast ortholog of frataxin, a mitochondrial human protein involved in Friedreich's ataxia, a severe neurodegenerative disease. 5 The frataxin family is highly conserved in both sequence and structure from bacteria to humans and is essential for life.…”