Protein Cold Denaturation as Seen From the Solvent

Abstract

Unlike most ordered molecular systems, globular proteins exhibit a temperature of maximum stability, implying that the structure can be disrupted by cooling. This cold denaturation phenomenon is usually linked to the temperature-dependent hydrophobic driving force for protein folding. Yet, despite the key role played by protein-water interactions, hydration changes during cold denaturation have not been investigated experimentally. Here, we use water-(17)O spin relaxation to monitor the hydration dynamics of the proteins BPTI, ubiquitin, apomyoglobin, and beta-lactoglobulin in aqueous solution from room temperature down to -35 degrees C. To access this temperature range without ice formation, we contained the protein solution in nonperturbing picoliter emulsion droplets. Among the four proteins, only the destabilized apomyoglobin was observed to cold denature. Ubiquitin was found to be thermodynamically stable at least down to -32 degrees C, whereas beta-lactoglobulin is expected to be unstable below -5 degrees C but remains kinetically trapped in the native state. When destabilized by 4 M urea, beta-lactoglobulin cold denatures at 10 degrees C, as found previously by other methods. As seen from the solvent, the cold-denatured states of apomyoglobin in water and beta-lactoglobulin in 4 M urea are relatively compact and are better described as solvent-penetrated than as unfolded. This finding challenges the popular analogy between cold denaturation and the anomalous low-temperature increase in aqueous solubility of nonpolar molecules. Our results also suggest that the reported cold denaturation at -20 degrees C of ubiquitin encapsulated in reverse micelles is caused by the low water content rather than by the low temperature.

Cold denaturation of monoclonal antibodies

“…The mechanism of such temperature-induced denaturation, although still not fully understood, is largely believed to be controlled by changes in the structure of water. 10,15,16 The phenomenon of cold denaturation is a thermodynamic consequence of the large positive ∆C p of unfolding for proteins, which results in curvature of the ∆G u versus temperature plot. 8 The cold denaturation temperature (T CD ) is dependent on a number of factors, e.g., the concentration of the protein, the presence of co-solutes, and the pH of the solution.…”

High-pressure NMR reveals close similarity between cold and alcohol protein denaturation in ubiquitin

“…3 shows the secondary shifts ΔδC α , ΔδC 0 , and ΔδN for the pressure-assisted cold-denatured state at 258 K, 2,500 bar. The negative secondary shifts for 13 C α and 13 C 0 in the region of residues 1-18 indicate a propensity for an extended β-type structure reminiscent of the native first β-hairpin. This is also supported by the 15 N secondary shifts, which are positive in this region and show a dip for residues 9-11, indicative of a β-turn.…”

“…2, see above). To characterize this effect we have also determined the chemical shifts of a 15 N-labeled nonapeptide as a function of pressure in the range from 1 to 2,500 bar and temperature from 258 to 298 K ( that the 15 N chemical shift of an exposed amide is much more influenced by pressure and temperature than that of a buried amide or the chemical shift of 13 C α and 13 C 0 nuclei. Presumably, the compression of the hydrogen bonds between the exposed amide and water is responsible for this behavior.…”

“…However, only in the case of the cold-shock protein of Bacillus caldolyticus, cold denaturation could be observed, but no structural data were presented (11). For ubiquitin, cold denaturation under normal pressure has not been observed down to temperatures of ∼233 K in small capillaries (12) or in micrometer-scale water-in-oil emulsions (13). In contrast, in a nanoscale reverse micelle system progressive heterogeneous disappearance of native state ubiquitin NMR resonances was already observed at temperatures below 253 K, but no new resonances of a colddenatured state could be detected (14).…”

Simulations of the confinement of ubiquitin in self-assembled reverse micelles

“…Protein cold denaturation is also believed to involve water penetration of the protein core. 57 Based on coordination number calculations, we define penetrating water and solvating water (see Analysis), and the number of these two kinds of waters in the URM and UBK systems are shown in Table II. There are, on average, two more water molecules penetrating in UBK than in URM, while there are twice as many solvating waters in UBK than in URM.…”