Cold temperature unfolding

As mentioned earlier, proteins are subject to cold denaturation because they exhibit maximal stability at temperatures greater than 0°C. The basis of this effect is the reduction in the stabilizing influence of hydrophobic interactions as temperature is reduced. Recall that the burial of hydrophobic side-chains in the folded protein is favored by entropy considerations (AS is positive), but that the enthalpy change associated with these burials is unfavorable (AH, too, is positive). Thus, as temperature decreases, there is less energy available to remove water from around hydrophobic groups in contact with the solvent. Furthermore, as temperature is reduced, the term [— TAS] takes on a smaller absolute value. For these reasons, the contribution of the hydrophobic effect to the net free energy of stabilization of a protein is reduced at low temperatures, and cold-induced unfolding of proteins (cold denaturation) may occur. 

From the stability curve for an N D transition shown in Figure 2 it is evident that there may be two points at which the standard Gibbs energy change A G (T) vanishes. This implies the existence of two transition temperatures T1/2, for heat denaturation, and for low temperature unfolding. This latter reciction is generally referred to as cold denaturation . Cold denaturation has been discussed by Brandts [20] on the basis of spectroscopic measurements, but was observed calorimetrically first by Privalov et al. [3],... 

Proteins unfolded by GdmHCl or urea will have a dominant conformation, Pn- At low temperatures we find about one-third of the residues in chemically denatured proteins in the Pn-helix conformation, with two-thirds in the form of the high-temperature ensemble. Since at least one-third of the residues in this ensemble are isolated Pn residues or in Pn helices of two or three residues, the total Pn content will be 50% or greater. The Pn content of cold- and acid-denatured proteins will be substantial, probably >40%, but not as large as in chemically denatured proteins. 

Beyond their practical value, extremophile enzymes present scientists with a fundamental puzzle. Fike all molecular characteristics, their exceptional stability must originate in their chemical structures. However, it is not yet certain what structural features determine these properties. What is known is that in their active folded form, cold-resistant enzymes appear to have relatively fewer structure-stabilizing interactions between different parts of the amino acid chain. As a result, they remain more flexible at a lower temperature than ordinary enzymes but unfold and lose their activity more quickly as the temperature is raised. Conversely, heat-resistant enzymes seem to have a larger number of... 

Intriguingly, the high value of A Cp leads to the phenomenon of cold denatura-tion as the temperature becomes lower, the enthalpy of unfolding decreases,... 

Fig. 9. Calculated overall free energy of stabilization (AGtota ) for yeast phos-phoglycerate kinase at pH 6.5 and 0.7 M GuHCl. This curve displays two zeros, corresponding to the temperatures of cold and heat denaturation. Also shown in the curve are the cooperative Gibbs free energies (AG ) associated with the uncompensated exposure of apolar surfaces on unfolding of each of the domains. For both domains, AG is positive for the heat denaturation and close to zero for the cold denaturation. This behavior results in a cooperative heat denaturation and a non-cooperative cold denaturation. [Reprinted from Freire el al. (1991).]...

Fig. 7. Microcalorimetric recording of the heat effect on cooling and subsequent heating of metmyoglobin solution at pH 3.83. The low temperature peaks correspond to heat release on cold denaturaton and heat absorption on subsequent renaturation of protein. The shift of these peaks in temperature is caused by slow kinetics of unfolding and folding of myoglobin structure at low temperature (for details, see Privalov et al 1986).

Hydrophobic effects are thus of practical interest. If we accept the goal of a simple, physical, molecularly valid explanation, then hydrophobic effects have also proved conceptually subtle. The reason is that hydrophobic phenomena are not tied directly to a simple dominating interaction as is the case for hydrophilic hydration of Na+, as an example. Instead hydrophobic effects are built up more collectively. In concert with this indirectness, hydrophobic effects are viewed as entropic interactions and exhibit counterintuitive temperature dependencies. An example is the cold denaturation of globular proteins. Though it is believed that hydrophobic effects stabilize compact protein structures and proteins denature when heated sufficiently, it now appears common for protein structures to unfold upon appropriate cooling. This entropic character of hydrophobic effects makes them more fascinating and more difficult. 

The unfolding process is a function of temperature. Many proteins have optimal stability in the temperature range 10-30°C. Loss of structure is expected both at low temperatures (cold denaturation) and at elevated temperatures. 

Figure 2. The elliptical temperature-pressure stability phase diagram characteristic for proteins. After Suzuki [2] and Hawley [4], Note the thermodynamic similarities between the cold, c, and pressure, p, unfolding and the contrast with heat, h, unfolding.

In this equation Ap is the compressibility factor difference (P =PV) and Aa the difference of the thermal expansion factor (a =aV) of the denatured and native states of proteins. An important assumption in the derivation of this equation is the temperature and pressure independence of Aa, AP and ACp. The AG=0 curve is an ellipse on the P-T plane and it describes the equilibrium border between the native and denatured state of the protein. This curve is known as the phase or stability diagram. This is visualized in Fig. 2. The diagram illustrates the interconnection between the cold, heat and pressure unfolding of proteins. 

There are several specific points in the elliptical curve The unfolding temperature at ambient pressure, T, the denaturation pressure pu at room temperature, and the cold denaturation temperature, Tc- They are given by the following equations ... 

Some proteins can show either two-state or multistate behavior depending on the conditions of denaturation. For example, heat dena-turation of phosphoglycerate kinase complies with the two-state approximation, but not its cold denaturation [58] this has been quantitatively analyzed as resulting from the existence of two domains, interacting mainly through hydrophobic contacts, that unfold almost independently at low temperature but behave as a single cooperative unit at high temperature, as a consequence of the temperature dependence of hydrophobic interactions [59,63]. 

At pH 2, BLG is monomeric its heat-induced unfolding is a two-state process, whereas its cold denaturation appears more complex [57], At neutral pH BLG dimerizes and the monomer-dimer equilibrium is shifted to the left as temperature increases. It has been re-... 

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