Cold denaturation temperature

Figure 13.9 Temperature response trace (at optimal Tm solution conditions) of AGu, for pepsinogen at pH 6, with and without 20% ethanol (EtOH). Labels shown are identified as Ted = cold denaturation temperature, Tms = temperature of maximum stability, and Tra...

Ts is the temperature of maximum stability, at which the AG(T) versus T curve passes through its maximum, i.e., AG(TS) > 0. At Tm, the maximum temperature of stability, AG (T) = 0. By necessity, Ts < Tm. There is another special point in the temperature stability (AG(T)) curve, however as the temperature stability curve resembles a inverted parabola, there must be a second intersection point with the line of minimum stability, AG(T) = 0. This intersection point is the minimum of the range of temperature stability and is called cold denaturation temperature, Tc. The cold denaturation temperature is practically never taken into consideration in the discussion of temperature stability of biocatalysts. One reason lies in its frequent inaccessibility often, Tc is below 0 °C and thus cannot be measured in water or any other mostly aqueous medium. 

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 ... 

The cold-denaturation temperature and heat-denature temperature are found by setting Equation (E6.2C) to zero. The values are found to be 218 K and 343 K, respectively. In between these temperatures the native protein is stable. Outside this range, the protein unfolds and becomes denatured. The maximum value for gd ga occurs at 278 K. This value can be found either by setting the derivative of Equation (E6.2G) equal to zero and solving for T, or by simply finding where the function reaches a maximum numerically (solver in Excel can be useful for this approach). 

The early stages of folding of barstar have been measured on the microsecond time scale by temperature jumping its cold-denatured state from 2 to 10°C.65,66 There is the fast formation of a folding intermediate (tm 200 fxs) with the peptidy 1-proline 48 bond trans, followed by the formation with ty2 60 ms of a second intermediate that is highly native-like because it binds to and inhibits barnase. The native-like intermediate then undergoes trans cis peptidyl-proline isomerization on the time scale of minutes to give the final native structure (equation 19.2). 

Figure 5 also illustrates other general features of globular protein stability. The positive overall ACp results in the existence of two temperatures at which AG° is zero. The low temperature point defines the so-called cold denaturation of the protein and the high temperature point defines the heat denaturation. Additionally, the curvature in AG° implies the existence of a temperature of maximal stability. This temperature occurs at the point where AS0 is equal to zero. The... 

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).]...

As seen in Fig. 10, the model accurately predicts the presence, location, and area of the cold and heat denaturation peaks. Under these conditions, the hierarchical partition function predicts a heat denaturation peak centered at 58°C and a cold denaturation peak centered at 4°C. The enthalpy change for the heat denaturation peak is 59 kcal mol-1 and the ACp is equal to 2.45 kcal K-1 mol-1. The experimental values reported by Privalov et al. (1986) are 57.5 and 3°C for the heat and cold denaturation transition temperatures, 53 kcal mol-1 for the enthalpy change, and 2.5 kcal K-1 mol-1 for ACp. Analysis of the theoretical curve indicates that it corresponds to a two-state transition, in agreement with the experimental data. The population of partially folded intermediates is never greater than 10-5 during the heat denaturation transition. 

One expects a significant amount of both the native and denatured protein structure in the vicinity of these two temperatures. The disruption of the native state on heating is usually called heat denaturation, since it proceeds with heat absorption and, consequently, with an increase in the molecular enthalpy and entropy. The disruption of the native structure on cooling, which we can call by analogy cold denaturation, should then proceed with a release of heat and, hence, with a decrease in enthalpy and entropy, because both of these functions have reversed their signs before reaching temperature 7 en. ... 

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. 

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. 

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