Unfolding temperature

FlGURE 7.7 Combined effects of two variables on conformational stability of globular proteins, (a) Denaturation (unfolding) temperature as a function of pH for papain (P), lysozyme (L), cytochrome C (C), parvalbumin (A), and myoglobin (M). (b) Effect of concentration of guanidinium chloride concentration and temperature on conformation of lysozyme at pH 1.7. (c) Effect of pressure (1 kbar= 10s Pa) and temperature on conformation of chymotrypsinogen. (d) Effect of pressure and pH on conformation of myoglobin (20°C). 

FIGURE 7.8 Effect of concentration of (a) various salts (Gu — guanidinium) and (b) various organic solutes on the denaturation (unfolding) temperature of ribonuclease A. In (b) water activity aw is used as the independent variable, rather than molar concentration (1 — aw) is about proportional to the mole fraction of solute. (After various sources, mainly von Hippel and Wong, J. Biol. Chem. 240 (1965) 3909.)... 

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 ... 

Consider that at low temperatures, a lubricant is a poor solvent for polymer chains. When the temperature increases, interactions between polymer chains decrease the space occupied by the polymer ball takes on greater volume and consequently, the viscosity decrease due to the lubricant temperature increase is compensated by the unfolding of the polymer chain and the result is a reduction of the difference between the viscosities at low and high temperature, and therefore an Increase in viscosity index. 

To facilitate conformational transitions in the before-mentioned adenylate kinase, Elamrani and co-workers scaled all atomic masses by a large factor thus allowing the use of a high effective simulation temperature of 2000K ([Elamrani et al. 1996]). To prevent protein unfolding, elements of secondary structure had to be constrained. 

The biologiccJ function of a protein or peptide is often intimately dependent upon the conformation(s) that the molecule can adopt. In contrast to most synthetic polymers where the individual molecules can adopt very different conformations, a protein usually exists in a single native state. These native states are found rmder conditions typically found in Uving cells (aqueous solvents near neutred pH at 20-40°C). Proteins can be unfolded (or denatured) using high-temperature, acidic or basic pH or certain non-aqueous solvents. However, this unfolding is often reversible cind so proteins can be folded back to their native structure in the laboratory. 

Folded proteins can be caused to spontaneously unfold upon being exposed to chaotropic agents, such as urea or guanidine hydrochloride (Gdn), or to elevated temperature (thermal denaturation). As solution conditions are changed by addition of denaturant, the mole fraction of denatured protein increases from a minimum of zero to a maximum of 1.0 in a characteristic unfolding isotherm (Fig. 7a). From a plot such as Figure 7a one can determine the concentration of denaturant, or the temperature in the case of thermal denaturation, required to achieve half maximal unfolding, ie, where... 

It should be noted that in almost all cases only one fold exists for any given sequence. The uniqueness of the native state arises from the fact that the interactions that stabilize the native strucmre significantly destabilize alternate folds of the same amino acid sequence. That is, evolution has selected sequences with a deep energy minimum for the native state, thus eliminating misfolded or partly unfolded structures at physiological temperatures. 

The positive sign of AG° means that the unfolding process is unfavorable that is, the stable form of the protein at 54.5°C is the folded form. On the other hand, the relatively small magnitude of AG° means that the folded form is only slightly favored. Figure 3.4 shows the dependence of AG° on temperature for... 

Eigure 3.5 presents the dependence of A.S ° on temperature for chymotryp-sinogen denaturation at pH 3. A positive A.S ° indicates that the protein solution has become more disordered as the protein unfolds. Comparison of the value of 1.62 kj/mol K with the values of A.S ° in Table 3.1 shows that the present value (for chymotrypsinogen at 54.5°C) is quite large. The physical significance of the thermodynamic parameters for the unfolding of chymotrypsinogen becomes clear in the next section. 

Section 6.1 considered the noncovalent binding energies that stabilize a protein strnctnre. However, the folding of a protein depends ultimately on the difference in Gibbs free energy (AG) between the folded (F) and unfolded (U) states at some temperature T ... 

Because the tertiary structure of a globular protein is delicately held together by weak intramolecular attractions, a modest change in temperature or pH is often enough to disrupt that structure and cause the protein to become denatured. Denaturation occurs under such mild conditions that the primary structure remains intact but the tertiary structure unfolds from a specific globular shape to a randomly looped chain (Figure 26.7). 

Fig. 32. Double-jump experiments of unfolding and refolding. The peptide [(Ala-Gly-Pro)s]3 in 50 ml phosphate buffer (pH 7.5) was incubated at 9.2 °C and quickly unfolded by a first temperature jump from 9.2 to 30 °C. This process took 25 s, the time needed to reach the final temperature. In a first experiment (curve A), the second jump back from 30 to 9.2 °C followed immediately after complete unfolding of the peptide, i.e. 25 s after the first jump. In a second and a third experiment (curve B, C), the time lapse between the first and the second jump was 75 and 125 s, respectively...

The immobilization procedure may alter the behavior of the enzyme (compared to its behavior in homogeneous solution). For example, the apparent parameters of an enzyme-catalyzed reaction (optimum temperature or pH, maximum velocity, etc.) may all be changed when an enzyme is immobilized. Improved stability may also accrue from the minimization of enzyme unfolding associated with the immobilization step. Overall, careful engineering of the enzyme microenvironment (on the surface) can be used to greatly enhance the sensor performance. More information on enzyme immobilization schemes can be found in several reviews (7,8). 

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