Denaturation transition

Figure 15.3 (a) Heat absorption in solutions of native RNase A (trace 1) and RNase A kept in 10% buffered formalin for 2 days (trace 2) and 6 days (trace 3) at pH 7.4 and 23°C. All samples were dialyzed against 75 mM potassium phosphate buffer (pH 7.4) prior to DSC. (b) Dependence of Td of the dialyzed RNase A samples on time of incubation in 10% buffered formalin at pH 7.4 and 23°C. (c) Heat absorption of solutions of formalin-treated RNase A fractions isolated by size-exclusion gel chromatography monomer (trace 1), dimmer (trace 2), and a mixture of oligomers with >5 cross-linked proteins (trace 3). Protein concentrations were 0.5 mg/mL. The thermal denaturation transition temperature (Td) is defined as the temperature of the maximum in the excess heat absorption trace associated with the protein s endothermic denaturation transition. See Rait et al.10 for details. 

For the AG of denaturation transition, the temperature (T) data were fitted to the Gibbs-Helmholtz equation ... 

Figure 12 illustrates how SEC may be used to monitor the oligomerization state of a monomeric two-stranded a-helical coiled coil (cf. Figure Id) in the presence of increasing concentrations of Gdn HC1 prior to and through the denaturation transition. 

The Calorimetrically Obtained van t Hoff Enthalpy In a manner analogous to that used to obtain the van t Hoff enthalpy from the fractional change in the optical absorbance, one can use the temperature dependence of the fractional enthalpy as a function of temperature to determine an effective enthalpy. We will adopt the notation to represent the total enthalpy associated with the denaturation transition. It can be obtained from an integration of the excess heat capacity, corrected for the baselines, as discussed before ... 

In all globular proteins studied, a significant increase in the heat capacity of the denatured protein relative to the native state has been observed in the vicinity of the denaturation transition. (This quantity is represented in... 

Fig. 3 Thermal denaturation transition of a DNA helix, (a) UV absorbance increases with temperature, following the unstacking of bases, following a sigmoidal shape. AD and Au are lower and upper baselines, also slightly dependent on temperature, (b) Fraction of single strands 6 extracted from data in (a), which defines the melting temperature corresponding to 9 = 0.5. Adapted with permission from [7]...

Fig. 8. Experimental ( — ) and theoretical heat capacity functions for the thermal folding/unfolding transition of phosphoglycerate kinase at pH 6.5 in the presence of 0.7 M GuHCl. The heat denaturation transition is characterized by a single peak, whereas the cold denaturation displays two peaks corresponding to the independent unfolding of the N and C domains. The experimental curve has been published before (Griko et al., 1989). As discussed in the text, the theoretical curve does not represent the best fit to the experimental data, but rather the calculated curve using structural information in conjunction with thermodynamic information for elementary interactions. [Reprinted from Freire et 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. 

Some 13 years after Carr s original observations, Kretsinger (1976), in his review of calcium-binding proteins, assumed that lysozyme can attach Ca(ll), as well as other cations. It was not until 1981 that binding of Ca(Il) to lysozyme was further studied. Imoto et al. (1981) determined the stability (association) constant (40 M" ) and found that lysozyme is inhibited in the presence of Ca(II), showing only 26% of the activity of the free enzyme toward hexa-AT-acetylglucosamine. Because of this inhibition, they predicted that Ca(II) binds near the catalytic carboxyls. Furthermore, Ca(II) shifts the native-denatured transition in lysozyme toward the native state, and thus has some preservative effect on the protein. 

Table I The melting temperatures (Tm) from the CD thermal denaturation studies are estimated from the temperature at which the slope of the first derivative of the uncorrected data was a minimum ty2 is the temperature of half-completion of the DSC thermal denaturation transition. A//cal is the calorimetric enthalpy. The DSC AAG°u = AG°u (mutant) - AG°u (wild-type), at the value of ti/2 for the wild-type protein (74.6°C).

Table II Summary of the B-sheet propensity data Tm is the midpoint of the thermal denaturation transition. Also shown are the Pb values for the probability of occurrence of each amino acid in B-sheet in proteins of known structure (12). A AAG value is reported for each mutant that is calculated relative to AG Ala GBi1X=AGB1X AGbia- This treatment assumes that within the transition region, A// is independent of temperature. Accordingly, AAG values are reported at a temperature that is within the transition region for all the mutants (60 C). For the standard, BIA, AG = -0.24 kcal/mol at 60 C. Experiments were performed in triplicate, and the results were averaged. The maximum error in the Tm is 0.4 C and in AG to be less than 5%.

Structural. Experimentally, one of the most noticeable features caused by the presence of organic cosolvents on protein structure is the decrease in the temperature at which denaturation occurs. Interestingly, for most of the enzymes thus far studied, at the 60 to 80% cosolvent concentration required in the cryosolvents, the midpoint of the thermal denaturation transition is usually in the —10° to + 10°C range in the pH region of catalytic activity. This means that in such solutions the enzymes are usually denatured at room temperature, but are in their... 

Table II. Effect of Cosolvents on the Midpoint (Tm) of the Reversible Native Denatured Transition of Ribonuclease A at pH4 2.8°...

The temperature of the thermal denaturation transition increases with decreasing hydration below 0.7 h. 4) The partial specific volumes of several proteins are the same in dilute solution and in the solid at hydration levels above 0.2 h. 5) The circular dlchrolsm spectrum of lysozyme in a film is closely similar to that in solution (30). 

Higher temperatures favor chemical modifications (irreversible) of the protein it is thus wiser to keep the final temperature as low as is compatible with the study of the denaturation transition, except of course if one were specifically interested in phenomena occurring at high temperature. 

The gross conformation of acetylated lysozyme is similar to that of the native enzyme, as has been shown by measurement of enzymic activity and fluorescence properties.However, protease digestion, t-butyl hypochlorite modification, and thermal denaturation experiments on native, acetylated, and guanidinated lysozymes have demonstrated that acetylation causes a small but significant shift of the (native denatured) transition to the right. It was concluded that the charge balance in the protein plays an important part in maintaining conformation. 

Discovery of latent heat and cooperative character of protein denaturation transition added much more food for thought for physicists working on protein folding. 

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