Thermal 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. 

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).]...

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... 

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). 

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. 

In contrast to the slow chemical exchange observed for the thermal denaturation (transition II), the spectral changes for the lower temperature transition I are consistent with more rapid chemical exchange between two (or possibly more) different tertiary conformations. 

Structured proteins have also been investigated by thermal analysis [40,41], denaturing resulting in an endotherm which is readily detected by differential scanning calorimetry (DSC). DSC of recombinant resilin in the swollen state showed no transitions over a wide temperature range (25°C-140°C), further evidence of the absence of any strucmre. This is in contrast to the strucmred proteins wool and bovine serum albumin, which show denamration endotherms at 145°C and 62°C, respectively (Figure 9.6). 

The van t Hoff plots for thermal denaturation of proteins are linear in the transition region, thus allowing the enthalpy change (AHm) of unfolding at the transition temperature (Tm) to be estimated. Because of the change in free energy in (AG) = 0 at Tm (reversible process), the entropy of unfolding (ASm) at the transition midpoint can be calculated from ... 

Figure 13.1 Microcalorimetry scans displaying Tm values for interleukin-1 receptor (IL-1R type I). The inlay displays the unfolding of IL-1R (I) showing the ACp measured as the baseline difference between the native (N) and denatured (D) states for two independent scans. Thermal unfolding of IL-1R (I) is composed of three cooperative unfolding transitions, labeled 1, 2, and 3.

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