Thermal unfolding

The accepted relationship for the temperature induced unfolding of a protein is 

The heat capacity change for unfolding of proteins has been foimd to be positive and to be related to the increase in solvent exposure of apolar side chains (1-3). In other words, a positive ACp is a result of the hydrophobic effect and a consequence is that the AG° (T) for unfolding of a protein will have a non-linear dependence on temperature, reaching a maximum at some temperature and showing both hi temperature and low temperature induced unfolding. 

A relationship for acid induced unfolding is given by equation 10. This is the simplest relationship of this type (16), and assumes that there are n equivalent acid dissociating groups on a protein that all have the same pK y in the unfolded state, and that they are all perturbed to have a pI. N in the N state that is at least 2 pH units lower than pKa u. If the pKa shift is not at least 2 pH imits, equation 11 should be used. If there are more than one type of perturbed amino acid residue and/or if the residues are perturbed by less than 2 pH units, then the equation must be expanded further to include several more fitting parameters, which usually makes fitting data untractable. 

Equation 10 includes as fitting parameters the free energy of rmfolding at neutral pH, AGS,un. the number of such assumed residues, n, and their pKa u in the 

The relationship for pressure, P, induced unfolding of proteins is given by equation 12, where AG°,un is again the value of the free energy change at 1 atmosphere pressure and AVun = Vu - Vn is the difference in volume of the unfolded and native states. 

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Fig. 14. Factor analysis loadings (first and second spectral components) for thermal unfolding of RNase A as monitored with amide F FTIR and far-UV ECD. In each case a pretransition is evident in the curves before the main transition at 55°C. This full band shape analysis can sense smaller variations and can be partitioned to give added insight. Since the main ECD change could be shown to be loss of intensity, the major structural change was unfolding of a helix. The frequency dispersion of the FTIR change showed that some /3-sheet loss accompanied this pretransitional helix unfolding, but that most sheet loss was in the main transition.

Privalov et al. (1989) also reported the temperature dependence of the ellipticity at 222 nm for the proteins studied at various pH values (Fig. 28). At the highest temperature studied (80°C), the 222 nm ellipticity value for the thermally unfolded, acid-unfolded, and Gdm-HCl-unfolded proteins appear to be converging, but show a range of 2000 deg cm2/dmol out of a total of 5000 deg cm2/dmol. (ApoMb is an exception in that, as noted before, the thermally denatured protein is apparently an associated /1-sheet. However, the acid- and Gdm HC1-unfolded forms of apoMb have similar [0] 222 values at 80°C.)... 

What is the predominant secondary structure in unfolded proteins In thermally unfolded proteins, there appears to be no predominant secondary structure. Individual residue conformations are distributed over the Pn, a, and f regions that constitute energy minima for single residues in aqueous solution (Section II,B). Still, since there is increasing evidence that the Pn conformation is at the global minimum, Pn conformers must be the most abundant in this ensemble. Short Pn- and o -helices and /1-strands will be present, but will rarely exceed two or three residues in length. 

Fig. 35. Far-UV (a) and near-UV (b) CD spectra of bovine Q -lactalbumin in various states. (1 and 2) The native state of the holo and apo forms, respectively (3) the A state (a) thermally unfolded state at 41° (4) and 78°C (5) (6) GdmCl-unfolded state, (b) Thermally unfolded state at 62.5°C (4) GdmCl-unfolded state (5). The open circles (holo) and squares (apo) are values derived by extrapolating refolding curves to zero time. From Kuwajima et al. (1985). Biochemistry 24, 874-881, with permission. 1985, American Chemical Society.

Studies of thermally denatured proteins remain technically challenging owing to the propensity of thermally unfolded proteins to aggregate. Despite this potential difficulty, small-angle scattering techniques have been employed in the characterization of a number of thermally unfolded states. 

An excluded-volume random-coil conformation will be achieved when the solvent quality exceeds the theta point, the temperature or denatu-rant concentration at which the solvent-monomer interactions exactly balance the monomer—monomer interactions that cause the polymer to collapse into a globule under more benign solvent conditions. A number of lines of small-angle scattering—based evidence are consistent with the suggestion that typical chemical or thermal denaturation conditions are good solvents (i.e., are beyond the theta point) and thus that chemically or thermally unfolded proteins adopt a near random-coil conformation. 

Of the 20 reduced or disulfide bond-free proteins for which the Rg of the chemically or thermally unfolded state has ben reported, 17 fall on a single curve (Fig. 4). Fitting the Rg of these 17 proteins produces a strong, statistically significant correlation (r 2 = 0.96) and an exponent, v = 0.61 d= 0.03, startlingly close to the expected value for an excluded-volume random coil chain. 

Navon A, Ittah V, Laity JH, et al. Local and long-range interactions in the thermal unfolding transition of bovine pancreatic ribonuclease A. Biochemistry 2001 40 93-104. 

Stelea SD, Pancoska P, Benight AS, et al. Thermal unfolding of ribonuclease A in phosphate at neutral pH deviations from the two-state model. Protein Sci. 2001 10 970-978. 

Chen, B.-L., and King, J. (1991). Thermal unfolding pathway for the thermostable P22 tailspike endorhamnosidase. Biochemistry 30, 6260-6269. 

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.

An explanation for these observances in the cases of ethanol and PEG may arise from hydrophobic interactions (i.e., methylene groups, methyl) with the unfolded state of the protein at elevated temperatures. This idea is supported by studies of the interaction of several alkylureas (methyl-, ACVdimethyl-, ethyl-, and butylureas) with the thermal unfolding of ribonuclease A, where it was shown... 

Welfle, K., R. Misselwitz, G. Hausdorf, W. Hohne, and H. Welfle. 1999. Conformation, pH-induced conformational changes, and thermal unfolding of anti-p24 (HIV-1) monoclonal antibody CB4-1 and its Fab and Fc fragments. Biochim Biophys Acta 1431 120-131. 

Zaiss, K. and R. Jaenicke. 1999. Thermodynamic study of phosphoglycerate kinase from Thermotoga maritima and its isolated domains reversible thermal unfolding monitored by differential scanning calorimetry and circular dichroism spectroscopy. Biochemistry 38 4633 -639. 

Liggins, J.R., F. Sherman, A.J. Mathews, and B.T. Nall. 1994. Differential scanning calorimetric study of the thermal unfolding transitions of yeast iso-1 and iso-2 cytochromes c and three composite isozymes. Biochemistry 33 9209-9219. 

KS590 displayed cooperative thermal unfolding, TEK42 remained stable in the range of 10-90°C. 

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