Thermal unfolding determination

Figure 17.3 Urea denaturation of bamase at 25°C. The open circles were measured directly at 25°C from the ratios [D]/[N], which were determined spectroscopically. The solid circles are values extrapolated to 25°C from thermal unfolding experiments at lower concentrations of urea and higher temperatures.

The studies discussed above gives an idea of conformational stability of proteins in ILs as inferred from the t measurement and melting temperature determination followed by various spectroscopic methods. However, stability of a protein/enzyme has contributions from both thermodynamic and kinetic parameters [55]. While thermal unfolding studies help to extract important thermodynamic parameters, stability of the proteins could also arise from the kinetic barrier to the conformational changes in the protein scaffold [55]. However, except for monellin [48], the thermodynamic stability in other cases has not been examined. Thus, in context of stability of proteins/enzymes in ILs, it is important and imperative to study and probe thermal unfolding as well as the thermodynamics and kinetics of the conformational change of the proteins in ILs. 

Figure 6 Characterization of receptor/chemokine complexes. (A) Analytical SEC traces of varying quality. The top trace has a sharp, symmetric peak while the peak in the bottom trace has a shoulder (indicated by arrow), which is a sign that the sample is partially aggregated. (B) Analytical SEC trace of the ACKR3/CXCL12 complex. (C) CPM measurements are used to determine the midpoint of thermal unfolding (Tm) of the receptor. Data can be plotted either as CPM fluorescence as a function of temperature (top) or as the derivative of the fluorescence (bottom). (D) CPM experiments with apo ACKR3 and the ACKR3/CXCL12 complex.

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... 

Fig. 12. Thermal denaturation for ribonuclease Tj as followed by VCD, from 20° to 65°C. The matrix descriptors determined for the native state and the unfolded high-temperature data are indicated. The values indicate a loss of the helix segment but maintenance of sheet segments. Also listed are the spectrally determined fractional contributions (FC) to the secondary structure. When combined with the segment analysis, this implies that the residual sheet segments must be very short. Reprinted with permission from Pancoska, P., et al. (1996). Biochemistry 35(40), 13094-13106, the American Chemical Society.

Neupogen), a four-helix bundle cytokine, is formulated at pH 4 but has been shown to maintain both thermal stability and tertiary structure at pH 2.60 In fact, the secondary structure of this molecule was shown to remain highly helical at pH 4 (Tm approximately 62°C) and 2 (Tm approximately 63°C) as compared to pH 7 (Tm approximately 55°C) where a less conformationally stable form was observed. In the same study, FTIR and CD data corroborated the tendency of the protein to unfold as measured by the loss of helical structure in the order pH 7 > pH 4 > pH 2. Moreover, after determining optimal pH conditions of thermostability, several studies have shown that excipient screening at such conditions can successfully predict the rank of formulation cocktails that offer the most favorable stability.14 23 31 56... 

Even extremophilic organisms and their proteins contain the same 20 amino acids with bonds similar to those in mesophiles. As the difference in free enthalpy between folded and unfolded states of globular proteins AG N >G is only about 45 15 kj mol-1 the sequence and structure of extremophilic proteins should differ from those of ordinary species. However, the main question, namely which properties cause the increase in denaturation temperature of thermostable proteins, is still debated (Rehaber, 1992). Theoretical and experimental analyses have shown that thermal stability is largely achieved by small but relevant changes at different locations in the structure involving electrostatic interactions and hydro-phobic effects (Karshikoff, 2001). There is no evidence for a common determinant or for just one effect causing thermostability. 

Other measures of protein flexibility have been found to correlate with thermal stability. One is resistance to proteolysis (Daniel et al., 1982 Fontana, 1988). Another is the quenching of buried tryptophan fluorescence by acrylamide, used in a study by Varley and Pain (1991). Both these processes are mediated by the same combination of local and global unfolding events that determine rates of hydrogen exchange. Their rates will depend on the ability of another molecule, acrylamide or a proteolytic enzyme, to penetrate into normally buried regions of the protein in order to either quench fluorescence or cleave peptide bonds. 

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