Three easy pieces

Abstract

Background: Differential scanning calorimetry is a powerful method that provides a complete thermodynamic characterization of the stability of a protein as a function of temperature. There are, however, circumstances that preclude a complete analysis of DSC data. The most common ones are irreversible denaturation transitions or transitions that take place at temperatures that are beyond the temperature limit of the instrument. Even for a protein that undergoes reversible thermal denaturation, the extrapolation of the thermodynamic data to lower temperatures, usually 25°C, may become unreliable due to difficulties in the determination of ΔCp.

Methods: The combination of differential scanning calorimetry and isothermal chemical denaturation allows reliable thermodynamic analysis of protein stability under less than ideal conditions.

Results and conclusions: This paper demonstrates how DSC can be used in combination with chemical denaturation to address three different scenarios: 1) estimation of an accurate ΔCp value for a reversible denaturation using as a test system the envelope HIV-1 glycoprotein gp120; 2) determination of the Gibbs energy of stability in the region in which thermal denaturation is irreversible using HEW lysozyme at different pH values; and, 3) determination of Gibbs energy of stability for a thermostable protein, thermolysin.

Keywords: Chemical denaturation; DSC; HEW lysozyme; HIV-1 gp120; ICD; Thermolysin.

Figure 1

The temperature dependence of the heat capacity function of gp120. The dashed lines are linear extrapolations of the baselines that correspond to the native and denatured states of gp120. This behavior is often seen in DSC of proteins and precludes accurate determination of ΔC_p.

Figure 2

The fraction of denatured gp120 obtained as a function of urea concentration for three different protein samples in PBS, pH 7.5, at 25 °C. The protein concentration was ~ 30 μg/mL. Analysis of the data yields ΔG = 7.30 ± 0.08 kcal/mol, m = 2.70 ± 0.05 kcal/(mol × M), and C1/2 = 2.71 ± 0.03 M.

Figure 3

The temperature dependence of the Gibbs energy of stability for gp120 and its enthalpic and entropic components. The values were calculated by combining data from DSC and ICD.

Figure 4

(a) The temperature dependence of the heat capacity function of HEW lysozyme at pH 7.5. The transition is irreversible as reflected by the lack of any transition in the repeated scan of the same sample. (b) Chemical denaturation and renaturation scans of lysozyme at pH 7.5, using GdnHCl as denaturant. The overlapping curves demonstrate the reversibility of the chemical denaturation of lysozyme. Both ICD experiments in the figure were performed in the presence of a constant concentration of 2 M urea (see method section 2.3 for details).

Figure 5

Gibbs energy of stability for lysozyme as a function of pH. The values in the region pH 4 – 7.5 indicated by the filled circles were determined by ICD. The values indicated by the filled squares below pH 4.0, where thermal denaturation of lysozyme is reversible, were calculated from values for ΔH° and ΔC_p determined by DSC in refs (16, 19) (see text for details).

Figure 6

GdnHCl denaturation of lysozyme at pH 7.5 in the presence of 1 (■), 2 (▲), and 3 M (●) urea. The protein concentration was ~ 30 μg/mL.

Figure 7

Global fit of the fraction denatured lysozyme as a function of the concentration of GdnHCl and urea in phosphate buffer, pH 7.5. The global fit is used to determine simultaneously ΔG°, m_GdnHCl and m_urea by non-linear least squares.

Figure 8

Chemical denaturation and renaturation scans of thermolysin using GdnHCl as denaturant at pH 7.5, (10 mM hepes, 100 mM NaCl, 10 mM CaCl₂) in the presence of a constant concentration of 3 M urea. The renaturation scans were recorded 11, 16, 24, and 48 hours times after the protein in high denaturant was diluted into wells containing buffer with different concentrations of denaturant. (For ease of viewing, the curve obtained after 16 hours has not been included in the figure.) b) C1/2 obtained from the renaturation scans recorded 11, 16, 24, and 48 hours after dilution of the denatured protein. The C1/2 value obtained from the denaturation scan has been added for comparison (48 h).

Figure 9

The fraction denatured thermolysin as a function of the concentration of GdnHCl in the presence of different urea concentrations: 2.5 (■), 3 (▲), and 3.5 M (●). The conditions are the same as described in the legend to Figure 8.

Figure 10

Global fit of the fraction of denatured thermolysin as a function of the concentration of GdnHCl and urea. As in Figure 7, the global fit allowed simultaneous determination of ΔG°, m_GdnHCl and m_urea.