Protein Stability by Chemical Denaturation

September 1, 2012

Quantifying protein stability is of fundamental importance in many areas of biomolecular research. The thermodynamic stability of a protein is reflected by a change in Gibbs free energy, ?G?, characterizing the equilibrium transition from a folded state, to an ensemble of unfolded, denatured states. In order to study ?G?, the folding equilibrium needs to be shifted towards the unfolded states which can be achieved by several different methods including the addition of chemical denaturants such as guanidinium chloride (GdnHCl).

Quantifying protein stability is of fundamental importance in many areas of biomolecular research. The thermodynamic stability of a protein is reflected by a change in Gibbs free energy, ΔG°, characterizing the equilibrium transition from a folded state, to an ensemble of unfolded, denatured states. In order to study ΔG°, the folding equilibrium needs to be shifted towards the unfolded states which can be achieved by several different methods including the addition of chemical denaturants such as guanidinium chloride (GdnHCl). Circular dichroism (CD) is a sensitive probe of protein conformation and is often used with chemical denaturation to obtain ΔG° in titration experiments (1). Here we present an automated CD (ACD) batch method that offers several advantages over such titrations:

  • Samples are prepared using an automated liquid handling system, which avoids lengthy equilibration times between CD measurements.

  • Mixing is performed outside the cuvette, allowing much reduced pathlengths (down to 0.1mm), thus extending the accessible wavelength range.

  • Automation significantly increases productivity and reduces human error.

  • Reduced exposure of sample to UV light.

Experimental Conditions

Experiments were performed in 50 mM glycine buffer, pH 2.9, 20 °C. The ACD autosampler was used to prepare samples in a 96 well plate from stock solutions of GdnHCl (7 mM) and Hen egg white lysozyme (10 mg/mL). The plate contained 31 different GdnHCl concentrations, ranging from 0 to 6 M in increments of 200 mM. Each sample was paired with a concentration-matched blank. CD and absorbance spectra (200–320 nm) were recorded in a fully automated, unattended fashion using a flow-through cell with a path length of 0.2 mm (<100 μL), a step size of 1 nm, and a spectral bandwidth of 1 nm. Raw CD and absorbance spectra were corrected by subtracting blank spectra under otherwise identical conditions. The GdnHCl concentration in each well was then accurately determined by refractometry. Blank-corrected CD spectra were normalised with respect to absorbance at 217 nm to account for any variations in protein concentration.

Results

Figure 1 shows the unfolding isotherm of lysozyme at 222 nm as a function of GdnHCl concentration. The data were fitted using weighted non-linear regression to obtain ΔG° and the m-value (the dependence of the Gibbs free energy change on denaturant concentration) which were 31.8 ± 0.62 kJ/mol and 9.8 ± 0.37 kJ/(mol M), respectively. The associated errors are standard deviations from five independent repeats, showing the excellent reproducibility of the technique.

Figure 1: Unfolding isotherm obtained by plotting the absorbance-normalized ellipticity at 222 nm as a function of GdnHCl concentration. Experimental data (red dots) with standard deviations (black bars) and fit (blue line).

Conclusion

The use of chemical denaturation to establish protein stability has been substantially improved by the use of automated CD. Many of the problems associated with performing traditional titration experiments are overcome by using an automated batch method. The technique has been used to obtain a precise ΔG° value for a protein unfolding transition.

References

(1) T.O. Street, N. Courtemanche, and D. Barrick, Protein folding and stability using denaturants, Methods in cell biology 84, 295–325 (2008).

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