Distribution, transition and thermodynamic stability of protein conformations in the denaturant-induced unfolding of proteins

Abstract

Background: Extensive and intensive studies on the unfolding of proteins require appropriate theoretical model and parameter to clearly illustrate the feature and characteristic of the unfolding system. Over the past several decades, four approaches have been proposed to describe the interaction between proteins and denaturants, but some ambiguity and deviations usually occur in the explanation of the experimental data.

Methodology/principal findings: In this work, a theoretical model was presented to show the dependency of the residual activity ratio of the proteins on the molar denaturant concentration. Through the characteristic unfolding parameters ki and Δmi in this model, the distribution, transition and thermodynamic stability of protein conformations during the unfolding process can be quantitatively described. This model was tested with the two-state unfolding of bovine heart cytochrome c and the three-state unfolding of hen egg white lysozyme induced by both guanidine hydrochloride and urea, the four-state unfolding of bovine carbonic anhydrase b induced by guanidine hydrochloride and the unfolding of some other proteins induced by denaturants. The results illustrated that this model could be used accurately to reveal the distribution and transition of protein conformations in the presence of different concentrations of denaturants and to evaluate the unfolding tendency and thermodynamic stability of different conformations. In most denaturant-induced unfolding of proteins, the unfolding became increasingly hard in next transition step and the proteins became more unstable as they attained next successive stable conformation.

Conclusions/significance: This work presents a useful method for people to study the unfolding of proteins and may be used to describe the unfolding and refolding of other biopolymers induced by denaturants, inducers, etc.

Figure 1. Denaturant-induced unfolding of a protein from its native state to completely unfolded state through n intermediate states.

N_D, I_Di and U_D denote the native state, intermediate state and completely unfolded state of a protein molecule, respectively. k _i indicates the thermodynamic equilibrium constant for the unfolding of the protein from one stable conformation state to the next. The symbol “” implies that the unfolding of the protein by the denaturant is only at a local thermodynamic equilibrium under a local denaturant concentration range.

Figure 2. Interactions between the protein and denaturant molecules in the denaturant-induced unfolding of proteins.

Figure 3. Residual activity ratios (r) of bovine heart cytochrome c exposed to different concentrations of guanidine hydrochloride or urea.

□: guanidine hydrochloride; Δ: urea. The concentration of bovine heart cytochrome c was 0.50 mg/mL, and the experimental temperature was 25°C.

Figure 4. Plots of ln(1/r−1) vs. ln[D] for the unfolding of bovine heart cytochrome c induced by guanidine hydrochloride and urea.

◊: guanidine hydrochloride; □: urea.

Figure 5. Molar fractions and of native and completely unfolded bovine heart cytochrome c exposed to different concentrations of guanidine hydrochloride (A) or urea (B).

◊: native bovine heart cytochrome c; □: completely unfolded bovine heart cytochrome c.

Figure 6. Residual activity ratios (r) of hen egg white lysozyme exposed to different concentrations of guanidine hydrochloride or urea.

Δ: guanidine hydrochloride; ○: urea. The concentration of hen egg white lysozyme was 0.50 mg/mL, and the experimental temperature was 25°C.

Figure 7. Plots of ln(1/r−1) vs. ln[D] (A) and ln[1/(r·k ₁·[D])−1] vs. ln[D] (B) for the unfolding of hen egg white lysozyme induced by guanidine hydrochloride and urea.

Δ: guanidine hydrochloride; ○: urea.

Figure 8. Molar fractions , and of native, intermediate and completely unfolded hen egg white lysozyme exposed to different concentrations of guanidine hydrochloride (A) or urea (B).

□: native egg white lysozyme; ○: intermediate egg white lysozyme; Δ: completely unfolded egg white lysozyme.

Figure 9. Residual activity ratios (r) of bovine carbonic anhydrase b exposed to different concentrations of guanidine hydrochloride or urea.

◊: guanidine hydrochloride; □: urea. The concentration of bovine carbonic anhydrase b was 0.50 mg/mL, and the experimental temperature was 25°C.

Figure 10. Molar fractions , , and of native, molten globule, pre-molten globule and completely unfolded bovine carbonic anhydrase b exposed to different concentrations of guanidine hydrochloride.

◊: native bovine carbonic anhydrase b; □: molten globule bovine carbonic anhydrase b; Δ: pre-molten globule bovine carbonic anhydrase b; ○: completely unfolded bovine carbonic anhydrase b.

Figure 11. Plots of lnk _i vs. Δm _i for the unfolding of some proteins induced by denaturants.

◊: guanidine hydrochloride; □: urea; Δ: methanol.