Table 1.
Equilibrium denaturation of wild-type and mutant CI2
| CI2 protein* | Predicted helicity¶, % | Determined, helicity†, % | mD-N‡, kcal mol−1 M−1 | [GdmCl]50%‡, M | ΔΔGD-NH2O§, kcal mol−1 |
|---|---|---|---|---|---|
| Wild type | 2.3 | 3.2 ± 1.1 | 1.90 ± 0.03 | 4.00 ± 0.01 | 0 |
| Mutations at the C terminus of the helix | |||||
| KR18 | 3.3 | n.d. | 2.34 ± 0.07 | 3.89 ± 0.01 | 0.21 ± 0.03 |
| KR18/DR23 | 10.0 | 8.1 ± 1.9 | 1.94 ± 0.03 | 3.03 ± 0.01 | 1.87 ± 0.04 |
| DA23 | 4.5 | 5.2 ± 1.0 | 1.87 ± 0.05 | 3.51 ± 0.01 | 0.96 ± 0.03 |
| Mutations in the protease-binding loop region | |||||
| RF48‖ | 1.70 ± 0.05 | 4.85 ± 0.08 | −1.65 ± 0.16 | ||
| RF48/FL50 | 1.88 ± 0.07 | 3.71 ± 0.03 | 0.56 ± 0.05 | ||
Equilibrium denaturation experiments with guanidinium chloride (GdmCl) were performed as described (6). The free energy of unfolding, ΔGD-N, varies according to denaturant concentration as follows: ΔGD-N = ΔGD-NH2O − mD-N [GdmCl].
Determined by titration of the CI2 peptides with trifluoroethanol in 10 mM sodium phosphate, pH 6.3, 25°C (34). This procedure gives the free energy of formation of the helix, ΔG, which is related to the equilibrium constant, K, by ΔG = −RT ln K. The fraction of formed helix is then defined as K/(K + 1).
mD-N is the denaturant dependence of the equilibrium unfolding transition and [GdmCl]50% is the denaturant concentration at which 50% of the protein is unfolded.
The change in free energy of equilibrium unfolding of the mutant and wild-type proteins, Δ(ΔGD-NH2O), was calculated using an average mD-N value of 1.94 kcal mol−1 M−1 (6).
The peptide helicity is calculated by the prediction program agadir (32, 33). The wild-type peptide sequence ELVGK-(SVEEAKKVILQDK)-PEAQ is the input sequence for the program and the conditions are: pH 6.3, 298 K, free N terminus and amidated C terminus. The helicity shown above is the average helicity in the helical region (indicated by the sequence in brackets), which corresponds to residues (12–24) in the intact CI2 protein. The residues shown in bold are those mutated in this study.
The determination of the stability of CI2 RF48 is not very reliable because the protein is very stable. Using the stronger denaturant guanidinium isothiocyanate (Gdm SCN) we derive a ΔΔGD-NH2O of −1.18 ± 0.20 kcal mol−1.