Table 4. Refolding rate constants and percentages of recovered activity from the reversible refolding experiment for the wild-type adGSTD4-4 enzyme and its engineered mutants.
The proteins were denatured initially with 4 M guanidinium chloride at room temperature for 10 min. Refolding rate constants (kref) were determined by non-linear regression analysis using a single-exponential equation (GraphPad Prism 4).
| Refolding experiment | ||
|---|---|---|
| Enzyme | Refolding rate constant (min−1) | % recovery |
| Wild-type | 0.54±0.02 | 20.7±0.67 |
| L33A | 0.85±0.05* | 18.6±0.55 |
| L33Y | nd | nd |
| L33F | nd | nd |
| L33I | 0.53±0.01 | 14.7±0.76* |
| L6A | 0.22±0.02* | 14.9±0.77* |
| T31A | 0.41±0.02* | 10.1±0.41* |
| I52A | nd | nd |
| I52L | 0.67±0.02* | 20.1±0.61 |
| E37A | 0.69±0.05* | 37.7±1.49* |
| E37Q | 0.74±0.03* | 14.0±0.32* |
| K40A | 0.26±0.02* | 14.2±0.95* |
| E42A | 0.32±0.02* | 31.5±1.44* |
| A35R | 0.40±0.01‡ | 29.6±0.64† |
ANOVA analysis revealed significant differences compared with the wild-type, where indicated: *P<0.001. The absence of a symbol indicates no significant difference compared with the wild-type. nd, not determined (low activity precluded measurement).
ANOVA analysis revealed significant differences compared with the wild-type, where indicated: †P<0.01. The absence of a symbol indicates no significant difference compared with the wild-type. nd, not determined (low activity precluded measurement).
ANOVA analysis revealed significant differences compared with the wild-type, where indicated: ‡P<0.05. The absence of a symbol indicates no significant difference compared with the wild-type. nd, not determined (low activity precluded measurement).