Cloned (Comment) | Organism |
---|---|
wild-type Dpo4 is fused to a C-terminal His6-tag and overexpressed in Escherichia coli | Saccharolobus solfataricus |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Saccharolobus solfataricus | - |
- |
- |
Purification (Comment) | Organism |
---|---|
- |
Saccharolobus solfataricus |
Synonyms | Comment | Organism |
---|---|---|
DNA polymerase IV | - |
Saccharolobus solfataricus |
Dpo4 | - |
Saccharolobus solfataricus |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
additional information | - |
wild-type enzyme exhibits a three-state cooperative unfolding profile, with an unfolding intermediate existing between the native and denatured states. The native state of wild-type Dpo4 persists from 26 to 87.5°C, and the unfolding intermediate is observed between 92 and 96.5 °C. Starting at 110 °C, the molar ellipticity approaches its highest value and does not change significantly in the temperature range of 110°C to 119°C. The Little Finger domain of the enzyme, which is only found in the Y-family DNA polymerases, is more thermostable than the polymerase core. The enzyme exhibits a three-state cooperative unfolding profile with an unfolding intermediate. The linker region between the Little Finger and Thumb domains of Dpo4 is a source of structural instability. Through site-directed mutagenesis, the interactions between the residues in the linker region and the Palm domain are identified to play a critical role in the formation of the unfolding intermediate. The secondary structure of Dpo4 is not altered when the temperature is increased from 26 to 87.5°C | Saccharolobus solfataricus |
88 | - |
Tm-value of mutant enzyme E100A/E235A/R240A: 88.3°C | Saccharolobus solfataricus |
89 | - |
Tm-value of mutant enzyme E100A: 88.9°C. Tm-value of mutant enzyme E100A/K148A: 89.4°C | Saccharolobus solfataricus |
90 | - |
Tm-value of mutant enzyme E235A/R240A or mutant enzyme K148A/E235A/R240A: 90.3°C. Tm-value of mutant enzyme E100A/K148A/E235A/R240A: 90.0°C | Saccharolobus solfataricus |
93 | - |
Tm-value of mutant enzyme K148A: 92.8°C | Saccharolobus solfataricus |
97 | - |
Tm-value of mutant enzyme R240A: 96.5°C | Saccharolobus solfataricus |
98 | - |
Tm-value of mutant enzyme P236A: 97.4°C | Saccharolobus solfataricus |
100 | - |
Tm-value of wild-type enzyme: 99.6°C | Saccharolobus solfataricus |