Single enzymes were found to either fully retain their activity or lose their activity when heated to the denaturation temperature. Previous study has shown that a small fraction of alkaline phosphatase in digital level became inactive at the denaturing temperature where as the surviving molecules retained full activity. In this work, we further studied this thermo-response with a model enzyme,β-glucuronidase, by using single molecule assays at its optimal temperature 37 °C and room temperature. The surface ofβ-glucuronidase was redesigned by using Rosetta suite of protein design tools. After comparing the activity and stability of resurfaced designs to the wild-type enzyme in bulk assays at room temperature and 37 °C, we found several pronounced thermoswitchable phenotypes in the resurfaced variants. Although in bulk assays it only shows activity is overall stronger in 37 °C than room temperature, a fraction of single molecules of these β-glucuronidase variants show a thermal switching behavior where enzymes that are completely inactive at RT gain activity at 37°C. More interestingly, the single molecule experiments also revealed reversible or irreversible thermal switching properties of a fraction of the population for these variants.

Thermoswitchability of β-Glucuronidase variants revealed through single-molecule studies

256th National Meeting (2018)

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