Temperature induced structural transitions from native to unfolded aggregated states of tobacco etch virus protease

• The thermal-induced unfolding of recombinant TEVp had been investigated.
• Thermal denaturation of TEVp involved a three-state transition.
• The transition from the native state to the stable intermediate was reversible.
• The transition from intermediate to denatured state was irreversible.
• Protein aggregations were found at higher temperature.

Tobacco etch virus protease (TEVp) is widely used to remove fusion tags from recombinant proteins because of its high and unique specificity. This work describes the conformational and the thermodynamic properties in the unfolding/refolding process of TEVp3M (three-point mutant: L56V/S135G/S219V) induced by temperature. With temperature increasing from 20 to 100 °C, the CD spectra showed a transition trend from α-helix to β-sheet, and the fluorescence emission, synchronous fluorescence, ANS and RLS spectroscopy consistently revealed that the temperature-induced unfolding process behaved in a three-state manner, for there was a relatively stable intermediate state observed around 50 °C. The reversibility of thermal unfolding of TEVp3M further showed that the transition from the native to the intermediate state was reversible (below 50 °C), however the transition from the intermediate to the unfolded state was irreversible (above 60 °C). Moreover, aggregates were observed above 60 °C as revealed by SDS–PAGE, Thioflavin-T fluorescence and Congo red absorbance.

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