Technical Notes

Evaluating unfolding reversibility using temperature cycle measurements

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Technology Brief 2 nanoDSF Evaluating unfolding reversibility using temperature cycle measurements With the Prometheus and PR.TimeControl Software The conformational stability of proteins is of central importance for a large number of commercial products, ranging from biopharmaceutics to industrial enzymes. While conventional stability measurements are routinely performed, e.g., determination of the unfolding temperature, or assessment of long-term stability, little is typically known about the reversibility of the unfolding process. This, however, is an important property of any protein, since an irreversible unfolding eventually leads to reduced long-term stability and thus to poor developability of pharmaceutical or industrial proteins. The new PR.TimeControl software for instruments of the Prometheus series enables easy evaluation of unfolding reversibility. This can be done either by repeat cycles between two defined temperatures ("temperature cycling"), or by a repeated increase in the maximum temperature for each cycle ("incremental cycling"). The latter measurement mode thereby allows for evaluating the reversibility of unfolding over a large temperature range to investigate unfolding reversibility of distinct protein domains. Here, several examples of temperature cycling experiments are presented: 1. Temperature cycling to evaluate protein refolding and refolding kinetics. In this experiment, two reversibly unfolding proteins were subjected to temperature cycles between 25 °C and 80 °C. Increase to 80 °C resulted in complete unfolding of the protein. Subsequent cooling to 25 °C triggered refolding of both proteins, indicated by a complete recovery of the F350/F330 value to the initial state. While protein 1 (green) displayed an instantaneous refolding process, the refolding kinetics of protein 2 (red) were much slower, indicated by the fact that ~20 minutes of cooling to 25 °C were required to reach the F350/F330 value of the folded state.

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