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.
