The reversibility of the protein folding threshold refers to the thermodynamic boundary within which a protein can spontaneously transition between its functional, folded state and an inactive, unfolded state without undergoing irreversible denaturation or aggregation. This reversible process typically depends on specific conditions, such as temperature, pH, and solvent, being maintained below a critical denaturation threshold.

• Thermal Thresholds: Proteins often exhibit a reversible folding/unfolding process within a specific thermal region. If the temperature remains below a certain threshold (e.g., 330 K / 56.85 °C  for some studies of hydrated lysozyme), the protein can return to its native state upon cooling.

• Time-Dependent Stability: If the protein is kept at a denaturation threshold temperature for an extended time, the process may shift from reversible folding to irreversible denaturation, often accompanied by aggregation.

Two-temperature cycling can be used to quantify the reversibility of protein unfolding. For example, after heating to 70°C in an acidic pH 5 buffer, Trastuzumab can partially refold (see Figure 2). DLS data shows that unfolded monomeric antibody does not aggregate but instead adopts an unfolded conformation with a larger hydrodynamic radius. Upon cooling back to 25°C, the antibodies largely refold, but refolding is incomplete. After each cycle, the cumulant radius increases slightly, indicating a gradual accumulation of protein in a less compact non-native conformation.