Chemical protein denaturation by chaotropes such as guanidine hydrochloride or urea is in contrast to thermal unfolding often a reversible process and can therefore be used to determine thermodynamic parameters of protein folding. Most importantly, the free energy change of the unfolding reaction (ΔG) and therefore the conformational stability of the protein of interest can be determined.

The Prometheus instruments are not only able to record thermal denaturation traces of proteins but also ideally suited to measure chemical unfolding in a very fast and efficient way using PR.PantaControl (Prometheus Panta) and PR.ChemControl (Prometheus NT.48 and NT.Plex) software. Measurements yield a full unfolding curve in less than one minute measuring time and automatically fits the data to extract ΔG and c50, the denaturant concentration at which 50% of the protein is unfolded.

Preparing unfolding reagents

We recommend purchasing guanidine hydrochloride and urea in highly pure forms, especially when working with intrinsic protein fluorescence as done with the Prometheus. Guanidine hydrochloride can also be obtained as highly pure and defined 8 M solutions. Urea and guanidine can be prepared as stock solutions in water or buffer. The solubility of urea and guanidine at 25°C is ~10.0 M and ~8.0 M, respectively.

Please note that solutions of urea are not stable (urea can decompose to cyanate and ammonium, resulting in covalent carbamylation of proteins in urea solutions). Thus, urea solutions should always be prepared freshly and used for a maximum of two days.

Preparing an unfolding series

When preparing a denaturant titration series manually, you can use the provided NanoTemper Excel^® Sheet to calculate a pipetting scheme (attached to this article). The commentary fields describe the necessary parameters in detail. It is recommended to prepare at least 24 dilution points: The Prometheus NT.Plex system measures 24, while Prometheus NT.48 can measure either 24 or 48 points. Importantly, the protein should be added last to the mixture. All samples should be efficiently mixed and incubated at least over night to ensure that the sample reaches the unfolding equilibrium even at low concentrations of denaturant. In case guanidine is used, the sample can be measured several times (e.g. after 1, 2 or 3 days) to ensure equilibrium is reached. To that end, store the prepared tubes and load new capillaries for re-measurement. In case the protein contains multiple free cysteine residues, the addition of DTT is recommended to prevent the formation of non-native disulfide bridges. It is important that the pH does not vary throughout the titration series, especially at concentrations close to the unfolding transition. The samples should be incubated at a defined temperature of e.g. 20 °C or 25 °C, depending on the temperature at which you want to determine ΔG.