Modulated Scanning Fluorimetry Can Quickly Assess Thermal Protein Unfolding Reversibility in Microvolume Samples

Hristo L Svilenov, Tim Menzen, Klaus Richter, Gerhard Winter

Determining the temperature at which the thermal unfolding of a protein starts becoming irreversible is relevant for many areas of protein research. Until now, published methods cannot determine, within a reasonable timeframe and with moderate sample consumption, the exposure temperature that starts causing irreversible protein unfolding. We present modulated scanning fluorimetry (MSF) and share a software (MSF Analyser) which can be used to derive non-reversibility curves of thermal protein unfolding from a series of incremental temperature cycles performed on only 10 µL sample, consuming as low as a few micrograms of protein. Further processing of the data can yield the onset temperature that starts causing non-reversible protein unfolding. The MSF method is based on the hardware of the already existing nanoDSF technology and can be applied to dozens of samples simultaneously. Here, we use MSF to study how solution pH affects the reversibility of thermal protein unfolding of several model proteins to show that the non-reversibility onset temperature (Tnr) is a unique biophysical parameter providing orthogonal information from thermal protein denaturation data and insights into the validity of thermal unfolding analysis in the context of equilibrium thermodynamics. We also show that MSF can be used to study enzyme stability after exposure to high temperatures. Besides, we demonstrate that protein thermal unfolding and non-reversibility can be affected in different ways upon modifications like PEG-ylation or labelling with fluorescent dyes. Finally, we show that MSF can be used to study the effect of various protein interactions on thermal protein unfolding reversibility. With the diverse examples in this work, we reveal how MSF can provide orthogonal information from thermal denaturation experiments that can bring benefits to various areas of protein research. The MSF Analyser software is available at

Mol Pharm March 2020