Fig. 3.
MST instruments yield comparable denaturant titrations to conventional fluorimeters. Changes in intrinsic fluorescence emission properties were recorded for chemical denaturant titrations of a range of different proteins (Table S1). Data were acquired using a standard AB2 fluorimeter (red circles, AB2 FES axis) and an NT.LabelFree instrument (blue squares, NT.LabelFree FES axis). The sigmoidal titrations were fitted to a function describing 2-state denaturation (red and blue lines, Eq. (3), Section 2.7) in order to determine [Denaturant]50%, mD–N and ΔGD−N0 (Table 2). Whilst MST instruments yielded titrations of similar quality to conventional spectrometers, the MST measurement strategy required only a fraction of the time, operator input and sample volumes (Table 1). For the majority of proteins tested here, the intrinsic fluorescence emission of the native state was more quenched than the corresponding denatured state. However, the reverse was true for E. coli cytochrome b562 F65W and the murine FBP28 WW domain. Consequently, the direction of the signal change accompanying the denaturation of these two proteins was inverted compared to the data for the other proteins shown here. This is an inherent property of these proteins and was independent of the instrument used.