Fig. 5.

Thermophoresis can be a useful orthogonal probe of protein denaturation. (A) Thermophoresis values of HBc (green circles) identified equivalent transitions to those identified using far-UV CD spectroscopy (black triangles), consistent with thermophoresis being an orthogonal probe of protein denaturation. Data were fitted to a 3-state equilibrium transition with a populated intermediate as previously described [10]. (B) Normalised thermophoresis time traces of SOD1 at a range of urea concentrations. A 3 K gradient was created between 5 and 35 s (‘Laser on’). SOD1 exhibited positive thermophoresis (i.e. it moved away from the higher temperature in the gradient) which caused an exponential decline of the fluorescence signal (Fig. 2). Denatured SOD1 (red lines) moved further than native protein (blue lines). There was a gradual transition from native to denatured protein, with both states being significantly populated at the transition midpoint (purple lines). Thermophoresis was reversible and the fluorescence signal returned to its initial value after the laser was switched off and the thermal gradient depleted. (C) Complementary techniques were used to probe chemical denaturation of SOD1. Thermophoresis values (green circles, derived from the data in b) yielded essential identical fitting parameters as far UV CD spectroscopy (black triangles). Under these conditions, no denaturation transition was evident using intrinsic fluorescence emission measured on the NT.LabelFree instrument (due to the wide bandpass emission filter in the NT.LabelFree instrument). All curves were fitted to Eq. (3) (see Section 2.7) which describes a two-state denaturation transition.