Loss of function in an ATP-depleted cell lysate upon increasing the temperature to 60 °C occurs in the case of thermo-labile mutant RAm1, but not for RA. (a) Experimental scheme: E. coli lysates expressing RA or RAm1 and depleted of ATP were incubated at 25 °C or 60 °C. (b) & (c) At the indicated time points, the concentration of functional RA was measured in the lysate by adding an excess of folding probe P1 (100 µM, 1 h incubation at 25 °C) and then the samples were subjected to SDS-PAGE (a scenario 1 experiment). Gels were directly visualized using a Bio-rad Gel Doc Imager employing UV illumination to quantify the fluorescence of the conjugate. (d) Using another aliquot, the specific activity of RA and RAm1 were measured by the functional assay at the time points indicated (described in supporting information experimental section (3)). Only the concentration of folded and functional RAm1 decreased upon application of a thermal stress, whereas RA was resistant to heat denaturation. FL = RA / RAm1-P1 conjugate fluorescence, WB = Western immunoblot.