Fig. 2. Purification and biophysical characterizations of CpxA^QTY.

a Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS–PAGE) image of purification of CpxA^QTY. Lane M, molecular weight markers; lane 1/2/3, the fractions of IMAC washed/eluted off by 50/80/300 mM imidazole, 25 mM Tris-HCl, 300 mM NaCl, 5% glycerol, pH = 8.0; lane 4, the collected fraction corresponding to the major peak of gel filtration, in 25 mM Tris-HCl, 150 mM NaCl, 50 mM arginine, 5% glycerol, pH = 7.5. The experiment was repeated three times independently with similar results and the representative result was shown. b Sedimentation velocity AUC data of CpxA^QTY. The fitted continuous c(s) distribution curve was shown. E, experimental; D, designed. c Thermostability of CpxA^QTY measured by nanoDSF. Technical triplicates of CpxA^QTY (red lines) were assayed and the fitted T_m values were indicated by red dashed lines. The control group (green lines) was set as only buffer, no proteins added. d Circular dichroism (CD) results of CpxA^QTY. CD spectra (Total, purple solid line) and its α-helix (red) and β-sheet (blue) component spectra. The secondary structure analysis was performed using JASCO multivariate secondary structure (SS) estimation program. The content of α-helix and β-sheet in the AlphaFold2 (AF2) model were determined using PyMOL. Source data are provided as a Source Data file.