PHAb10 and PHAb11 are thermostable dimers.
(a) Overall structure of PHAb8 monomer. (b) Dimeric structure of PHAb10. Chain A is colored in green, Chain B is colored in magenta, and the gray stick denotes the rotation axis. The electrostatic surface of PHAb10 with two potential substrate binding cavities are shown (negative = red; positive = blue). (c) Dimeric structure of PHAb11. Chain A is colored in wheat, Chain B is colored in green-cyan, and the red stick denotes the rotation axis. The electrostatic surface of PHAb11 with two potential substrate binding cavities is also shown (negative = red; positive = blue). (d) Superimposition of monomeric PHAb8, PHAb10, and PHAb11. PHAb8 is shown in light blue, PHAb10 in green, and PHAb11 in wheat. Residues involved in the catalytic triad are shown in sticks. (e-f) Residual bactericidal activity of PHAb10, PHAb11, and their truncation variants. Each truncation variant is treated at 100°C for 1 hour, stored at 25°C for 1 hour, and then tested for bactericidal activity by a log-killing assay. Dash lines represent the limit of detection and data below the limit of detection is not shown. (g) Thermal unfolding curves of PHAb8, PHAb10, and PHAb11 from 20 to 100°C as determined by nanoDSF. Values on the y-axis represent the first derivative of the fluorescence ratio at 350 nm and 330 nm. Peaks represent the transition temperature of each protein. (h-j) Circular dichroism spectra of PHAb8 (h), PHAb10 (i), and PHAb11 (j) at 90°C and temperatures close to their transition temperatures. (k-o) Circular dichroism spectra of PHAb10, PHAb11, and their truncation variants before and after heat treatment. Each domain fragment is treated at 100°C for 1 hour, stored at 25°C for additional hour prior to CD detection.