Fig. 4
From: An iterative computational design approach to increase the thermal endurance of a mesophilic enzyme
Real-time differential interference contrast (DIC) microscopy of PDC variants showing three distinct phases. PDC variants were evaluated during a heating ramp (1 °C/min) from room temperature to 60 °C followed by a temperature hold at 60 °C. In phase 1, the protein is in solution with no visible birefringence. In phase 2, the protein becomes visible, forming an alternative conformation that appears to be soluble as it is still moving in solution. In phase 3, all of the protein has aggregated into a semicontinuous lattice of precipitated protein. The graph displays the temperature at which each of the PDC variant transitions between phases. Wild-type PDC (WT) is shown in gray, PDC1.01 in yellow, PDC1.10 in blue, PDC2.01 in red, PDC2.02 in green, and PDC2.03 in purple. PDC1.01 (yellow) and PDC1.10 (blue) were combined to generate PDC2.02 (green). PDC2.01 (red) and PDC1.10 (blue) were combined to generate PDC2.03 (purple). During the 60 °C experiments, PDC2.03 protein never formed a lattice of precipitated protein to enter phase 3. *Variant PDC1.10 includes the mutation A189K. Variant 2.02 was evaluated with and without the mutation A189K (see Additional file 1: Figure S3 and Fig. 3c, respectively). Thermal stability was the same with and without the mutation, and thus A189K was not included in variant 2.03