Figure 5.
Determination of functional thermotolerance. A. Scheme for measuring temperature-dependent function. TPADO was diluted into a working condition into MOPS buffer then incubated in a thermocycler for 10 minutes at a series of elevated temperatures. The temperature in the thermocycler was then dropped back down to the reaction temperature (28°C) for an additional 10 minutes. The incubated protein was added to reaction wells containing TPARED, substrates NADH and TPA, and a buffer. Reactions were monitored in real-time by change in NADH λmax = 340 nm. This process is repeated for each temperature and can be repeated incubating both TPADO and TPARED. B. Specific activity plots showing the molar consumption of NADH over time for the first minute of reaction for 28°C (dark green, hollow circle), 40°C (light green, filled circle), 50°C (orange, square), and 55°C (red, diamond). C. Relative initial velocity (Vi) for samples when TPADO is inculbated (red, solid line) and when both TPADO and TPARED are incubated (gray, dashed line) where the functional melting temperature (TM) is indicated by arrows.