Figure 5. CD measurements of EcR1 and Mutant 9 with increasing temperature

The protein concentration of each sample was 3 mM, and the buffer was 300 mM NaCl, 50 mM NaPO₄, pH 7. With increasing temperature, each protein began to denature as indicated by the decreasing magnitudes of the CD signals. Sharp changes in the magnitudes of the CD signals at 45 °C for Mutant 9 and at 60 °C for EcR1 suggested that the proteins undergo a transition from a principally native state to a mostly denatured state near these temperatures. There was a residual CD signal in both proteins even at high temperatures (85-90 °C). This signal may represent some remaining secondary structure, perhaps b-strand, which has been reported in other proteins at high temperature.