acquired by monitoring the CD signal intensity at 215 nm over the
range 20–90 C, as described in
detail in the experimental section. All curves shown are consistent
with a simple two state equilibrium between folded and unfolded
forms. Y65R and MNEI progressively unfold into a random coil conformation,
as indicated by the disappearance of the negative band
at 215 nm and by the appearance of a strong negative band with a
maximum just below 200 nm. At pH 3.3 the parent protein
resulted more stable in solution, as Tm values are about 81.63 C
and 75.99 C for MNEI and Y65R, respectively. the transition from b-sheet to random coil
at this pH could be completely reversed if the temperature was
lowered back to 25 C within a few minutes. Upon increasing the
pH by about two units, from 3.3 to 5.1, we found again the
wild-type protein to be more stable than the mutant, because
the Tm values are 82.78 C and 80.13 C for MNEI and Y65R, respectively.
Heat denaturation curves registered at pH 7.0 gave Tm values
of 72.28 C and 66.36 C for MNEI and Y65R, respectively,
confirming that the higher thermal stability of MNEI also occurs
at neutral pH. Analysis of these data indicates that the parent protein
MNEI preserves its superior thermal stability within the pH
range 3.3–7.0 and that at pH 5.1 both proteins possess the maximum
relative value of Tm, also showing the lowest difference in
thermal stability between them.