Thermal Stability: ΔΔG_H2O
Gibbs Free Energy of Folding/Unfolding (ΔG)
Fluorescence Light Scattering,Spectrofluorimetry
10 mM MES, 2 mM EDTA, 90 mM NaCl, 10 mM DTT
25 to 60°C at 0.5°C/min
excitation/emission at 500 nm
Equilibrium unfolding of IL-l/3 monitored by fluorescence exhibited two transitions. The unusual biphasic fluorescence response to denaturant necessitated an alternative means of calculating "native" base lines. This was accomplished by extrapolation of native base lines from the data obtained following the sharp increase
in fluorescence to the beginning of the cooperative unfolding transition (usually in the range of 0.5-1.0 M GdnHC1).
Fluorescence yields were converted to the fraction of unfolded protein, F_app (calculated by extrapolation of unfolded and native base lines through the transition zone) determined (34). F_app data were converted to ΔG values by nonlinear least squares fitting to a two-state model using NLIN (SAS Institute, Cary, NC).