Name:

Thermal Stability: ΔΔG_H2O

Category:

Stability/Folding

Property:

Gibbs Free Energy of Folding/Unfolding (ΔG)

Units:

kcal/mol

Measurement Technique:

Fluorescence Light Scattering,Spectrofluorimetry

Buffers:

10 mM MES, 2 mM EDTA, 90 mM NaCl, 10 mM DTT

Temperature:

25 to 60°C at 0.5°C/min

pH:

6.5

Protein Concentration:

1 mg/ml

Wavelength:

excitation/emission at 500 nm

Ionic Strength:

None

Details:

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).