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Abstract

Measuring protein conformational stability is one key to solving the protein folding problem. The conformational stability is the free energy change of the unfolding reaction, F |[harr]| U, under ambient conditions, |[Delta]|G U = GU - GF. Traditional methods of measuring |[Delta]|G U are solvent (urea or guanidinium chloride (GdmCl)) or thermal denaturation. Solvent denaturation curves are generally analyzed using the linear extrapolation method (LEM): |[Delta]|G = |[Delta]|G U(H2O) - m[denaturant]|[nbsp]||[nbsp]||[nbsp]||[nbsp]||[nbsp]|(1) where m is a measure of the dependence of |[Delta]|G on denaturant, and |[Delta]|GU (H2O) is an estimate of the conformational stability that assumes that the linear dependence of |[Delta]|G on denaturant observed in the transition region continues to 0 M denaturant. Thermal denaturation experiments yield the melting temperature, Tm, the enthalpy change at T m, |[Delta]|Hm, and the heat capacity change, |[Delta]|C p, which can then be used to calculate |[Delta]|GU at any temperature T, |[Delta]|GU(T), with the Gibbs–Helmholtz equation: |[Delta]|GU(T) = |[Delta]|Hm(1 - T/Tm) + |[Delta]|Cp[T - Tm - T ln (T/Tm)]|[nbsp]||[nbsp]||[nbsp]||[nbsp]||[nbsp]|(2)