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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 ↔ U, under ambient conditions, ∆G U = G U -G F . Traditional methods of measuring ∆G U are solvent (urea or guanidinium chloride (GdmCl)) or thermal denaturation 1 . Solvent denaturation curves are generally analyzed using the linear extrapolation method (LEM):where m is a measure of the dependence of ∆G on denaturant, and ∆G U (H 2 O) is an estimate of the conformational stability that assumes that the linear dependence of ∆G on denaturant observed in the transition region continues to 0 M denaturant. Thermal denaturation experiments yield the melting temperature, T m , the enthalpy change at T m , ∆H m , and the heat capacity change, ∆C p , which can then be used to calculate ∆G U at any temperature T, ∆G U (T), with the Gibbs-Helmholtz equation:Both methods require an extrapolation from denaturing to ambient conditions. This is worrisome because the ensemble of denatured states in the presence of a denaturant or at higher temperatures may be different under ambient conditions. When a protein is placed in D 2 O, the amide hydrogens begin to exchange with deuterium, and the rate constants for each individual amide can be measured using nuclear magnetic resonance (NMR). It has long been recognized that protein conformational stabilities can potentially be estimated from the rate constants of exchange of the most slowly exchanging amide hydrogens 1 . Some recent studies support this idea 2-5 , whereas other studies disagree 6-8 . We show here that if the effects of D 2 O and proline isomerization are considered, the conformational stabilities calculated from hydrogen exchange rate constants are in excellent agreement with those determined by traditional methods. Measuring ∆G HXThe rate of exchange can be described by:where k op and k cl are the rate constants for the structural opening and closing reaction, and k rc is the rate constant for exchange from the open state 9 . Under EX2 conditions where k cl >> k rc , the rate constant for exchange, k ex = (k op /k cl )k rc = K op k rc where K op is the equilibrium constant for structural opening. The free energy change for structural opening, ∆G HX , is then given by:We give the average of the three largest ∆G HX values calculated using equation 4 for a variety of proteins (Table 1). The ∆G HX values are always higher than the ∆G U (H 2 O) and ∆G U (T) values. Denatured states in native state conditionsIn folded proteins, most peptide bonds have a trans conformation, but 0.03% of the Xaa-nonPro bonds and 5.2% of the Xaa-Pro bonds have a cis conformation 10 . In unfolded proteins, an equilibrium will be reached with the Xaa-nonPro bonds almost exclusively in the trans conformation, and 6-38% of the Xaa-Pro bonds in the cis conformation depending on identity of Xaa 11 (Table 2). For Xaa-Pro residues in the unfolded state, the average percentage cis = 12.6 ± 7.5, which is less than the 20% cis c...

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