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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 ↔ 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...

Protein Folding—How and Why: By Hydrogen Exchange, Fragment Separation, and Mass Spectrometry

“…After earlier suggestions supported mainly by temperature dependence studies (53, 54), the calibration of unprotected HX rates (3, 11, 45) (k ch in Equation 1) made it possible to compute unfolding free energy based on HX rates of the slowest hydrogens in any given protein (Equation 3). The agreement of these values with global stability measured by standard methods as well as their large dependence on denaturant and temperature revealed that proteins experience transient global unfolding/refolding reactions even under stable native conditions (4, 20, 28). One consequence is that proteins may spontaneously unfold and refold many times, in whole or in part, during their lifetime, as considered below.…”

Structural Mass Spectrometry of the αβ-Tubulin Dimer Supports a Revised Model of Microtubule Assembly

“…Second, GDP-tubulin exhibits a step in the denaturation curve with a midpoint of approximately 0.125 M urea. Third, as H/D exchange correlates with protein conformational stability (39), GTP-tubulin is more stable than either GDP-tubulin or GMPCPP-tubulin up to approximately 0.5 M urea. Fourth, GMPCPP-tubulin is markedly less stable than GTP-tubulin over most of the concentration range but most notably in the absence of urea.…”

Multiple Ligand-Bound States of a Phosphohexomutase Revealed by Principal Component Analysis of NMR Peak Shifts

“…This can account for the mix of hydrogen bonds being stabilized, destabilized, or unaffected by ligand binding that is stabilizing to the ground state 26 . Estimations of global folding stability using the amide groups most protected from HX 25 are not, however, affected by localized accelerations of HX. The folding stability estimated for the entire enzyme suggests that the X1P-bound state is globally stabilized by ~0.6 kcal/mol over the E P state.…”

“…Conditions of pH 7.4 and 35 °C ensured that the HX behavior of the X1P complex occurred in the bimolecular EX2 regime, where the residues with the largest ΔG HX can be used to estimate folding stability by the method of ref. 25. Both subsecond HX and slow hydrogen-deuterium exchange (HDX) of E P +X1P were measured, revealing rate constants and ΔG HX for 201 amide groups.…”