Interleukin-1β folding between pH 5 and 7: Experimental evidence for three-state folding behavior and robust transition state positions late in folding

The thermodynamic stability and folding kinetics of the all beta-sheet protein interleukin-1beta were measured between 0 and 4 M GdmC1 concentrations and pH 5-7. Native interleukin-1beta undergoes a 3.5 kcal/mol decrease in thermodynamic stability, DeltaG(NU)(H2O) as pH is increased from 5 to 7. The native state parameter m(NU), measuring protein destabilization/[GdmCl], remains constant between pH 5 and 7, indicating that the solvent-exposed surface area difference between the native state and unfolded ensemble is unchanged across this pH range. Similarly, pH changes between 5 and 7 decrease only the thermodynamic stability, DeltaG(H2O), and not the m-values, of the kinetic intermediate and transition states. This finding is shown to be consistent with transition state configurations which continue to be the high-energy configurations of the transition state in the face of changing stability conditions. A three-state folding mechanism U reversible arrow I reversible arrow N is shown to be sufficient in characterizing IL-1beta folding under all conditions studied. The m-values of refolding transitions are much larger than the m-values of unfolding transitions, indicating that that the fast, T-2 (U reversible arrow I), and slow, T-1 (I reversible arrow N), transition states are highly similar to the intermediate I and native state N, respectively. Many of the folding properties of interleukin-1beta are shared among other members of the beta-trefoil protein family, although clear differences can exist.