The Unfolded State of the C-Terminal Domain of the Ribosomal Protein L9 Contains Both Native and Non-Native Structure

摘要

Interest in the structural and dynamic properties of unfolded proteins has increased innrecent years owing to continued interest in protein folding andmisfolding.Knowledge of the unfoldednstate under native conditions is particularly important for obtaining a complete picture of the proteinnfolding process. The C-terminal domain of protein L9 is a globular R, β protein with an unusualmixednparallel and antiparallel β-strand structure. The folding kinetics and equilibrium unfolding of CTL9nstrongly depend on pH, and follow a simple two state model. Both the native and the unfolded statencan be significantly populated at pH3.8 in the absence of denaturant, allowing the native state and thenunfolded state to be characterized under identical conditions. Backbonen15nN,n13nC,n1nH and side-chainn13nCβ,n1nHβ chemical shifts, amide protonNOEs, and 15nNR2 relaxation rates were obtained for the twonconformational states at pH 3.8. All the data indicate that the pH 3.8 native state is well folded and isnsimilar to the native state at neutral pH. There is significant residual structure in the pH 3.8 unfoldednstate. The regions corresponding to the two native state R-helices show strong preference to populatenhelicaljandψangles.The segment that connectsR-helix 2 and β-strand 2 has a significant tendency tonformnon-native R-helical structure.Comparisonwith the pH2.0 unfolded state and the urea unfoldednstate indicates that the tendency to adopt both native and non-native helical structure is stronger at pHn3.8, demonstrating that the unfolded state of CTL9 under native-like conditions is more structured.nThe implications for the folding of CTL9 are discussed