The unfolded state of the C-terminal domain of the ribosomal protein L9 contains both native and non-native structure

Interest in the structural and dynamic properties of unfolded proteins has increased in recent years owing to continued interest in protein folding and misfolding. Knowledge of the unfolded state under native conditions is particularly important for obtaining a complete picture of the protein folding process. The C-terminal domain of protein L9 is a globular α, β protein with an unusual mixed parallel and antiparallel β-strand structure. The folding kinetics and equilibrium unfolding of CTL9 strongly depend on pH, and follow a simple two state model. Both the native and the unfolded state can be significantly populated at pH 3.8 in the absence of denaturant, allowing the native state and the unfolded state to be characterized under identical conditions. Backbone ¹⁵N, ¹³C, ¹H and side-chain ¹³C_β, ¹H_β chemical shifts, amide proton NOEs, and ¹⁵N R₂ relaxation rates were obtained for the two conformational states at pH 3.8. All the data indicate that the pH 3.8 native state is well folded and is similar to the native state at neutral pH. There is significant residual structure in the pH 3.8 unfolded state. The regions corresponding to the two native state α-helices show strong preference to populate helical φ and ψ angles. The segment that connects α-helix 2 and β-strand 2 has a significant tendency to form non-native α-helical structure. Comparison with the pH 2.0 unfolded state and the urea unfolded state indicates that the tendency to adopt both native and non-native helical structure is stronger at pH 3.8, demonstrating that the unfolded state of CTL9 under native-like conditions is more structured. The implications for the folding of CTL9 are discussed.