The N-Terminal Domain of Ribosomal Protein L9 Folds via a Diffuse and Delocalized Transition State.

The N-terminal domain of L9 (NTL9) is a 56-residue mixed α-β protein that lacks disulfides, does not bind cofactors, and folds reversibly. NTL9 has been widely used as a model system for experimental and computational studies of protein folding and for investigations of the unfolded state. The role of side-chain interactions in the folding of NTL9 is probed by mutational analysis. ϕ-values, which represent the ratio of the change in the log of the folding rate upon mutation to the change in the log of the equilibrium constant for folding, are reported for 25 point mutations and 15 double mutants. All ϕ-values are small, with an average over all sites probed of only 0.19 and a largest value of 0.4. The effect of modulating unfolded-state interactions is studied by measuring ϕ-values in second- site mutants and under solvent conditions that perturb unfolded-state energetics in a defined way. Neither of these alterations significantly affects the distribution of ϕ-values. The results, combined with those of earlier studies that probe the role of hydrogen-bond formation in folding and the burial of surface area, reveal that the transition state for folding contains extensive backbone structure and buries a significant fraction of hydrophobic surface area, but lacks well developed side-chain-side-chain interactions. The folding transition state for NTL9 does not contain a specific "nucleus" consisting of a few key residues; rather, it involves extensive backbone hydrogen bonding and partially formed structure delocalized over almost the entire domain. The potential generality of these observations is discussed.