Abstract
Despite the important role of the unfolded states in protein stability, folding, and aggregation, they remain poorly understood due to the lack of residue-specific experimental data. Here, we explore features of the unfolded state of the NTL9 protein by applying all-atom replica-exchange simulations to the two fragment peptides NTL9(1-22) and NTL9(6-17). We found that while NTL9(6-17) is unstructured, NTL9(1-22) transiently folds as various β-hairpins, a fraction of which contain a native β-sheet. Interestingly, despite a large number of charged residues, the formation of backbone hydrogen bonds is concomitant with hydrophobic but not electrostatic contacts. Although the fragment peptides lack a proposed specific contact between Asp(8) and Lys(12), the individually weak, nonspecific interactions with lysines together stabilize the charged Asp(8), leading to a pKa shift of nearly 0.5 units, in agreement with the NMR data. Taken together, our data suggest that the unfolded state of NTL9 likely contains a β-hairpin in segment 1-22 with sequence-distant hydrophobic contacts, thus lending support to a long-standing hypothesis that the unfolded states of proteins exhibit native-like topology with hydrophobic clusters.