Residual Structure of Unfolded Ubiquitin as Revealed by Hydrogen/Deuterium-Exchange 2D NMR

The characterization of residual structures persistent in unfolded proteins in concentrated denaturant solution is currently an important issue in studies of protein folding, because the residual structure present, if any, in the unfolded state may form a folding initiation site and guide the subsequent folding reactions. Here, we thus studied the hydrogen/deuterium (H/D)-exchange behavior of unfolded ubiquitin in 6.0 M guanidinium chloride at pH 2.6 and 20°C. We employed a dimethylsulfoxide (DMSO)-quenched H/D-exchange NMR technique with the use of spin desalting columns, which allowed us to make a quick medium exchange from 6.0 M guanidinium chloride to a quenching DMSO solution. The technique is particularly effective for studies of the H/D-exchange kinetics of unfolded proteins in concentrated denaturant. By the backbone resonance assignment of the hetero-nuclear single quantum coherence spectrum of 15N-labeled ubiquitin in the DMSO solution, we successfully investigated the H/D-exchange kinetics of 27 identified peptide amide groups in the ubiquitin sequence. Although most of these amide groups were not protected, the four amide groups of Ile3, Val5, Ile13 and Leu73 were weakly but significantly protected with a protection factor of 2.5-3.0, indicating that there were residual structures in unfolded ubiquitin and that these amide groups were 60-67% hydrogen-bonded by the residual structures. We show that the first native β-hairpin, composed of residues 2-16 in the native ubiquitin structure, is partially structured even in 6.0 M guanidinium chloride and that the amide group of Leu73 is protected by a nonnative hydrogen-bonding interaction. From comparison with the previous folding studies of ubiquitin, it is concluded that the residual native β-hairpin in unfolded ubiquitin forms a folding initiation site and guides the subsequent folding reactions of the protein.