Direct observation of dipolar couplings and hydrogen bonds across a beta-hairpin in 8 M urea

A detailed, quantitative description of the unfolded states of proteins at atomic resolution has been elusive due to enormous experimental and theoretical problems resulting from the huge number of degrees of freedom of an unfolded structural ensemble. In particular, direct long-range information has been extremely sparse. Here we show that such long-range information can be obtained by NMR with high sensitivity and precision from H-N-H-N residual dipolar couplings (RDCs) and hydrogen bond (Hbonds) scalar couplings for an unfolded, perdeuterated (amide protonated) protein (urea-denatured ubiquitin at pH 2.5). Besides numerous sequential contacts, the RDCs reveal the persistence of nativelike structure in ubiquitin's first beta-hairpin. This native-like structure is confirmed by the direct detection of H-bonds via (h3)J(NC') H-bond scalar couplings as well as by chemical shifts, (3)J(HNHA) couplings, and relaxation rates. A quantitative analysis suggests that despite 25% native backbone torsion angles indicated by the chemical shifts, the H-bonds of the hairpin are formed to a much lesser degree in urea.