The study of intermediates in the protein folding pathway provides a wealth of
information about the energy landscape. The intermediates also frequently
initiate pathogenic fibril formations. While observing the intermediates is
difficult due to their transient nature, extreme conditions can partially unfold
the proteins and provide a glimpse of the intermediate states. Here, we observe
the high resolution structure of a hydrophobic core mutant of Ubiquitin at an
extreme acidic pH by nuclear magnetic resonance (NMR) spectroscopy. In the
structure, the native secondary and tertiary structure is conserved for a major
part of the protein. However, a long loop between the beta strands β3 and β5
is partially unfolded. The altered structure is supported by fluorescence data
and the difference in free energies between the native state and the
intermediate is reflected in the denaturant induced melting curves. The unfolded
region includes amino acids that are critical for interaction with cofactors as
well as for assembly of poly-Ubiquitin chains. The structure at acidic pH
resembles a late folding intermediate of Ubiquitin and indicates that upon
stabilization of the protein's core, the long loop converges on the core in the
final step of the folding process.
