Characterization of a partially denatured state of a protein by two-dimensional NMR: reduction of the hydrophobic interactions in ubiquitin.

A stable, partially structured state of ubiquitin, the A-state, is formed at pH 2.0 in 60% methanol/40% water at 298 K. Detailed characterization of the structure of this state has been carried out by 2D NMR spectroscopy. Assignment of slowly exchanging amide resonances protected from the solvent in the native and A-state shows that gross structural reorganization of the protein has not occurred and that the A-state contains a subset of the interactions present in the native state (N-state). Vicinal coupling constants and NOESY data show the presence of the first two strands of the five-strand beta-sheet that is present in the native protein and part of the third beta-strand. The hydrophobic face of the beta-sheet in the A-state is covered by a partially structured alpha-helix, tentatively assigned to residues 24-34, that is considerably more flexible than the alpha-helix in the N-state. There is evidence for some fixed side-chain--side-chain interactions between these two units of structure. The turn-rich area of the protein, which contains seven reverse turns and a short piece of 3(10) helix, does not appear to be structured in the A-state and is approaching random coil.