HETERONUCLEAR 3-DIMENSIONAL NMR-SPECTROSCOPY OF A PARTIALLY DENATUREDPROTEIN - THE A-STATE OF HUMAN UBIQUITIN

Human ubiquitin is a 76-residue protein that serves as a protein degra dation signal when conjugated to another protein. Ubiquitin has been s hown to exist in at least three states: native (N-state), unfolded (U- state), and, when dissolved in 60% methanol:40% water at pH 2.0, parti ally folded (A-state). If the A-state represents an intermediate in th e folding pathway of ubiquitin, comparison of the known structure of t he N-state with that of the A-state may lead to an understanding of th e folding pathway. Insights into the structural basis for ubiquitin's role in protein degradation may also be obtained. To this end we deter mined the secondary structure of the A-state using heteronuclear three -dimensional NMR spectroscopy of uniformly N-15-enriched ubiquitin. Se quence-specific H-1 and N-15 resonance assignments were made for more than 90% of the residues in the A-state. The assignments were made by concerted analysis of three-dimensional H-1-N-15 NOESY-HMQC and TOCSY- HMQC data sets. Because of H-1 chemical shift degeneracies, the increa sed resolution provided by the N-15 dimension was critical. Analysis o f short- and long-range NOEs indicated that only the first two strands of beta-sheet, comprising residues 2-17, remain in the A-state, compa red to five strands in the N-state. NOEs indicative of an alpha-helix, comprising residues 25 33, were also identified. These residues were also helical in the N-state. In the N-state, residues in this helix we re in contact with residues from the first two strands of beta-sheet. It is likely, therefore, that residues 1-33 comprise a folded domain i n the A-state of ubiquitin. On the basis of H-1alpha chemical shifts a nd weak short-range NOEs, residues 34-76 do not adopt a rigid secondar y structure but favor a helical conformation. This observation may be related to the helix-inducing effects of the methanol present. The sec ondary structure presented here differs from and is more thorough than that determined previously by two-dimensional H-1 methods [Harding et al. (1991) Biochemistry, 30, 3120 3128].