Heteronuclear three-dimensional NMR spectroscopy of a partially denatured protein: The A-state of human ubiquitin

Abstract

Human ubiquitin is a 76-residue protein that serves as a protein degradation signal when conjugated to another protein. Ubiquitin has been shown 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, partially folded (A-state). If the A-state represents an intermediate in the folding pathway of ubiquitin, comparison of the known structure of the N-state with that of the A-state may lead to an understanding of the folding pathway. Insights into the structural basis for ubiquitin's role in protein degradation may also be obtained. To this end we determined the secondary structure of the A-state using heteronuclear three-dimensional NMR spectroscopy of uniformly 15N-enriched ubiquitin. Sequence-specific 1H and 15N 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 1H-15N NOESY-HMQC and TOCSY-HMQC data sets. Because of 1H chemical shift degeneracies, the increased resolution provided by the 15N dimension was critical. Analysis of short- and long-range NOEs indicated that only the first two strands of beta-sheet, comprising residues 2-17, remain in the A-state, compared 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 were in contact with residues from the first two strands of beta-sheet. It is likely, therefore, that residues 1-33 comprise a folded domain in the A-state of ubiquitin. On the basis of 1H alpha chemical shifts and weak short-range NOEs, residues 34-76 do not adopt a rigid secondary structure but favor a helical conformation. This observation may be related to the helix-inducing effects of the methanol present. The secondary structure presented here differs from and is more thorough than that determined previously by two-dimensional 1H methods [Harding et al. (1991) Biochemistry, 30, 3120-3128].

Structural and dynamic characterization of the urea denatured state of the immunoglobulin binding domain of streptococcal protein G by multidimensional heteronuclear NMR spectroscopy

“…In contrast to other denatured proteins that have been studied by NMR (Neri et al, 1992a(Neri et al, , 1992b(Neri et al, , 1992cLogan et al, 1993Logan et al, , 1994Stockman et al, 1993;Arcus et al, 1994;, the chemical shift, 'H-IH NOE and coupling constant data show no evidence for the presence of any residual partially populated structure in the urea-unfolded state of GB1 at pH 2. Thus, the unfolded state of GB1 characterized in this paper probably represents the closest example of a random coil unfolded protein observed to date.…”

“…Residual structure has also been found in the acid-denatured state of barnase (Arcus et al, 1994), the acid-denatured molten globule states of human ubiquitin (Stockman et al, 1993) and a-lactalbumin (Alexandrescu et al, 1993), a recombinant model of the reduced unfolded state of bovine pancreatic trypsin inhibitor (Lumb & Kim, 1994), a deletion mutant of staphylococcal nuclease (Alex- (Kraulis, 1991) and the coordinates are taken from Gronenborn et al (1991). andrescu et al, 1994;, and the denatured form of a destabilizing mutant of staphylococcal nuclease .…”

Comprehensive structural and dynamical view of an unfolded protein from the combination of single-molecule FRET, NMR, and SAXS

“…1), comprising the native helix in the region of residues 21-31 as well as in the C-terminal half. The location of both the β-hairpin and helical propensities are reminiscent of ubiquitin's A-state induced at low pH and ∼60% methanol (28,66,67). However, in the A-state, these secondary structure elements have significantly higher average populations (67).…”

Autonomous folding of a peptide corresponding to the N‐terminal β‐hairpin from ubiquitin

“…exhibit C a H shifts whose pattern and relative magnitude approximately mirror~40-100%! those found in the native structure~Harding et al, 1991;Stockman et al, 1993!. On the other hand, the C-terminal segment in the A-state is not at all native like, but is composed almost entirely of non-native helical structurẽ Stockman et al, 1993!. To assess the extent to which the native-like structures in the N-terminal part of the molecule might be interdependent for their stability, peptide fragments corresponding to residues 1-21 and 1-35 were examined under the same mixed solvent conditions.…”