Structure and properties of a dimeric N-terminal fragment of human ubiquitin

J Mol Biol. 2001 Dec 7;314(4):773-87. doi: 10.1006/jmbi.2001.5181.

Abstract

Previous peptide dissection and kinetic experiments have indicated that in vitro folding of ubiquitin may proceed via transient species in which native-like structure has been acquired in the first 45 residues. A peptide fragment, UQ(1-51), encompassing residues 1 to 51 of ubiquitin was produced in order to test whether this portion has propensity for independent self-assembly. Surprisingly, the construct formed a folded symmetrical dimer that was stabilised by 0.8 M sodium sulphate at 298 K (the S state). The solution structure of the UQ(1-51) dimer was determined by multinuclear NMR spectroscopy. Each subunit of UQ(1-51) consists of an N-terminal beta-hairpin followed by an alpha-helix and a final beta-strand, with orientations similar to intact ubiquitin. The dimer is formed by the third beta-strand of one subunit interleaving between the hairpin and third strand of the other to give a six-stranded beta-sheet, with the two alpha-helices sitting on top. The helix-helix and strand portions of the dimer interface also mimic related features in the structure of ubiquitin. The structural specificity of the UQ(1-51) peptide is tuneable: as the concentration of sodium sulphate is decreased, near-native alternative conformations are populated in slow chemical exchange. Magnetization transfer experiments were performed to characterize the various species present in 0.35 M sodium sulphate, namely the S state and two minor forms. Chemical shift differences suggest that one minor form is very similar to the S state, while the other experiences a significant conformational change in the third strand. A segmental rearrangement of the third strand in one subunit of the S state would render the dimer asymmetric, accounting for most of our results. Similar small-scale transitions in proteins are often invoked to explain solvent exchange at backbone amide proton sites that have an intermediate level of protection.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Chromatography, Gel
  • Dimerization
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Nuclear Magnetic Resonance, Biomolecular
  • Peptide Fragments / chemistry*
  • Peptide Fragments / metabolism*
  • Protein Binding
  • Protein Structure, Quaternary
  • Protein Structure, Secondary
  • Structure-Activity Relationship
  • Ubiquitin / chemistry*
  • Ubiquitin / metabolism
  • Ultracentrifugation

Substances

  • Peptide Fragments
  • Ubiquitin