Conformational changes associated with post-translational modifications of pro143 in Skp1 of dictyostelium -a dipeptide model system

Chamini V. Karunaratne, Thomas K. Weldeghiorghis, Christopher M. West, Carol M. Taylor

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Prolyl hydroxylation and subsequent glycosylation of the E3SCF ubiquitin ligase subunit Skp1 affects its conformation and its interaction with F-box proteins and, ultimately, O2-sensing in the organism. Taking a reductionist approach to understand the molecular basis for these effects, a series of end-capped Thr-Pro dipeptides was synthesized, tracking the sequential post-translational modifications that occur in the protein. The conformation of the pyrrolidine ring in each compound was gauged via coupling constants (3JHα,Hβ) and the electronegativity of the Cγ-substituents by chemical shifts (13C). The equilibrium between the cis-trans conformations about the central prolyl peptide bond was investigated by integration of signals corresponding to the two species in the 1H NMR spectra over a range of temperatures. These studies revealed an increasing preference for the trans-conformation in the order Pro < Hyp < [α-(1,4)GlcNAc]Hyp. Rates for the forward and reverse reactions, determined by magnetization transfer experiments, demonstrated a reduced rate for the trans-to-cis conversion and a significant increase in the cis-to-trans conversion upon hydroxylation of the proline residue in the dipeptide. NOE experiments suggest that the Thr side chain pushes the sugar away from the pyrrolidine ring. These effects, which depended on the presence of the N-terminal Thr residue, offer a mechanism to explain altered properties of the corresponding full-length proteins.

Original languageEnglish (US)
Pages (from-to)15170-15175
Number of pages6
JournalJournal of the American Chemical Society
Volume136
Issue number43
DOIs
StatePublished - Oct 29 2014
Externally publishedYes

Fingerprint

Dictyostelium
Dipeptides
Hydroxylation
Post Translational Protein Processing
Conformations
F-Box Proteins
Ligases
Ubiquitin
Proteins
Glycosylation
Proline
Electronegativity
Peptides
Temperature
Chemical shift
Sugars
Magnetization
Experiments
Nuclear magnetic resonance
pyrrolidine

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Conformational changes associated with post-translational modifications of pro143 in Skp1 of dictyostelium -a dipeptide model system. / Karunaratne, Chamini V.; Weldeghiorghis, Thomas K.; West, Christopher M.; Taylor, Carol M.

In: Journal of the American Chemical Society, Vol. 136, No. 43, 29.10.2014, p. 15170-15175.

Research output: Contribution to journalArticle

Karunaratne, Chamini V. ; Weldeghiorghis, Thomas K. ; West, Christopher M. ; Taylor, Carol M. / Conformational changes associated with post-translational modifications of pro143 in Skp1 of dictyostelium -a dipeptide model system. In: Journal of the American Chemical Society. 2014 ; Vol. 136, No. 43. pp. 15170-15175.
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