Modulation of protein stability by O-glycosylation in a designed Gc-MAF analog

Justin Spiriti, Federica Bogani, Arjan van der Vaart, Giovanna Ghirlanda

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

The post-translational modification of proteins by the covalent attachment of carbohydrates to specific side chains, or glycosylation, is emerging as a crucial process in modulating the function of proteins. In particular, the dynamic processing of the oligosaccharide can correlate with a change in function. For example, a potent macrophage-activating factor, Gc-MAF, is obtained from serum vitamin D binding protein (VDBP) by stepwise processing of the oligosaccharide attached to Thr 420 to the core α-GalNAc moiety. In previous work we designed a miniprotein analog of Gc-MAF, MM1, by grafting the glycosylated loop of Gc-MAF on a stable scaffold. GalNAc-MM1 showed native-like activity on macrophages (Bogani 2006, J. Am. Chem. Soc. 128 7142-43). Here, we present data on the thermodynamic stability and conformational dynamics of the mono- and diglycosylated forms. We observed an unusual trend: each glycosylation event destabilized the protein by about 1 kcal/mol. This effect is matched by an increase in the mobility of the glycosylated forms, as evaluated by molecular dynamics simulations. An analysis of the solvent-accessible surface area shows that glycosylation causes the three-helix bundle to adopt conformations in which the hydrophobic residues are more solvent exposed. The number of hydrophobic contacts is also affected. These two factors, which are ultimately explained with a change in occupancy for conformers of specific side chains, may contribute to the observed destabilization.

Original languageEnglish
Pages (from-to)157-167
Number of pages11
JournalBiophysical Chemistry
Volume134
Issue number3
DOIs
Publication statusPublished - May 1 2008

Keywords

  • De novo design
  • GalNAc
  • Molecular dynamics
  • O-glycosylation
  • Thermodynamic stability

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Organic Chemistry

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