A surface exposed O-linked galactose residue destabilises the structure of a folded helix-loop-helix dimer.
A 42-residue glycopeptide Tn-15 and the corresponding reference polypeptide Thr-15 were designed and synthesized to provide a model system for the study of how glycosylation affects the stability of a molten globule-like protein. Tn-15 and Thr-15 fold into hairpin helix-loop-helix motifs that dimerise to form four-helix bundles and the only difference between the sequences is that Tn-15 carries an O-linked N-acetylgalactosamine residue at the side chain of threonine-15 whereas the sequence Thr-15 is unglycosylated. An analysis of the mean residue ellipticities at 222 nm of the two polypeptides and of the alpha-H chemical shift deviations from random coil values showed that glycosylation reduced the helical content of the polypeptides and increased the dissociation constant of the helix-loop-helix dimer to form monomers. The pH dependencies of the helical content of Tn-15 and Thr-15 differed as that of Thr-15 was largely unaffected by pH in the range from pH 4 to pH 10, whereas Tn-15 lost almost half of the helical content at pH 4 upon raising the pH to 10. No single amino acid residue was found to ionize in a way that could explain the observed pH dependence of Tn-15. The temperature dependence of the mean residue ellipticity of Tn-15 revealed a surprising decrease in helicity at 278 K in comparison with that at 293 K, reminiscent of cold denaturation, that was not observed for the reference four-helix bundle Thr-15.
Vijayalekshmi
Sarojini
S
Department of Chemistry, Linköping University, 581 83 Linköping, Sweden.
George
Shaji K
SK
Andersson
Linda K
LK
Kihlberg
Jan
J
Baltzer
Lars
L
D000116:Acetylgalactosamine / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002942:Circular Dichroism; D019281:Dimerization; D005690:Galactose / Q000031:analogs & derivatives* / Q000737:chemistry; D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D018257:Helix-Loop-Helix Motifs; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry; D013696:Temperature
D000596:Amino Acids; D006020:Glycopeptides; D010455:Peptides; D000116:Acetylgalactosamine; D005690:Galactose