title: NMR structure determination of a segmentally labeled glycoprotein using in vitro glycosylation.
abstract: Although there is great interest in three-dimensional structures of glycoproteins and complex oligosaccharides, their structural determination have been hampered by inhomogeneous and incomplete glycosylation, poor expression, low tendency to crystallize, and severe chemical shift overlap. Using segmental labeling of the glycan and the protein component by in vitro glycosylation, we developed a novel method of NMR structural determination that overcomes some of these problems. Highly homogeneously glycosylated proteins in milligram amounts can be obtained. This allowed the determination of the structure of an N-linked glycoprotein from Campylobacter jejuni. The glycosylation acceptor site was found to be in a flexible loop. The presented methodology extends the observable NOE distance limit of oligosaccharides significantly over 4 A, resulting in a high number of distance restraints per glycosidic linkage. A well-defined glycan structure was obtained.
authors.affiliation: Institute of Molecular Biology and Biophysics, ETH Zurich, CH-8093 Zurich, Switzerland.
authors.forename: Vadim
authors.initials: V
authors.lastname: Slynko
authors.forename: Mario
authors.initials: M
authors.lastname: Schubert
authors.forename: Shin
authors.initials: S
authors.lastname: Numao
authors.forename: Michael
authors.initials: M
authors.lastname: Kowarik
authors.forename: Markus
authors.initials: M
authors.lastname: Aebi
authors.forename: Frédéric H-T
authors.initials: FH
authors.lastname: Allain
mesh_terms: D000595:Amino Acid Sequence; D016123:Campylobacter jejuni / Q000737:chemistry; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry*; D006031:Glycosylation; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D013237:Stereoisomerism
chemical_list: D006023:Glycoproteins