NMR structure determination of a segmentally labeled glycoprotein using in vitro glycosylation.
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.
Slynko
Vadim
V
Institute of Molecular Biology and Biophysics, ETH Zurich, CH-8093 Zurich, Switzerland.
Schubert
Mario
M
Numao
Shin
S
Kowarik
Michael
M
Aebi
Markus
M
Allain
Frédéric H-T
FH
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
D006023:Glycoproteins