A single N-acetylgalactosamine residue at threonine 106 modifies the dynamics and structure of interferon α2a around the glycosylation site.

Enzymatic addition of GalNAc to isotopically labeled IFNα2a produced in Escherichia coli yielded the O-linked glycoprotein GalNAcα-[(13)C,(15)N]IFNα2a. The three-dimensional structure of GalNAcα-IFNα2a has been determined in solution by NMR spectroscopy at high resolution. Proton-nitrogen heteronuclear Overhauser enhancement measurements revealed that the addition of a single monosaccharide unit at Thr-106 significantly slowed motions of the glycosylation loop on the nanosecond time scale. Subsequent addition of a Gal unit produced Gal(β1,3)GalNAcα-[(13)C,(15)N]IFNα2a. This extension resulted in a further decrease in the dynamics of this loop. The methodology used here allowed the first such description of the structure and dynamics of an O-glycoprotein and opens the way to the study of this class of proteins.