BIOL 255 |
| Glycosylation, the most frequent post-translational modification of proteins, is elusive to structural biology due to poor receptivity to structural methods and difficult sample preparation. We used (1H,15N) NMR to characterize the glycoprotein RNase B relative to its pre-glycosylated form, the enzyme RNase A. We assigned ~90% of backbone amide resonances in RNase B by comparison with RNase A temperature- and pH-dependent chemical shifts. This transferred-assignment method avoids the double (13C,15N) labeling typically required for assignment, which would be prohibitively expensive for glycoproteins. Subsequently, we investigated chemical-shift perturbations in RNase B relative to A to assess oligosaccharide-induced changes. We hypothesize that dynamic response to the modification is responsible for reduced enzymatic activity in the glycoprotein. This is supported by shift perturbations at a catalytic residue (H119) 15 Å distant from oligosaccharide attachment, and at several sites on the opposite side of the protein that exhibit functionally relevant dynamics in RNase A. |
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Frontiers in Chemical Biology
5:00 PM-7:00 PM, Wednesday, August 22, 2007 BCEC -- Exhibit Hall - B2, Poster
Division of Biological Chemistry |