Stabilizing Impact of N-Glycosylation on the WW Domain Depends Strongly on the Asn-GlcNAc Linkage

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• 作者：Brijesh K. Pandey ; Sebastian Enck ; Joshua L. Price
• 刊名：ACS Chemical Biology
• 出版年：2013
• 出版时间：October 18, 2013
• 年：2013
• 卷：8
• 期：10
• 页码：2140-2144
• 全文大小：272K
• 年卷期：v.8,no.10(October 18, 2013)
• ISSN：1554-8937

文摘

N-glycans play important roles in many cellular processes and can increase protein conformational stability in specific structural contexts. Glycosylation (with a single GlcNAc) of the reverse turn sequence Phe-Yyy-Asn-Xxx-Thr at Asn stabilizes the Pin 1 WW domain by 鈭?.85 卤 0.12 kcal mol^鈥?. Alternative methods exist for attaching carbohydrates to proteins; some occur naturally (e.g., the O-linkage), whereas others use chemoselective ligation reactions to mimic the natural N- or O-linkages. Here, we assess the energetic consequences of replacing the Asn linkage in the glycosylated WW domain with a Gln linkage, with two natural O-linkages, with two unnatural triazole linkages, and with an unnatural 伪-mercaptoacetamide linkage. Of these alternatives, only glycosylation of the triazole linkages stabilizes WW, and by a smaller amount than N-glycosylation, highlighting the need for caution when using triazole- or 伪-mercaptoacetamide-linked carbohydrates to mimic native N-glycans, especially where the impact of glycosylation on protein conformational stability is important.