Department of Chemistry and Biochemistry, Brigham Young University , Provo, Utah 84602, United States.
ACS Chem Biol. 2013 Oct 18;8(10):2140-4. doi: 10.1021/cb4004496. Epub 2013 Aug 20.
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 -0.85 ± 0.12 kcal mol(-1). 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 α-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 α-mercaptoacetamide-linked carbohydrates to mimic native N-glycans, especially where the impact of glycosylation on protein conformational stability is important.
N-糖基化在许多细胞过程中发挥着重要作用，并能在特定的结构环境中增加蛋白质构象的稳定性。在天冬酰胺（Asn）处的苯丙氨酸-yyy-天冬酰胺-xxx-苏氨酸反向转弯序列上的糖基化（单个 GlcNAc）通过-0.85 ± 0.12 kcal mol(-1)稳定 Pin 1 WW 结构域。存在将碳水化合物连接到蛋白质的替代方法；有些是自然发生的（例如 O 连接），而其他方法则使用化学选择性连接反应来模拟天然的 N-或 O 连接。在这里，我们评估了用 Gln 连接、两个天然 O 连接、两个非天然三唑连接和一个非天然α-巯基乙酰胺连接替代糖基化 WW 结构域中天冬酰胺连接的能量后果。在这些替代方案中，只有三唑连接的糖基化稳定了 WW，并且稳定程度小于 N-糖基化，这突出表明在使用三唑或α-巯基乙酰胺连接的碳水化合物模拟天然 N-聚糖时需要谨慎，尤其是在糖基化对蛋白质构象稳定性的影响很重要的情况下。
