Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Proc Natl Acad Sci U S A. 2011 Aug 23;108(34):14127-32. doi: 10.1073/pnas.1105880108. Epub 2011 Aug 8.
Cotranslational N-glycosylation can accelerate protein folding, slow protein unfolding, and increase protein stability, but the molecular basis for these energetic effects is incompletely understood. N-glycosylation of proteins at naïve sites could be a useful strategy for stabilizing proteins in therapeutic and research applications, but without engineering guidelines, often results in unpredictable changes to protein energetics. We recently introduced the enhanced aromatic sequon as a family of portable structural motifs that are stabilized upon glycosylation in specific reverse turn contexts: a five-residue type I β-turn harboring a G1 β-bulge (using a Phe-Yyy-Asn-Xxx-Thr sequon) and a type II β-turn within a six-residue loop (using a Phe-Yyy-Zzz-Asn-Xxx-Thr sequon) [Culyba EK, et al. (2011) Science 331:571-575]. Here we show that glycosylating a new enhanced aromatic sequon, Phe-Asn-Xxx-Thr, in a type I' β-turn stabilizes the Pin 1 WW domain. Comparing the energetic effects of glycosylating these three enhanced aromatic sequons in the same host WW domain revealed that the glycosylation-mediated stabilization is greatest for the enhanced aromatic sequon complementary to the type I β-turn with a G1 β-bulge. However, the portion of the stabilization from the tripartite interaction between Phe, Asn(GlcNAc), and Thr is similar for each enhanced aromatic sequon in its respective reverse turn context. Adding the Phe-Asn-Xxx-Thr motif (in a type I' β-turn) to the enhanced aromatic sequon family doubles the number of proteins that can be stabilized by glycosylation without having to alter the native reverse turn type.
共翻译后 N-糖基化可以加速蛋白质折叠、减缓蛋白质展开并增加蛋白质稳定性，但这些能量效应的分子基础尚不完全清楚。在治疗和研究应用中，对天然位点的蛋白质进行 N-糖基化可能是稳定蛋白质的一种有用策略，但如果没有工程设计指南，通常会导致蛋白质能量学发生不可预测的变化。我们最近引入了增强型芳香序列基元作为一组可移植的结构基元，这些基元在特定的反向转弯环境中通过糖基化得到稳定：一个包含 G1β-突环的五残基 I 型β-转角（使用 Phe-Yyy-Asn-Xxx-Thr 序列基元）和一个六残基环内的 II 型β-转角（使用 Phe-Yyy-Zzz-Asn-Xxx-Thr 序列基元）[CulybaEK等人，（2011）科学 331:571-575]。在这里，我们表明在 I 型'β-转角中糖基化新的增强型芳香序列基元 Phe-Asn-Xxx-Thr 可以稳定 Pin1 WW 结构域。比较在相同的 WW 结构域中糖基化这三个增强型芳香序列基元的能量效应表明，对于与具有 G1β-突环的 I 型β-转角互补的增强型芳香序列基元，糖基化介导的稳定性最大。然而，在各自的反向转弯环境中，每个增强型芳香序列基元中三部分相互作用（苯丙氨酸、天冬酰胺（GlcNAc）和苏氨酸）引起的稳定性部分相似。在增强型芳香序列基元家族中添加 Phe-Asn-Xxx-Thr 基元（在 I 型'β-转角中）可以使两倍数量的蛋白质通过糖基化稳定化，而无需改变天然的反向转弯类型。
