Chemical synthesis of glycoproteins with the specific installation of gradient enriched 15N-labeled amino acids for getting insight into glycoprotein behavior
Chemical synthesis of glycoproteins with the specific installation of gradient enriched 15N-labeled amino acids for getting insight into glycoprotein behavior
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通过特定安装梯度富集 15N 标记氨基酸来化学合成糖蛋白，以深入了解糖蛋白行为
DOI：
10.1002/chem.201606049
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发表时间：
2017
期刊：
影响因子：
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通讯作者：
Yasuhiro Kajihara
中科院分区：
文献类型：
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作者：
Nguyen Minh Hien;Masayuki Izumi;Hajime Sato;Ryo Okamoto;Yasuhiro Kajihara
阐明寡糖对糖蛋白性质的影响，如局部构象变化、稳定性和动力学，仍然具有挑战性。本文提出了一种新的合成糖蛋白酰胺骨架的部分15 N-标记方法。使用固相肽合成（SPPS）和天然化学连接（NCL），在蛋白质骨架的特定位置插入13个15 N标记的氨基酸，同时有意改变15 N原子的富集。这个想法基于1H-15 N HSQC信号的强度，结合经典的同源TOCSY和NOESY方法，甚至可以区分相同类型的氨基酸，从而可以理解合成同质糖蛋白的局部构象的动力学。结果表明，连接双触角复合型或高甘露糖型寡糖不会干扰蛋白质构象。然而，T1值表明，寡糖影响动力学的局部构象。变温圆二色性（CD）光谱和T1值清楚地表明，寡糖似乎抑制蛋白质波动，换句话说，稳定蛋白质结构。这种对寡糖行为的深入了解表明了对糖蛋白及其受体之间结合亲和力的一些进一步影响。
Elucidating the effects of oligosaccharides on glycoprotein properties, such as local conformational changes, stability, and dynamics, has still been challenging. In this paper, a novel partial15N‐labeling method for the amide backbone of a synthetic glycoprotein is proposed. Using solid‐phase peptide synthesis (SPPS) and native chemical ligation (NCL), thirteen15N‐labeled amino acids were inserted at specific positions of the protein backbone, while intentionally varying the enrichment of15N atoms. This idea discriminated even the same type of amino acid based on the intensities of1H‐15N HSQC signals, combined with classic homonuclear TOCSY and NOESY methods, thus allowing for understanding the dynamics of the local conformation of a synthetic homogeneous glycoprotein. Results suggested that the attachment of an oligosaccharide of either a bi‐antennary complex‐type or a high‐mannose‐type did not disturb protein conformation. However,T1values suggested that the oligosaccharide influenced dynamics at the local conformation. Temperature‐varied circular dichroism (CD) spectra andT1values clearly indicated that oligosaccharides appeared to inhibit protein fluctuation or, in other words, stabilize protein structure. This insight into oligosaccharide behavior suggests some further effects on binding affinity between a glycoprotein and its receptor.