The N-degron pathway, formerly the N-end rule pathway, is a protein degradation process that determines the half-life of proteins based on their N-terminal residues. In contrast to the well-established in vivo studies over decades, in vitro studies of this pathway, including biochemical characterization and high-resolution structures, are relatively limited. In this study, we have developed a unique fusion technique using microtubule-associated protein 1A/1B light chain 3B, a key marker protein of autophagy, to tag the N terminus of the proteins involved in the N-degron pathway, which enables high yield of homogeneous target proteins with variable N-terminal residues for diverse biochemical studies including enzymatic and binding assays and substrate identification. Intriguingly, crystallization showed a markedly enhanced probability, even for the N-degron complexes. To validate our results, we determined the structures of select proteins in the N-degron pathway and compared them with the Protein Data Bank-deposited proteins. Furthermore, several biochemical applications of this technique were introduced. Therefore, this technique can be used as a general tool for the in vitro study of the N-degron pathway.

中文翻译：

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LC3B融合技术在涉及N-德格龙途径的蛋白质的生化和结构研究中的用途。

N-degron途径（以前称为N-末端规则途径）是一种蛋白质降解过程，可根据其N-末端残基确定蛋白质的半衰期。与数十年来建立的体内研究相比，该途径的体外研究（包括生化特征和高分辨率结构）相对有限。在这项研究中，我们已经开发了一种独特的融合技术，使用自噬的关键标记蛋白微管相关蛋白1A / 1B轻链3B标记N-degron途径中涉及的蛋白的N末端，从而实现高产量具有可变N末端残基的均质靶蛋白用于各种生化研究，包括酶和结合测定以及底物鉴定。有趣的是，结晶显示出明显提高的概率，即使是N-degron配合物 为了验证我们的结果，我们确定了N-degron途径中选定蛋白质的结构，并将其与蛋白质数据库存储的蛋白质进行了比较。此外，介绍了该技术的几种生化应用。因此，该技术可以用作N-德格龙途径的体外研究的通用工具。