Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706-1396, United States.
J Am Chem Soc. 2012 Aug 1;134(30):12658-67. doi: 10.1021/ja3039486. Epub 2012 Jul 17.
Deamidation of asparagine and glutamine is the most common nonenzymatic, post-translational modification. Deamidation can influence the structure, stability, folding, and aggregation of proteins and has been proposed to play a role in amyloid formation. However there are no structural studies of the consequences of deamidation on amyloid fibers, in large part because of the difficulty of studying these materials using conventional methods. Here we examine the effects of deamidation on the kinetics of amyloid formation by amylin, the causative agent of type 2 diabetes. We find that deamidation accelerates amyloid formation and the deamidated material is able to seed amyloid formation by unmodified amylin. Using site-specific isotope labeling and two-dimensional infrared (2D IR) spectroscopy, we show that fibers formed by samples that contain deamidated polypeptide contain reduced amounts of β-sheet. Deamidation leads to disruption of the N-terminal β-sheet between Ala-8 and Ala-13, but β-sheet is still retained near Leu-16. The C-terminal sheet is disrupted near Leu-27. Analysis of potential sites of deamidation together with structural models of amylin fibers reveals that deamidation in the N-terminal β-sheet region may be the cause for the disruption of the fiber structure at both the N- and C-terminal β-sheet. Thus, deamidation is a post-translational modification that creates fibers that have an altered structure but can still act as a template for amylin aggregation. Deamidation is very difficult to detect with standard methods used to follow amyloid formation, but isotope-labeled IR spectroscopy provides a means for monitoring sample degradation and investigating the structural consequences of deamidation.
脱酰胺作用是最常见的非酶促翻译后修饰之一。脱酰胺作用可以影响蛋白质的结构、稳定性、折叠和聚集，并且已经被提出在淀粉样纤维形成中发挥作用。然而，由于使用传统方法研究这些材料的困难，目前还没有关于脱酰胺作用对淀粉样纤维结构影响的结构研究。在这里，我们研究了脱酰胺作用对胰岛淀粉样多肽（2 型糖尿病的致病物质）形成淀粉样纤维的动力学的影响。我们发现脱酰胺作用加速了淀粉样纤维的形成，并且脱酰胺的物质能够通过未修饰的胰岛淀粉样多肽引发淀粉样纤维的形成。使用定点同位素标记和二维红外（2D IR）光谱，我们表明，含有脱酰胺多肽的样品形成的纤维含有较少量的β-折叠。脱酰胺作用导致 N 端β-折叠区（Ala-8 和 Ala-13 之间）的破坏，但β-折叠在 Leu-16 附近仍然保留。C 端片层在 Leu-27 附近被破坏。脱酰胺作用的潜在位点分析以及胰岛淀粉样纤维的结构模型表明，N 端β-折叠区域的脱酰胺作用可能是纤维结构在 N 端和 C 端β-折叠处均被破坏的原因。因此，脱酰胺作用是一种翻译后修饰，它产生的纤维具有改变的结构，但仍然可以作为胰岛淀粉样多肽聚集的模板。脱酰胺作用很难用标准方法检测到，这些方法通常用于跟踪淀粉样纤维的形成，但同位素标记的红外光谱为监测样品降解和研究脱酰胺作用的结构后果提供了一种手段。
