Kheterpal I, Williams A, Murphy C, Bledsoe B, Wetzel R
Graduate School of Medicine, University of Tennessee Medical Center,1924 Alcoa Highway, Knoxville, Tennessee 37920, USA.
Biochemistry. 2001 Oct 2;40(39):11757-67. doi: 10.1021/bi010805z.
Although the gross morphology of amyloid fibrils is fairly well understood, very little is known about how the constituent polypeptides fold within the amyloid folding motif. In the experiments reported here, we used trypsin and chymotrypsin to conduct limited proteolysis studies on synthetic amyloid fibrils composed of the Alzheimer's disease peptide Abeta(1-40). In both reactions, the extreme N-terminal proteolytic fragment is released from fibrils as rapidly as it is from the Abeta monomer, while other proteolytic fragments are generated much more slowly. Furthermore, aggregated material isolated by centrifugation of intermediate digestion time points from both proteases contains, in addition to full-length material, peptides that possess mature C-termini but truncated N-termini. These data strongly suggest that the N-terminal region of Abeta is not involved in the beta-sheet network of the amyloid fibril, while the C-terminus is essentially completely engaged in protective-presumably beta-sheet-structure. In both digests, release of the extreme N-terminal fragments of Abeta(1-40) reaches plateau values corresponding to about 80% of the total available Abeta. This suggests that there are two classes of peptides in the fibril: while the majority of Abeta molecules have an exposed N-terminus, about 20% of the peptides have an N-terminus that is protected from proteolysis within the fibril structure. The most likely cause of this heterogeneity is the lateral association of protofilaments into the fibril structure, which would be expected to generate a unique environment for those Abeta N-termini located at protofilament packing interfaces and/or in the interior core region between the packed protofilaments. This suggests that the N-terminal region of Abeta, while not directly involved in the beta-sheet network of the fibril, may contribute to fibril stability by participating in protofilament packing.
尽管淀粉样蛋白纤维的总体形态已得到较好的理解，但对于组成多肽在淀粉样蛋白折叠基序中如何折叠却知之甚少。在本文报道的实验中，我们使用胰蛋白酶和胰凝乳蛋白酶对由阿尔茨海默病肽β-淀粉样蛋白（1-40）组成的合成淀粉样蛋白纤维进行了有限的蛋白水解研究。在这两种反应中，极端N端蛋白水解片段从纤维中释放的速度与从β-淀粉样蛋白单体中释放的速度一样快，而其他蛋白水解片段的产生则要慢得多。此外，通过离心两种蛋白酶中间消化时间点分离得到的聚集物，除了全长物质外，还含有具有成熟C端但N端截短的肽段。这些数据强烈表明，β-淀粉样蛋白的N端区域不参与淀粉样蛋白纤维的β-折叠网络，而C端基本上完全参与了保护性的——推测是β-折叠结构。在两种消化反应中，β-淀粉样蛋白（1-40）极端N端片段的释放达到平台值，约占总可用β-淀粉样蛋白的80%。这表明纤维中有两类肽：虽然大多数β-淀粉样蛋白分子的N端是暴露的，但约20%的肽的N端在纤维结构中受到保护，免受蛋白水解。这种异质性最可能的原因是原纤维横向缔合形成纤维结构，这预计会为位于原纤维堆积界面和/或堆积原纤维之间内部核心区域的那些β-淀粉样蛋白N端创造一个独特的环境。这表明β-淀粉样蛋白的N端区域虽然不直接参与纤维的β-折叠网络，但可能通过参与原纤维堆积来促进纤维的稳定性。
