Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, 1 Pasteur St., 02-093 Warsaw, Poland.
Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, 1 Pasteur St., 02-093 Warsaw, Poland; Bioinformatics Laboratory, Mossakowski Medical Research Centre, Polish Academy of Sciences, 5 Pawinskiego St., 02-106 Warsaw, Poland.
Int J Biol Macromol. 2020 May 1;150:894-903. doi: 10.1016/j.ijbiomac.2020.02.153. Epub 2020 Feb 15.
The so-called 'H-fragment' of insulin is an extremely amyloidogenic double chain peptide consisting of the N-terminal parts of A-chain and B-chain linked by a disulfide bond between Cys-7A and Cys-7B. Here, we conduct a detailed investigation of the self-association behavior of H-fragment monomers into amyloid-like fibrils using kinetic assays, infrared spectroscopy, circular dichroism (CD), atomic force microscopy (AFM) and molecular dynamics (MD) simulations. Unlike the intact predominantly α-helical insulin, H-fragment remains in a disordered state in aqueous solutions. Its aggregation accelerates with acidification of the environment leading, at pH 1.9, to the formation of thin and structurally homogenous fibrils with the infrared features typical for parallel β-sheet conformation. According to time-lapse AFM morphological analysis both secondary nucleation and fragmentation are involved in later stages of H-fibrils' self-assembly. Based on the low nucleation order (two) obtained from the global fitting of kinetic data, realistic all-atom MD simulations of pairs of interacting H-fragment monomers were subsequently carried out. The molecular self-association scenario emerging from these simulations implicates the intrinsic conformational instability of H-monomer in its tendency to aggregate and form intermolecular β-sheet structure. Our findings provide the new mechanistic context for studies of insulin misfolding and aggregation.
所谓的胰岛素“H 片段”是一种极其淀粉样的双链肽，由 A 链和 B 链的 N 端部分通过 Cys-7A 和 Cys-7B 之间的二硫键连接而成。在这里，我们使用动力学测定、红外光谱、圆二色性（CD）、原子力显微镜（AFM）和分子动力学（MD）模拟等方法，对 H 片段单体自组装成类似淀粉样纤维的行为进行了详细研究。与完整的主要为 α-螺旋的胰岛素不同，H 片段在水溶液中仍处于无序状态。其聚集随着环境的酸化而加速，在 pH 1.9 时，形成具有典型平行 β-折叠构象的红外特征的薄而结构均匀的纤维。根据时移 AFM 形态分析，在 H 纤维自组装的后期阶段，涉及二次成核和片段化。根据从动力学数据全局拟合获得的低成核顺序（两个），随后对相互作用的 H 片段单体对进行了真实的全原子 MD 模拟。这些模拟中出现的分子自组装情景表明 H-单体的固有构象不稳定性导致其聚集并形成分子间 β-折叠结构的趋势。我们的研究结果为胰岛素错误折叠和聚集的研究提供了新的机制背景。
