Insulin aggregation poses a significant problem in pharmacology and medicine as it occurs during prolonged storage of the hormone and in vivo at insulin injection sites. We have recently shown that dominant forces driving the self-assembly of insulin fibrils are likely to arise from intermolecular interactions involving the N-terminal segment of the A-chain (ACC_1–13). Here, we study how proline substitutions within the pilot GIVEQ sequence of this fragment affect its propensity to aggregate in both neutral and acidic environments. In a reasonable agreement with in silico prediction based on the Cordax algorithm, proline substitutions at positions 3, 4, and 5 turn out to be very effective in preventing aggregation according to thioflavin T-fluorescence-based kinetic assay, infrared spectroscopy, and atomic force microscopy (AFM). Since the valine and glutamate side chains within this segment are strongly involved in the interactions with the insulin receptor, we have focused on the possible implications of the Q → P substitution for insulin’s stability and interactions with the receptor. To this end, comparative molecular dynamics (MD) simulations of the Q5P mutant and wild-type insulin were carried out for both free and receptor-bound (site 1) monomers. The results point to a mild destabilization of the mutant vis à vis the wild-type monomer, as well as partial preservation of key contacts in the complex between Q5P insulin and the receptor. We discuss the implications of these findings in the context of the design of aggregation-resistant insulin analogues retaining hormonal activity.

中文翻译：

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从 A 链 N 末端片段衍生的高度淀粉样蛋白肽的脯氨酸扫描中获得抗聚集胰岛素设计的线索

胰岛素聚集在药理学和医学中造成了重大问题，因为它发生在激素的长期储存期间和体内胰岛素注射部位。我们最近表明，驱动胰岛素原纤维自组装的主导力量可能来自涉及 A 链 N 末端片段 (ACC _1-13 ) 的分子间相互作用。在这里，我们研究了该片段的先导 GIVEQ 序列中的脯氨酸取代如何影响其在中性和酸性环境中聚集的倾向。与基于 Cordax 算法的计算机预测合理一致，根据基于硫代黄素 T 荧光的动力学测定、红外光谱和原子力，第 3、4 和 5 位的脯氨酸取代对于防止聚集非常有效显微镜（AFM）。由于该片段内的缬氨酸和谷氨酸侧链与胰岛素受体的相互作用密切相关，因此我们重点关注 Q→P 取代对胰岛素稳定性和与受体相互作用的可能影响。为此，对 Q5P 突变体和野生型胰岛素的游离和受体结合（位点 1）单体进行了比较分子动力学 (MD) 模拟。结果表明突变体相对于野生型单体有轻微的不稳定，并且 Q5P 胰岛素和受体之间的复合物中的关键接触部分保留。我们在设计保留激素活性的抗聚集胰岛素类似物的背景下讨论了这些发现的含义。