Dec Robert, Jaworek Michel W, Dzwolak Wojciech, Winter Roland
Physical Chemistry I - Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn Street 4a, 44227 Dortmund, Germany.
Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Pasteur Street 1, 02-093 Warsaw, Poland.
J Am Chem Soc. 2023 Feb 10. doi: 10.1021/jacs.2c12611.
Disease-associated progression of protein dysfunction is typically determined by an interplay of transition pathways leading to liquid-liquid phase separation (LLPS) and amyloid fibrils. As LLPS introduces another layer of complexity into fibrillization of metastable proteins, a need for tunable model systems to study these intertwined processes has emerged. Here, we demonstrate the LLPS/fibrillization properties of a family of chimeric peptides, ACCK, in which the highly amyloidogenic fragment of insulin (ACC) is merged with oligolysine segments of various lengths (K, = 8, 16, 24, 32, 40). LLPS and fibrillization of ACCK are triggered by ATP through Coulombic interactions with K fragments. ACCK and ACCK form fibrils after a short lag phase without any evidence of LLPS. However, in the case of the three longest peptides, ATP triggers instantaneous LLPS followed by the disappearance of droplets occurring with the formation of amyloid fibrils. The kinetics of the phase transition and the stability of mature co-aggregates are highly sensitive to ionic strength, indicating that electrostatic interactions play a pivotal role in selecting the LLPS-fibrillization transition pathway. Densely packed ionic interactions that characterize ACCK-ATP fibrils render them highly sensitive to hydrostatic pressure due to solvent electrostriction, as demonstrated by infrared spectroscopy. Using atomic force microscopy imaging of rapidly frozen samples, we demonstrate that early fibrils form within single liquid droplets, starting at the droplet/bulk interface through the formation of single bent fibers. A hypothetical molecular scenario underlying the emergence of the LLPS-to-fibrils pathway in the ACCK-ATP system has been put forward.
与疾病相关的蛋白质功能障碍进展通常由导致液-液相分离（LLPS）和淀粉样原纤维的转变途径之间的相互作用决定。由于LLPS为亚稳态蛋白质的纤维化引入了另一层复杂性，因此出现了对可调节模型系统来研究这些相互交织过程的需求。在这里，我们展示了一族嵌合肽ACCK的LLPS/纤维化特性，其中胰岛素的高度淀粉样生成片段（ACC）与不同长度的寡聚赖氨酸片段（K，n = 8、16、24、32、40）融合。ACCK的LLPS和纤维化由ATP通过与K片段的库仑相互作用触发。ACCK和ACCK在短暂的延迟期后形成原纤维，没有任何LLPS的迹象。然而，对于三种最长的肽，ATP触发瞬时LLPS，随后液滴消失并形成淀粉样原纤维。相变动力学和成熟共聚物的稳定性对离子强度高度敏感，表明静电相互作用在选择LLPS-纤维化转变途径中起关键作用。如红外光谱所示，ACCK-ATP原纤维所特有的密集堆积离子相互作用使其对静水压力高度敏感。通过对快速冷冻样品的原子力显微镜成像，我们证明早期原纤维在单个液滴内形成，从液滴/本体界面开始，通过形成单根弯曲纤维。我们提出了ACCK-ATP系统中LLPS-to-原纤维途径出现的一个假设分子情景。
