Liu Chang, Dan Yoav, Yun Ji, Adler-Abramovich Lihi, Luo Jinghui
PSI Center for Life Sciences, PSI, Villigen 5232, Switzerland.
Department of Oral Biology, The Goldschleger School of Dental Medicine, Faculty of Medical & Health Sciences, Tel-Aviv University, Tel-Aviv 6997801, Israel.
ACS Nano. 2025 Apr 8;19(13):13250-13263. doi: 10.1021/acsnano.5c00433. Epub 2025 Mar 26.
Peptide self-assembly is fundamental to various biological processes and holds significant potential for nanotechnology and biomedical applications. Despite progress in understanding larger-scale assemblies, the early formation of low-molecular-weight oligomers remains poorly understood. In this study, we investigate the aggregation behavior of the self-assembling diphenylalanine (FF) peptide and its analogs. Utilizing single-nanopore analysis, we detected and characterized the low-molecular-oligomer formation of FF, --butoxycarbonyl-diphenylalanine (BocFF), fluorenylmethyloxycarbonyl-diphenylalanine (FmocFF), and fluorenylmethyloxycarbonyl-pentafluoro-phenylalanine (Fmoc-F-Phe) in real time. This approach provided detailed insights into the early stages of peptide self-assembly, revealing the dynamic behavior and formation kinetics of low-molecular-weight oligomeric species. Analysis revealed that the trimer is the key nucleus for FF, while the dimer is the primary nucleus for FmocFF and Fmoc-F-Phe aggregation, whereas both the dimer and trimer serve as nuclei for BocFF. Mass photometry was employed to track the evolution of these oligomers, revealing the transition from low- to high-molecular-weight species, thereby elucidating intermediate phases in the aggregation process. Transmission electron microscopy and Fourier transform infrared spectroscopy were further employed to characterize the final assembly states, offering high-resolution imaging of morphological structures and detailed information on secondary structures. Based on these analyses, we constructed a comprehensive graph that correlates the entire aggregation processes of the tested self-assembling peptides across multiple scales. This integrative approach provides a holistic understanding of peptide self-assembly, particularly in the formation of low-molecular-weight oligomers toward mature supramolecular structures. These findings shed light on their assembly pathways and structural properties, advancing our understanding of their assembly pathways for nanotechnology and biomedical applications.
肽的自组装是各种生物过程的基础，在纳米技术和生物医学应用中具有巨大潜力。尽管在理解更大规模的组装方面取得了进展，但低分子量寡聚物的早期形成仍知之甚少。在本研究中，我们研究了自组装二苯丙氨酸（FF）肽及其类似物的聚集行为。利用单纳米孔分析，我们实时检测并表征了FF、叔丁氧羰基 - 二苯丙氨酸（BocFF）、芴甲氧羰基 - 二苯丙氨酸（FmocFF）和芴甲氧羰基 - 五氟苯丙氨酸（Fmoc - F - Phe）的低分子寡聚物形成。这种方法为肽自组装的早期阶段提供了详细的见解，揭示了低分子量寡聚体物种的动态行为和形成动力学。分析表明，三聚体是FF的关键核，而二聚体是FmocFF和Fmoc - F - Phe聚集的主要核，而二聚体和三聚体都是BocFF的核。采用质量光度法跟踪这些寡聚物的演变，揭示从低分子量到高分子量物种的转变，从而阐明聚集过程中的中间阶段。进一步利用透射电子显微镜和傅里叶变换红外光谱来表征最终的组装状态，提供形态结构的高分辨率成像和二级结构的详细信息。基于这些分析，我们构建了一个综合图表，将测试的自组装肽在多个尺度上的整个聚集过程关联起来。这种综合方法提供了对肽自组装的全面理解，特别是在低分子量寡聚物向成熟超分子结构形成过程中。这些发现揭示了它们的组装途径和结构特性，推进了我们对其在纳米技术和生物医学应用中组装途径的理解。
