Wychowaniec Jacek K, Šrejber Martin, Zeng Niting, Smith Andrew M, Miller Aline F, Otyepka Michal, Saiani Alberto
Department of Materials, Manchester Institute of Biotechnology, School of Natural Sciences, Faculty of Science and Engineering, The University of Manchester UK.
AO Research Institute Davos Clavadelerstrasse 8 Davos 7270 Switzerland
RSC Adv. 2024 Nov 27;14(50):37419-37430. doi: 10.1039/d4ra07065h. eCollection 2024 Nov 19.
Self-assembling peptides remain persistently interesting objects for building nanostructures and further assemble into macroscopic structures, hydrogels, at sufficiently high concentrations. The modulation of self-assembling β-sheet-forming peptide sequences, with a selection from the full library of amino acids, offers unique possibility for rational tuning of the resulting nanostructured morphology and topology of the formed hydrogel networks. In the present work, we explored how a known β-sheet-disassembling amino acid, proline (P), affects the self-assembly and gelation properties of amphipathic peptides. For this purpose, we modified the backbone of a known β-sheet-forming peptide, FEFKFEFK (F8, F = phenylalanine, E = glutamic acid, and K = lysine), with P to form three sequences: FEFKPEFK (FP), FEFKPEFKF (KPE) and FEFEPKFKF (EPK). The replacement of F by P in the hydrophobic face resulted in the loss of the extended β-sheet conformation of the FP peptide and no gelation at concentration as high as 100 mg mL, compared to typical 5 mg mL concentration corresponding to F8. However, by retaining four hydrophobic phenylalanine amino acids in the sequences, hydrogels containing a partial β-sheet structure were still formed at 30 mg mL for KPE (pH 4-10) and EPK (pH 2-5). TEM, AFM, small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) revealed that KPE and EPK peptides self-assemble into nanoribbons and twisted nanofibers, respectively. Molecular dynamics confirmed that the single amino acid replacement of F by P prevented the assembly of the FP peptide with respect to the stable β-sheet-forming F8 variant. Moreover, additional prolongation by F in the KPE variant and shuffling of the polar amino acid sequence in the EPK peptide supported aggregation capabilities of both variants in forming distinct shapes of individual aggregates. Although the overall number of amino acids is the same in both KPE and EPK, their shifted charge density (, the chemical environment in which ionic groups reside) drives self-assembly into distinct nanostructures. The investigated structural changes can contribute to new material designs for biomedical applications and provide better understanding in the area of protein folding.
自组装肽一直是构建纳米结构并在足够高浓度下进一步组装成宏观结构（即水凝胶）的有趣研究对象。通过从完整的氨基酸文库中进行选择来调节形成β-折叠的自组装肽序列，为合理调控所得纳米结构的形态以及所形成水凝胶网络的拓扑结构提供了独特的可能性。在本工作中，我们探究了一种已知的能拆解β-折叠的氨基酸——脯氨酸（P），是如何影响两亲性肽的自组装和凝胶化性质的。为此，我们用P修饰了一种已知的形成β-折叠的肽FEFKFEFK（F8，F = 苯丙氨酸，E = 谷氨酸，K = 赖氨酸）的主链，形成了三个序列：FEFKPEFK（FP）、FEFKPEFKF（KPE）和FEFEPKFKF（EPK）。在疏水面上用P取代F导致FP肽失去了延伸的β-折叠构象，并且在高达100 mg/mL的浓度下都没有凝胶化，而F8对应的典型凝胶化浓度为5 mg/mL。然而，通过在序列中保留四个疏水的苯丙氨酸氨基酸，对于KPE（pH 4 - 10）和EPK（pH 2 - 5），在30 mg/mL时仍形成了含有部分β-折叠结构的水凝胶。透射电子显微镜（TEM）、原子力显微镜（AFM）、小角X射线散射（SAXS）和广角X射线散射（WAXS）表明，KPE和EPK肽分别自组装成纳米带和扭曲的纳米纤维。分子动力学证实，相对于稳定的形成β-折叠的F8变体，用P单氨基酸取代F阻止了FP肽的组装。此外，KPE变体中F的额外延长以及EPK肽中极性氨基酸序列的重排支持了这两种变体形成不同形状的单个聚集体的聚集能力。虽然KPE和EPK中的氨基酸总数相同，但它们电荷密度的变化（即离子基团所处的化学环境）驱动它们自组装成不同的纳米结构。所研究的结构变化有助于生物医学应用的新材料设计，并为蛋白质折叠领域提供更好的理解。
