The ultrashort peptide N-fluorenylmethoxycarbonyl-phenylalanyl-phenylalanine (FmocFF) has been largely investigated due to its ability to self-assemble into fibrils (100 nm−μm scale) that can form a sample-spanning gel network. The initiation of the gelation process requires either a solvent switch (water added to dimethyl sulfoxide) or a pH-switch (alkaline to neutral) protocol, both of which ensure the solubility of the peptide as a necessary step preceding gelation. While the respective gel phases are well understood in structural and material characteristics terms the pregelation conditions are known to a lesser extent. The question we asked is to what extent the gel-forming fibrils are already partially formed, i.e., oligomers or protofibrils. Focusing on the pregelation conditions for the pH-switch method, we investigated the self-assembly of soluble FmocFF aggregates in alkaline pH by UV circular dichroism, IR, vibrational circular dichroism, and ¹H NMR spectroscopy for different peptide concentrations and more systematically as a function of temperature. The temperature dependence of the UVCD spectra of FmocFF in H₂O and D₂O revealed a complicated isotope effect that affects the peptide backbone and fluorene conformations in peptide aggregates differently. Moreover, we found that the melting of formed aggregates depends on peptide concentration in a nonmonotonic way. At 20 mM the UVCD data revealed the population of at least two different thermodynamic intermediate states, which seem to differ in terms of the relative arrangement of the fluorene moiety. The IR spectrum of this sample at room temperature indicates an antiparallel β-sheet arrangement, as suggested earlier in the literature. However, we show that this interpretation can only be valid if one invokes a nondispersive redshift of the two amide I’ bands in a locally crystalline environment. The respective vibrational circular dichroism spectrum of the amide I’ region is consistent with a left-handed helically twisted structure of the formed aggregates. A comparison of our data with spectra of the aqueous gel phase suggests that fibrils in the latter resemble the ones at alkaline pH probed by our experiments.

中文翻译：

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Fmoc-二苯丙氨酸在碱性 pH 下的有序聚集体作为原纤维形成和肽凝胶化的前体


超短肽 N-芴基甲氧基羰基-苯丙酰-苯丙氨酸 （FmocFF） 因其能够自组装成可以形成样品跨样凝胶网络的原纤维（100 nm-μm 尺度）而受到广泛研究。凝胶化过程的开始需要溶剂切换（向二甲基亚砜中加入水）或 pH 切换（碱性到中性）方案，这两者都可以确保肽的溶解度，这是凝胶化前的必要步骤。虽然在结构和材料特性方面对各自的凝胶相有很好的了解，但对预凝胶化条件的了解程度较小。我们问的问题是，形成凝胶的原纤维在多大程度上已经部分形成，即低聚物或原原纤维。着眼于 pH 切换法的预凝胶化条件，我们通过 UV 圆二色谱、IR、振动圆二色性和 ¹H NMR 波谱研究了可溶性 FmocFF 聚集体在碱性 pH 值中的自组装，针对不同的肽浓度，以及更系统地作为温度的函数。FmocFF 在 H₂O 和 D₂O 中的 UVCD 光谱的温度依赖性揭示了复杂的同位素效应，该效应对肽聚集体中的肽骨架和芴构象产生不同的影响。此外，我们发现形成的聚集体的熔化以非单调的方式取决于肽的浓度。在 20 mM 时，UVCD 数据揭示了至少两种不同热力学中间态的种群，它们似乎在芴部分的相对排列方面有所不同。该样品在室温下的红外光谱表明β片反平行排列，如文献前面所述。 然而，我们表明，只有在局部结晶环境中调用两个酰胺 I' 带的非色散红移时，这种解释才有效。酰胺 I' 区的相应振动圆二色性光谱与形成聚集体的左旋螺旋扭曲结构一致。将我们的数据与凝胶水相的光谱进行比较表明，后者中的原纤维类似于我们实验探测的碱性 pH 值下的原纤维。