Ordered Aggregates\nof Fmoc-Diphenylalanine at Alkaline\npH as a Precursor of Fibril Formation and Peptide Gelation

The ultrashort peptide <i>N</i>-fluorenylmethoxycarbonyl-phenylalanyl-phenylalanine\n(FmocFF) has been largely investigated due to its ability to self-assemble\ninto fibrils (100 nm−μm scale) that can form a sample-spanning\ngel network. The initiation of the gelation process requires either\na solvent switch (water added to dimethyl sulfoxide) or a pH-switch\n(alkaline to neutral) protocol, both of which ensure the solubility\nof the peptide as a necessary step preceding gelation. While the respective\ngel phases are well understood in structural and material characteristics\nterms the pregelation conditions are known to a lesser extent. The\nquestion we asked is to what extent the gel-forming fibrils are already\npartially formed, i.e., oligomers or protofibrils. Focusing on the\npregelation conditions for the pH-switch method, we investigated the\nself-assembly of soluble FmocFF aggregates in alkaline pH by UV circular\ndichroism, IR, vibrational circular dichroism, and <sup>1</sup>H NMR\nspectroscopy for different peptide concentrations and more systematically\nas a function of temperature. The temperature dependence of the UVCD\nspectra of FmocFF in H<sub>2</sub>O and D<sub>2</sub>O revealed a\ncomplicated isotope effect that affects the peptide backbone and fluorene\nconformations in peptide aggregates differently. Moreover, we found\nthat the melting of formed aggregates depends on peptide concentration\nin a nonmonotonic way. At 20 mM the UVCD data revealed the population\nof at least two different thermodynamic intermediate states, which\nseem to differ in terms of the relative arrangement of the fluorene\nmoiety. The IR spectrum of this sample at room temperature indicates\nan antiparallel β-sheet arrangement, as suggested earlier in\nthe literature. However, we show that this interpretation can only\nbe valid if one invokes a nondispersive redshift of the two amide\nI’ bands in a locally crystalline environment. The respective\nvibrational circular dichroism spectrum of the amide I’ region\nis consistent with a left-handed helically twisted structure of the\nformed aggregates. A comparison of our data with spectra of the aqueous\ngel phase suggests that fibrils in the latter resemble the ones at\n alkaline pH probed by our experiments.