Formation of peptide-based oligomers in dimethylsulfoxide: identifying the precursor of fibril formation.

The well-studied dipeptide fluorenylmethyloxycarbonyl-di-phenylalanine (FmocFF) forms a rigid hydrogel upon dissolving in dimethylsulfoxide (DMSO) and dilution in H2O. Here, we explored the pre-aggregation of the peptide in pure DMSO by vibrational spectroscopies, X-ray powder diffraction and dynamic light scattering. Our results show an equilibrium between a dominant population of amorphous oligomers (on a length scale of 2 nm) and a small number of protofibrils/fibrils (on a length scale of 30 nm in the centimolar and of 200 nm in the sub-molar region). To probe the mechanism underlying the formation of these protofilaments, we measured the 1H-NMR, IR and visible Raman spectra of DMSO containing different FmocFF concentrations, ranging between 10 and 300 mM. Our data reveal that interpeptide hydrogen bonding leads to the self-assembly of FmocFF in the centimolar region, while π-π stacking between Fmoc-groups is observed above 100 mM. The high 3J(HNHCα) coupling constant of the N-terminal amide proton indicates that the Fmoc end-cap of the peptide locks the N-terminal residue into a conformational ensemble centered at a φ-value of ca. -120°, which corresponds to a parallel ß-sheet type conformation. The 3J(HNHCα) coupling constant of the C-terminal residue is indicative of a polyproline II (pPII)/ßt mixture. Our results suggest that the gelation of FmocFF caused by the addition of a small amount of water to DMSO mixtures is facilitated by the formation of disordered protofibrils in pure DMSO.

Exploring the gel phase of cationic glycylalanylglycine in ethanol/water. II. Spectroscopic, kinetic and thermodynamic studies.

HYPOTHESIS: Recently, we reported a three-dimensional phase diagram for the gelation of cationic tripeptide glycylalanylglycine (GAG) in water-ethanol mixtures. We showed that the gel strength reaches an optimum for a peptide concentration of 200 mM and ethanol/water mixtures with ca. 55-60 mol% ethanol. An increase of the ethanol fraction causes a substantial upshift of the gel's softening temperature which is indicative of a reduced peptide solubility. We expect the formation of long crystalline fibrils which form the sample spanning network of the gel phase to precede the gelation process and that the fibril microstructure depends on the rate and concentration of peptide. EXPERIMENTS: We used UV circular dichroism (UVCD) spectroscopy to probe the kinetics of GAG fibril formation as a function of peptide concentration and ethanol fraction. We provide experimental evidence for the notion that the utilized CD signal reflects the three-dimensional assembly of peptides rather than a two-dimensional sheet structure. UVCD was also used to probe the melting of GAG fibrils with increasing temperature. FTIR and vibrational circular dichroism (VCD) spectroscopy were employed to characterize the structure of sheets with which the observed fibrils were formed. FINDINGS: Fibrilization and gelation kinetics occur on a very similar time scale for very short gelation times (<7 min) observed at high peptide concentrations and/or ethanol fractions. Otherwise, gelation proceeds significantly slower than fibrilization. The trends in the UVCD spectral response parallel the trends in the storage modulus as a function of peptide concentration and ethanol fraction. IR and VCD profiles of amide I' reveal that fibril structure and the respective chirality are both affected by peptide concentration and solvent composition. At high ethanol fractions, the VCD changes its sign suggesting a conversion from phase II to phase I. Generally, the latter is obtained only at temperatures below 15 °C. Altogether, our results reveal how GAG fibrilization and gelation are interrelated and how the gel properties can be tuned by changing the composition of the ternary GAG/water/ethanol mixture.