Influence of central sidechain on self-assembly of glycine-x-glycine peptides.

Low molecular weight gelators (LMWGs) are the subject of intense research for a range of biomedical and engineering applications. Peptides are a special class of LMWG, which offer infinite sequence possibilities and, therefore, engineered properties. This work examines the propensity of the GxG peptide family, where x denotes a guest residue, to self-assemble into fibril networks via changes in pH and ethanol concentration. These triggers for gelation are motivated by recent work on GHG and GAG, which unexpectedly self-assemble into centimeter long fibril networks with unique rheological properties. The propensity of GxG peptides to self-assemble, and the physical and chemical properties of the self-assembled structures are characterized by microscopy, spectroscopy, rheology, and X-ray diffraction. Interestingly, we show that the number, length, size, and morphology of the crystalline self-assembled aggregates depend significantly on the x-residue chemistry and the solution conditions, i.e. pH, temperature, peptide concentration, etc. The different x-residues allow us to probe the importance of different peptide interactions, e.g. π-π stacking, hydrogen bonding, and hydrophobicity, on the formation of fibrils. We conclude that fibril formation requires π-π stacking interactions in pure water, while hydrogen bonding can form fibrils in the presence of ethanol-water solutions. These results validate and support theoretical arguments on the propensity for self-assembly and leads to a better understanding of the relationship between peptide chemistry and fibril self-assembly. Overall, GxG peptides constitute a unique family of peptides, whose characterization will aid in advancing our understanding of self-assembly driving forces for fibril formation in peptide systems.

Forbidden Secondary Structures Found in Gel-Forming Fibrils of Glycylphenylalanylglycine.

The zwitterionic l-tripeptide glycylphenylalanylglycine self-assembles into very long crystalline fibrils in an aqueous solution, which causes the formation of an exceptionally strong gel phase (G' ∼ 5 × 106 Pa). The Rietveld refinement analysis of its powder X-ray diffraction (PXRD) pattern reveals a unit cell with four peptides forming a P212121 space group and adopting an inverse polyproline II conformation, that is, a right-handed helical structure that occupies the "forbidden" region of the Ramachandran plot. This unusual structure is stabilized by a plethora of intermolecular interactions facilitated by the large number of different functional groups of the unblocked tripeptide. Comparisons of simulated and experimental Fourier transform infrared and vibrational circular dichroism (VCD) amide I' profiles corroborate the PXRD structure. Our experimental setup reduces the sample to a quasi-two-dimensional network of fibrils. We exploited the influence of this reduced dimensionality on the amide I VCD to identify the main fibril axis. We demonstrate that PXRD, vibrational spectroscopy, and amide I simulations provide a powerful toolset for secondary structure and fibril axis determination.

Concentration Dependence of a Hydrogel Phase Formed by the Deprotonation of the Imidazole Side Chain of Glycylhistidylglycine.

Upon deprotonation of its imidazole group at ∼pH 6, the unblocked tripeptide glycylhistidylglycine (GHG) self-assembles into very long crystalline fibrils on a 10-1000 µm scale which are capable of forming a volume spanning network, that is, hydrogel. The critical peptide concentration for self-assembly at a pH of 6 lies between 50 and 60 mM. The fraction of peptides that self-assemble into fibrils depends on the concentration of deprotonated GHG. While IR spectra seem to indicate the formation of fibrils with standard amyloid fibril ß-sheet structures, vibrational circular dichroism spectra show a strongly enhanced amide I' signal, suggesting that the formed fibrils exhibit significant chirality. The fibril chirality appears to be a function of peptide concentration. Rheological measurements reveal that the rate of gelation is concentration-dependent and that there is an optimum gel strength at intermediate peptide concentrations of ca. 175 mM. This paper outlines the unique properties of the GHG gel phase which is underlain by a surprisingly dense fibril network with an exceptionally strong modulus that make them potential additives for biomedical applications.

The impact of thermal history on the structure of glycylalanylglycine ethanol/water gels.

This work revisits several open questions regarding the mechanisms of GAG fibril formation and structure as a function of temperature. The authors recently hypothesized that there is a solubility limit of GAG in ethanol/water that induces self-assembly. In other words, not all peptides can participate in fibrillization and some fraction is still soluble in solution. We show via FTIR spectroscopy that, indeed, free peptides are still present in solution after fibril formation, strongly supporting the solubility model. Furthermore, previous work showed GAG self-assembled into right-handed (phase I) or left-handed (phase II) chiral structures depending on temperature. In this study, we analyze the crystalline structure of phase I and II gels via WAXS and SAXS to compare their crystalline structures and order. Rheological measurements were used to investigate the response of the fibrillar network to temperature. They reveal that the ability of the peptide to self-assemble depends on the solubility at a given temperature and not on thermal history. Furthermore, the gel softening point, the linear viscoelastic gel microstructure, and relaxation spectrum are very similar between phase I and phase II. Overall, the temperature only affects the chirality of the fibrils and the formation kinetics.

The tripeptide GHG as an unexpected hydrogelator triggered by imidazole deprotonation.

The tripeptide glycyl-histidyl-glycine (GHG) self-assembles into long, crystalline fibrils forming a strong hydrogel (G'∼ 50 kPa) above a critical concentration of 40 mM upon the deprotonation of its imidazole group. Spectroscopic data reveal a mixture of helically twisted ß-sheets and monomers to coexist in the gel phase.

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.

Exploring the gel phase of cationic glycylalanylglycine in ethanol/water. I. Rheology and microscopy studies.

HYPOTHESIS: The cationic tripeptide glycylalanylglycine (GAG) self-assembles into long, thick crystalline fibrils in an ethanol/water solution. At sufficiently high concentrations, the fibrils form a volume spanning hydrogel network. We report an extensive rheology and microscopy-based study regarding the self-assembly of GAG in ethanol/water solutions to understand the conditions for fibril formation as well as the thermal stability for future developments of this material. EXPERIMENTS: By systematically varying GAG concentration and ethanol fraction, we observe a two-dimensional fibril aggregate phase diagram. Microscopy studies shed light on the shape and size of fibrils as well as the macroscopic packing depending on conditions. The kinetics and evolution of the macroscopic fibril microstructure was investigated using rheology. FINDINGS: The mechanism of fibril formation is put into the context of a solubility framework, where ethanol reduces peptide solubility and induces self-assembly. The rate of fibril formation and strength of the gel can be controlled by peptide concentration and ethanol fraction. The faster rate of fibril formation leads to inhomogeneous packing of fibrils denoted by discrete dense fibril clusters. The solubility of the fibrils can be manipulated by temperature making the gel thermo-switchable, a property of interest for biomedical systems.

Exploring the thermal reversibility and tunability of a low molecular weight gelator using vibrational and electronic spectroscopy and rheology.

Cationic glycylalanylglycine (GAG) self-assembles into a gel in a 55 mol% ethanol/45 mol% water mixture. The gel exhibits a network of crystalline fibrils grown to lengths on a 10-4-10-5 m scale (Farrel et al., Soft Matter, 2016, 12, 6096-6110). Rheological data are indicative of a rather strong gel with storage moduli in the 10 kPa regime. Spectroscopic data revealed the existence of two gel phases; one forms below T = 15 °C (phase I) while the other one forms in a temperature range between 15 °C and the melting temperature of ca. 35 °C (phase II). We explored the reformation of the cationic GAG gel in 55 mol% ethanol/45 mol% water after thermal annealing by spectroscopic and rheological means. Our data reveal that even a short residence time of 5 minutes in the sol phase at 50 °C produced a delay of the gelation process and a gel of lesser strength. These observations suggest that the residence time at the annealing temperature can be used to adjust the strength of both gel phases. Our spectroscopic data show that the annealing process does not change the chirality of peptide fibrils in the two gel phases and that the initial aggregation state of the reformation process is by far more ordered for phase I than it is for phase II. In the gel phases of GAG/ethanol/water mixtures, ethanol seems to function as a sort of catalyst that enables the self-assembly of the peptide in spite of its low intrinsic propensity for aggregation.