Laminated Morphology of Nontwisting β-Sheet Fibrils Constructed via Peptide Self-Assembly

A synthetic peptide has been de novo designed that self-assembles into β-sheet fibrils exhibiting a nontwisted, stacked morphology. The stacked morphology is constituted by 2.5 nm wide filaments that laterally associate to form flat fibril laminates exceeding 50 nm in width and micrometers in length. The height of each fibril is limited to the length of exactly one peptide monomer in an extended β-strand conformation, approximately 7 nm. Once assembled, these highly ordered, 2-D structures are stable over a wide range of pH and temperature and exhibit characteristics similar to those of amyloid fibrils. Furthermore, the rate of assembly and degree of fibril lamination can be controlled with kinetic parameters of pH and temperature. Finally, the presence of a diproline peptide between two β-sheet-forming strands in the peptide sequence is demonstrated to be an important factor in promoting the nontwisting, laminated fibril morphology

“Click” Chemistry in a Supramolecular Environment: Stabilization of Organogels by Copper(I)-Catalyzed Azide−Alkyne [3 + 2] Cycloaddition

Organogels are thermoreversible, viscoelastic (soft) materials consisting of low molecular weight compounds which self-assemble into fibers, often of micrometer lengths and nanometer diameters. The installation of terminal azide and alkyne functional groups on the end of a standard alkylamide-based organogelator was found to cause a modest disruption in the gelation properties of the molecule. Cross-linking of those groups by the copper(I)-catalyzed azide−alkyne cycloaddition reaction produced thermoreversible materials of substantially greater gelation temperatures and mechanical rigidity. These results highlight the ability of azides and alkynesparticipants in the most commonly used “click” reactionto function as innocuous precursors to meaningful covalent interactions in materials science

Reversible Stiffening Transition in β-Hairpin Hydrogels Induced by Ion Complexation

We have previously shown that properly designed lysine and valine-rich peptides undergo a random coil to β-hairpin transition followed by intermolecular self-assembly into a fibrillar hydrogel network only after the peptide solutions are heated above the intramolecular folding transition temperature. Here we report that these hydrogels also undergo a stiffening transition as they are cooled below a critical temperature only when boric acid is used to buffer the peptide solution. This stiffening transition is characterized by rheology, dynamic light scattering, and small angle neutron scattering. Rheological measurements show that the stiffening transition causes an increase in the hydrogel storage modulus (G‘) by as much as 1 order of magnitude and is completely reversible on subsequently raising the temperature. Although this reversible transition exhibits rheological properties that are similar to polyol/borax solutions, the underlying mechanism does not involve hydroxyl−borate complexation. The stiffening transition is mainly caused by the interactions between lysine and boric acid/borate anion and is not driven by the changes in the secondary structure of the β-hairpin peptide. Addition of glucose to boric acid and peptide solution disrupts the stiffening transition due to competitive glucose−borate complexation

Sequence-Dependent Gelation Kinetics of β-Hairpin Peptide Hydrogels

The gelation kinetics of four β-hairpin oligopeptides that have been designed to exhibit responsive behavior to changes in environmental conditions, such as pH, ionic strength and temperature, are characterized using multiple particle tracking microrheology and circular dichroism (CD) spectroscopy. The peptides, predominantly an alternating sequence of valine and lysine residues, differ by a point substitution of a single amino acid near a type II′ β-turn sequence. The rate of gelation becomes faster for point substitutions which reduce the total charge of the peptide. Similarly, increasing the ionic strength reduces or screens intra- and intermolecular electrostatic repulsions, again leading to faster gelation kinetics. CD measurements show that the concentration of folded peptide at the gel point decreases as the gelation kinetics become slower, possibly indicating a relationship between the assembly rate and the resulting gel microstructure. Finally, a model is developed based on the electrostatic barrier to peptide folding and association which agrees semiquantitatively with the microrheology results. This represents a first step toward understanding the role of peptide charge and physicochemical conditions in the self-assembly of these peptide hydrogelators

Tuning the pH Responsiveness of β-Hairpin Peptide Folding, Self-Assembly, and Hydrogel Material Formation

A design strategy to control the thermally triggered folding, self-assembly, and subsequent hydrogelation of amphiphilic β-hairpin peptides in a pH-dependent manner is presented. Point substitutions of the lysine residues of the self-assembling peptide MAX1 were made to alter the net charge of the peptide. In turn, the electrostatic nature of the peptide directly influences the solution pH at which thermally triggered hydrogelation is permitted. CD spectroscopy and oscillatory rheology show that peptides of lower net positive charge are capable of folding and assembling into hydrogel material at lower values of pH at a given temperature. The pH sensitive folding and assembling behavior is not only dependent on the net peptide charge, but also on the exact position of substitution within the peptide sequence. TEM shows that these peptides self-assemble into hydrogels that are composed of well-defined fibrils with nonlaminated morphologies. TEM also indicates that fibril morphology is not influenced by making these sequence changes on the hydrophilic face of the hairpins. Rheology shows that the ultimate mechanical rigidity of these peptide hydrogels is dependent on the rate of folding and self-assembly. Peptides that fold and assemble faster afford more rigid gels. Ultimately, this design strategy yielded a peptide MAX1(K15E) that is capable of undergoing thermally triggered hydrogelation at physiological buffer conditions (pH 7.4, 150 NaCl, 37 °C)

Thermally Reversible Hydrogels via Intramolecular Folding and Consequent Self-Assembly of a <i>de Novo</i> Designed Peptide

A small de novo designed peptide (MAX3) is described that exhibits complete thermoreversible self-assembly into a hydrogel network. Importantly, a prerequisite to hydrogelation is that the peptide must first fold into a conformation conducive to self-assembly. At ambient temperature, MAX3 is unfolded, resulting in a low viscosity aqueous solution. On increasing the temperature, the peptide undergoes a unimolecular folding event, affording an amphiphilic β-hairpin that consequently self-assembles into a hydrogel network. Increasing the temperature serves to dehydrate the nonpolar residues of the unfolded peptide and trigger folding via hydrophobic collapse. Cooling the resultant hydrogel results in β-hairpin unfolding and consequent complete dissolution of the hydrogel. The temperature at which folding and consequent self-assembly into a rigid hydrogel occur can be tuned by altering the hydrophobicity of the peptides

Responsive Hydrogels from the Intramolecular Folding and Self-Assembly of a Designed Peptide

A general peptide design is presented that links the pH-dependent intramolecular folding of β-hairpin peptides to their propensity to self-assemble, affording hydrogels rich in β-sheet. Chemical responsiveness has been specifically engineered into the material by linking intramolecular folding to changes in solution pH, and mechanical responsiveness, by linking hydrogelation to self-assembly. Circular dichroic and infrared spectroscopies show that at low pH individual peptides are unstructured, affording a low-viscosity aqueous solution. Under basic conditions, intramolecular folding takes place, affording amphiphilic β-hairpins that intermolecularly self-assemble. Rheology shows that the resulting hydrogel is rigid but is shear-thinning. However, quick mechanical strength recovery after cessation of shear is observed due to the inherent self-assembled nature of the scaffold. Characterization of the gelation process, from the molecular level up through the macroscopic properties of the material, suggests that by linking the intramolecular folding of small designed peptides to their ability to self-assemble, responsive materials can be prepared. Cryo-transmission electron and laser scanning confocal microscopies reveal a water-filled porous scaffold on both the nano- and microscale. The environmental responsiveness, morphology, and peptidic nature make this hydrogel a possible material candidate for biomedical and engineering technology

Light-Activated Hydrogel Formation via the Triggered Folding and Self-Assembly of a Designed Peptide

Photopolymerization can be used to construct materials with precise temporal and spatial resolution. Applications such as tissue engineering, drug delivery, the fabrication of microfluidic devices and the preparation of high-density cell arrays employ hydrogel materials that are often prepared by this technique. Current photopolymerization strategies used to prepare hydrogels employ photoinitiators, many of which are cytotoxic and require large macromolecular precursors that need to be functionalized with moieties capable of undergoing radical cross-linking reactions. We have developed a simple light-activated hydrogelation system that employs a designed peptide whose ability to self-assemble into hydrogel material is dependent on its intramolecular folded conformational state. An iterative design strategy afforded MAX7CNB, a photocaged peptide that, when dissolved in aqueous medium, remains unfolded and unable to self-assemble; a 2 wt % solution of freely soluble unfolded peptide is stable to ambient light and has the viscosity of water. Irradiation of the solution (260 < λ < 360 nm) releases the photocage and triggers peptide folding to produce amphiphilic β-hairpins that self-assemble into viscoelastic hydrogel material. Circular dichroic (CD) spectroscopy supports this folding and self-assembly mechanism, and oscillatory rheology shows that the resulting hydrogel is mechanically rigid (G‘ = 1000 Pa). Laser scanning confocal microscopy imaging of NIH 3T3 fibroblasts seeded onto the gel indicates that the gel surface is noncytotoxic, conducive to cell adhesion, and allows cell migration. Lastly, thymidine incorporation assays show that cells seeded onto decaged hydrogel proliferate at a rate equivalent to cells seeded onto a tissue culture-treated polystyrene control surface

Curvature-Coupled Hydration of Semicrystalline Polymer Amphiphiles Yields flexible Worm Micelles but Favors Rigid Vesicles: Polycaprolactone-Based Block Copolymers

Crystallization processes are in general sensitive to detailed conditions, but the present understanding of underlying mechanisms is insufficient. A crystallizable chain within a diblock copolymer assembly, for example, is expected to couple curvature to crystallization and thereby impact rigidity as well as preferred morphology, and yet the effects on dispersed phases have remained unclear. The hydrophobic polymer polycaprolactone (PCL) is semicrystalline in bulk (Tm = 60 °C) and is shown here to generate flexible worm micelles or rigid vesicles in water from several dozen poly(ethylene oxide)-based diblocks (PEO−PCL). Despite the fact that “worms” have a mean curvature between that of vesicles and spherical micelles, “worms” are seen only within a narrow, process-dependent wedge of morphological phase space that is deep within the vesicle phase. Fluorescence imaging shows worms are predominantly in one of two states − either entirely flexible with dynamic thermal undulations or fully rigid; only a few worms appear rigid at room temperature (T ≪ Tm), indicating suppression of crystallization by both curvature and PCL hydration. Worm rigidification, which depends on molecular weight, is also prevented by copolymerization of caprolactone with just 10% racemic lactide that otherwise has little impact on bulk crystallinity. In contrast to worms, vesicles of PEO−PCL are always rigid and typically leaky. Defects between crystallite domains induce dislocation-roughening with focal leakiness although select PEO−PCLwhich classical surfactant arguments would predict make wormsyield vesicles that retain encapsulant and appear smooth, suggesting a gel or glassy state. Hydration in dispersion thus tends to selectively soften high curvature microphases