Molecular structure of monomorphic peptide fibrils within a kinetically trapped hydrogel network

Abstract

Most, if not all, peptide-and protein-based hydrogels formed by self-assembly can be characterized as kinetically trapped 3D networks of fibrils. The propensity of disease-associated amyloidforming peptides and proteins to assemble into polymorphic fibrils suggests that cross-β fibrils comprising hydrogels may also be polymorphic. We use solid-state NMR to determine the molecular and supramolecular structure of MAX1, a de novo designed gelforming peptide, in its fibrillar state. We find that MAX1 adopts a β-hairpin conformation and self-assembles with high fidelity into a double-layered cross-β structure. Hairpins assemble with an in-register Syn orientation within each β-sheet layer and with an Anti orientation between layers. Surprisingly, although the MAX1 fibril network is kinetically trapped, solid-state NMR data show that fibrils within this network are monomorphic and most likely represent the thermodynamic ground state. Intermolecular interactions not available in alternative structural arrangements apparently dictate this monomorphic behavior.M AX1 is a 20-residue peptide designed de novo to fold into an amphiphilic β-hairpin that self-assembles to form a fibrillar network within a self-supporting hydrogel (1). The MAX1 gel exhibits shear thin-recovery rheological behavior (2), is cytocompatible toward mammalian cells, yet is inherently antimicrobial (3) and thus has applications in tissue engineering and drug delivery. In addition to exploring the utility of the gel, we seek to understand the mechanism of gelation, the macroscale morphology of its fibrillar network, and the underlying molecular structure of its fibrils.MAX1 contains two segments of alternating lysine and valine residues, connected by a four-residue turn-forming segment. When initially dissolved in water, electrostatic repulsions among protonated lysine sidechains lead to an ensemble of monomeric random coil conformations (1). Peptide folding and self-assembly, leading to gelation (Fig. 1), can be triggered by attenuating electrostatic repulsions, by adjusting the solution pH and/or ionic strength. Increasing the solution temperature also drives hydrophobic collapse of valine sidechains, further favoring MAX1 assembly. According to circular dichroism (1), cryo-transmission electron microscopy (TEM) (4), small-angle neutron scattering (5), and dynamic light scattering coupled with rheological measurements (4), soon after the triggering event, peptides assemble into branched clusters of β-sheet-rich, semiflexible nanofibrils throughout the solution. Individual clusters contain dangling fibril ends that grow and interpenetrate neighboring clusters as the network evolves. Multiple particle tracking microrheology shows that the time at which the fibril network percolates the entire sample volume, defining the gel point, is less than 1 min at 1% (wt/vol) peptide (6). In this mechanism of gelation, the growing fibrils become kinetically trapped in the evolving network as they percolate the sample volume. Fibrils do not precipitate, but rather ...

Beta-hairpin hydrogels as scaffolds for high-throughput drug discovery in three-dimensional cell culture

“…2D). Tagging of MAX8 with IKVAV or YIGSR, both ligands that are normally observed in laminin protein and support neuronal differentiation [13, 36], increased the proliferation of ONS-76 cells when compared to unmodified MAX8 (Fig. 2C).…”

“…Top center, folded peptide viewed looking down at folded β-hairpin. After folding, hairpins undergo hydrophobic collapse and hydrogen bonding into nanofibrils with a hydrophobic core of valine side chains and diameter of ~3 nm [13, 15]. Bottom center, a view along a fibril axis with the hydrophobic valine core.…”

“…Nanofibrillar hydrogels are well-suited as a 3D cell culture scaffold due to their similarity to the extracellular matrix; when properly designed, synthetic hydrogels can mimic the physical and biological properties of in vivo cell environments [7–12]. β-hairpin hydrogels consist of peptides that fold into β-hairpin conformation and then undergo hydrophobic collapse and hydrogen bonding into nanofibrils with a hydrophobic core [13]. We used MAX8 [14] (Fig.…”

Insights into protein misfolding and aggregation enabled by solid-state NMR spectroscopy

“…As with their naturally occurring counterparts, structural studies of these designer amyloids benefit from valuable insights enabled by ssNMR [23, 114–118]. In most cases, these amyloid- or cross-β-based materials are based on short amyloidogenic peptides that may be modified with acyl chains or by cross-linking [23, 115, 116].…”

Enzymatic Control of the Conformational Landscape of Self‐Assembling Peptides

“…[3] Solid state NMR shows that these designed peptides fold into a well-defined hairpin conformation in their self-assembled state and that hairpins are arranged within each fibril to form a bilayer cross-β structure. [4] Bilayer formation is driven by the burial and association of hydrophobic amino acid side chains within the core of the fibril forming an extremely dry interface void of water. Importantly, formation of this dry core represents the thermodynamic driving force for peptide assembly and is essential for stabilizing the folded conformation of the β-hairpin in its self-assembled state.…”