Structural Polymorphism in a Self-Assembled Tri-Aromatic Peptide System

Abstract

Self-assembly is a process of key importance in natural systems and in nanotechnology. Peptides are attractive building blocks due to their relative facile synthesis, biocompatibility, and other unique properties. Diphenylalanine (FF) and its derivatives are known to form nanostructures of various architectures and interesting and varied characteristics. The larger triphenylalanine peptide (FFF) was found to self-assemble as efficiently as FF, forming related but distinct architectures of plate-like and spherical nanostructures. Here, to understand the effect of triaromatic systems on the self-assembly process, we examined carboxybenzyl-protected diphenylalanine (z-FF) as a minimal model for such an arrangement. We explored different self-assembly conditions by changing solvent compositions and peptide concentrations, generating a phase diagram for the assemblies. We discovered that z-FF can form a variety of structures, including nanowires, fibers, nanospheres, and nanotoroids, the latter were previously observed only in considerably larger or co-assembly systems. Secondary structure analysis revealed that all assemblies possessed a β-sheet conformation. Additionally, in solvent combinations with high water ratios, z-FF formed rigid and self-healing hydrogels. X-ray crystallography revealed a "wishbone" structure, in which z-FF dimers are linked by hydrogen bonds mediated by methanol molecules, with a 2-fold screw symmetry along the c-axis. All-atom molecular dynamics (MD) simulations revealed conformations similar to the crystal structure. Coarse-grained MD simulated the assembly of the peptide into either fibers or spheres in different solvent systems, consistent with the experimental results. This work thus expands the building block library for the fabrication of nanostructures by peptide self-assembly.

Keywords: diphenylalanine; peptide nanotubes; self-assembly; self-assembly mechanism; structural polymorphism; toroids.

Figure 1

Structural analysis of z-FF assemblies. (a) Chemical structure of the z-FF molecule. (b-f) HR-SEM imaging of z-FF assemblies formed in (b) ethanol stock, 5 mg/mL, 50% ethanol; (c) ethanol stock, 0.5 mg/mL, 100% ethanol; (d) HFIP stock, 10 mg/mL, 50% ethanol; (e) methanol stock, 5 mg/mL, 50% methanol; and (f) methanol stock, 5 mg/mL, 25% methanol. Scale bar: 1 μm.

Figure 2

Secondary structure analysis of z-FF assemblies. (a) FTIR spectra of z-FF at representative conditions. (b) CD spectra of z-FF at representative conditions.

Figure 3

Rheological properties of z-FF hydrogels. (a) z-FF gels. (b) Time sweep rheology showing G′ and G″ (solid and dashed lines, respectively) of 10 and 5 mg/mL gels (black and gray lines, respectively). (c) Time sweep for thixotropic properties.

Figure 4

z-FF crystal structure. (a) A z-FF dimer with H-bonds bridged by methanol. (b) “Wishbone” structure stacking between unit cells. (c) Schematic representation of the “wishbone” structure. (d) View down the c-axis, rotated by 10°, of a high-order assembly. A 2-fold screw axis symmetry element is seen in light blue.

Figure 5

All-atom MD simulations of the self-assembly of 20, 27, and 50 z-FF molecules in methanol (left panel, middle panel, and right panel, respectively). All simulations started from disordered states. (a) The probability density function (PDF) of end-to-end distance of z-FF molecules. The structures of two representative z-FF peptides with their end-to-end distance located at the two peaks are also shown. (b) The free energy surface of z-FF oligomers as a function of the centroid distance and the angle between two aromatic rings of intermolecular Z1−Z1, F2−F2, and F3−F3 pairs.

Figure 6

Multiple microsecond CG-MD simulations of the self-assembly of 300, 600, and 900 z-FF molecules in ethanol and an ethanol−water mixture. All simulations were initiated from disordered states. (a) The z-FF assemblies generated at t = 1 μs of CG-MD simulations in ethanol (upper panel) and in an ethanol−water mixture (lower panel). (b,c) Representative CG-MD self-assembly process of 900 z-FF molecules leading to (b) the formation of spheres in ethanol and (c) the formation of fibrils in an ethanol−water mixture. (d) The free energy surface of z-FF assemblies as a function of the centroid distance and the angle between two aromatic rings of inter/intra-molecular ZZ, ZF, and FF pairs. A parallel z-FF dimer from the z-FF spheres/fibrils in the bottom right shows the parallel/T-shaped stacking pattern between two inter/intra-molecular FF aromatic rings. (e,f) Normalized solvent accessible surface area (SASA) of z-FF assemblies in (e) ethanol and (f) an ethanol−water mixture as a function of simulation time. The SASAs include total, hydrophobic, and hydrophilic SASA.