Self-Assembly of Aromatic Amino Acid Enantiomers into Supramolecular Materials of High Rigidity

Most natural biomolecules may exist in either of two enantiomeric forms. Although in nature, amino acid biopolymers are characterized by l-type homochirality, incorporation of d-amino acids in the design of self-assembling peptide motifs has been shown to significantly alter enzyme stability, conformation, self-assembly behavior, cytotoxicity, and even therapeutic activity. However, while functional metabolite assemblies are ubiquitous throughout nature and play numerous important roles including physiological, structural, or catalytic functions, the effect of chirality on the self-assembly nature and function of single amino acids is not yet explored. Herein, we investigated the self-assembly mechanism of amyloid-like structure formation by two aromatic amino acids, phenylalanine (Phe) and tryptophan (Trp), both previously found as extremely important for the nucleation and self-assembly of aggregation-prone peptide regions into functional structures. Employing d-enantiomers, we demonstrate the critical role that amino acid chirality plays in their self-assembly process. The kinetics and morphology of pure enantiomers is completely altered upon their coassembly, allowing to fabricate different nanostructures that are mechanically more robust. Using diverse experimental techniques, we reveal the different molecular arrangement and self-assembly mechanism of the dl-racemic mixtures that resulted in the formation of advanced supramolecular materials. This study provides a simple yet sophisticated engineering model for the fabrication of attractive materials with bionanotechnological applications.


Supplementary Note:
We simulated pure left-handed enantiomer crystal of 12 x 12 x 12 amino acids to further examine a possible directionality in crystal growth of Phe. On each facet of the crystal, the central 10 x 10 amino acids in the first layer were free and amino acids in deeper layers were frozen (Methods).
These facets ( Figure S14) were denoted as 1) "top bilayer plane" (facet 1 in Figure 3 and colored yellow in Figure S13), it contacts the solvent through apolar aromatic side groups and when extended it forms additional bilayers, 2) "parallel to zipper plane" (facet 2 in Figure 3 and colored green in Figure S13), its normal vector is parallel to the aromatic zipper and when it is extended the aromatic zipper lengthens, and 3) "orthogonal to zipper plane" (facet 3 in Figure 3 and colored orange in Figure S13), its normal vector is at an angle with the aromatic zipper's direction (angle depends on unit cell parameters) and is orthogonal to a vector normal to the zwitterion layer. The results in Figure S14 reveal that Phe amino acids in the left handed crystal have significant mobility in facets both parallel and orthogonal to the aromatic zipper (facet 2 and 3 in Figure 3, respectively).
In order to quantify the strength of the Phe binding on each facet, we calculated nonbonding interaction energies of free amino acids 1) with other amino acids on the same facet (Table S1) and 2) with the remaining amino acids in the crystal (Table S2). In the left handed crystal, free amino acids in the top bilayer have strong interactions with other mobile amino acids as well as with the constrained amino acids, due to hydrogen bonding networks through zwitterions. Free amino acids in the facet parallel to the aromatic zipper are most mobile due to their relatively weak interactions with the constrained amino acids in the crystal. At the same time, free amino acids in the facet orthogonal to the aromatic zipper have the strongest interactions with the constrained amino acids. They have a significant mobility, due to their weak interactions with other mobile amino acids on the same facet. Once the crystal is forming, amino acids on the facet parallel to the aromatic zipper will most likely assemble and stay intact. Amino acids on the facet orthogonal to the aromatic zipper are less likely to assemble. Once they assemble, they will bind relatively strongly to the crystal, thus contributing to the stability of the crystal, though they would not tend to propagate in this direction.
We simulated two crystals (100% L Trp, 50/50% L/D Trp) of 12 x 12 x 12 amino acids to further examine a possible directionality in crystal growth of Trp, using the same methods as with the Phe crystals. These facets ( Figure S16) had the same nomenclature as the Phe crystal, i.e. 1) "top bilayer plane" (facet 1 in Figure 7 and colored yellow in Figure S15), it contacts the solvent through apolar indole side groups and when extended it forms additional bilayers, 2) "parallel to zipper plane" (facet 2 in Figure 7 and colored green in Figure S15), its normal vector is parallel to the aromatic zipper and when it is extended the aromatic zipper lengthens, and 3) "orthogonal to zipper plane" (facet 3 in Figure 7 and colored orange in Figure S15), its normal vector is at an angle with the aromatic zipper direction (angle depends on unit cell parameters) and it is normal to the zwitterion layer. The results in Figure S16 reveal that Trp in the left handed crystal has a significant mobility only in the facet orthogonal to the aromatic zipper (facet 3 in Figure 7 (top)), whereas in the mixed enantiomer crystal Trp, has a significant mobility only in the facet parallel to the aromatic zipper (facet 2 in Figure 7, bottom). The crystals should experience slower growth on these facets. However, a pure enantiomer crystal becomes also twisted (Figure 7), which can prevent further crystallization in the folded directions.
We calculated nonbonding interaction energies of free amino acids in order to quantify the strength of Trp binding on each facet 1) with other amino acids on the same facet (Table S3) and 2) with the remaining amino acids in the crystal (Table S4). In the left handed crystal, free amino acids in the top bilayer have strong interactions with other mobile amino acids and also with the constrained amino acids, due to hydrogen bonding networks through zwitterions. At the same time, free amino acids in the facet orthogonal to the aromatic zipper have the weakest interactions with the constrained amino acids, thus they have a significant mobility. In the mixed enantiomer crystal, one side of a given aromatic zipper has one chirality and the other side has the opposite chirality. Tables S3 to S4 reveal that, due to steric effects, amino acids with the same chirality have a stronger affinity to each other, but amino acids with the opposite chirality have weaker affinities. Moreover, amino acids in the facet orthogonal to the aromatic zipper have strong interactions within the same facet and weak interactions with the remaining amino acids. The opposite is true for amino acids in the facet parallel to the aromatic zipper: they have weak affinities to other amino acids within the same facet and strong affinities to amino acids in the remainder of the crystal.     Figures (b, d) have mixed chirality within each facet, whereas figure (f) has homogenous chirality. Scale bar represents 1 nm.