Selective addition of amino acids to R(FR) 3 templated by (FE) 4

Throughout this study, we use substrate peptides (analogous to primer strands in DNA replication) that are the charge complement of their template peptide. For example, the template (FE) 4 (Ac-FEFEFEFE-NH 2 , Table 1) has a net negative charge at neutral pH, while substrate R(FR) 3 (RFRFRFR-NH 2 ) has a net positive charge and one phenylalanine residue less than the template (Fig. 1a). Additionally, the template peptides have an N-terminal acetylation and all peptides have a C-terminal amide. Both (FE) 4 and R(FR) 3 are soluble separately above 1 mM (Supplementary Fig. 1). However, a mixture of 100 μM of each peptide in 50 mM phosphate at pH 7.4 yields a precipitate that has many hallmarks of an amyloid including an intense amide C = O stretch at 1625 cm−1 in its infrared spectrum and fibril morphology in transmission electron micrographs (Supplementary Figs. 1 and 2). Phenylalanine was activated with carbonyldiimidazole (CDI) and then added in stoichiometric amounts to the R(FR) 3 /(FE) 4 aggregate or to soluble R(FR) 3 . After 18 h, the reactions were solubilized with guanidine and analyzed by reverse-phase HPLC. Surprisingly, the yield of the phenylalanine addition product (FR) 4 was much higher in the aggregated sample (51%), compared to the soluble control (2.7%; Fig. 1b). The low yield in the soluble reaction was not simply due to a non-reactive soluble peptide: we also tested four other amino acids (D, G, V and R, Fig. 1c–f) similarly activated with CDI, and R(FR) 3 reacts with aspartate to form DR(FR) 3 (33% yield) nearly as efficiently as does R(FR) 3 /(FE) 4 (35% yield, Fig. 1c). Interestingly, despite the efficiency of phenylalanine addition to the co-aggregate, double additions of phenylalanine were not detected in the stochiometric reaction. While glycine did yield double additions, its reaction with R(FR) 3 /(FE) 4 strongly favored single additions relative to soluble R(FR) 3 (Fig. 1d). Taking additionally into account the outcomes of the valine and arginine reactions (Fig. 1 and Supplementary Table 1) these results indicate that the R(FR) 3 /(FE) 4 co-aggregated amyloid is sequence-specific for the addition of hydrophobic amino acids, enhancing only single additions to the substrate.

Table 1 Selected peptides used in this study Full size table

Fig. 1 Single addition reactions with the R(FR) 3 /(FE) 4 amyloid. a Schematic model of the amyloid-templated addition reaction, depicting just three layers of the repetitive amyloid structure in which the phenylalanine is represented by squares and the arginine and aspartate by blue and red circles respectively. The blue shading highlights the sites of addition in the substrate peptide. b–f, Reverse-phase HPLC chromatograms of the addition reactions with phenylalanine (b), aspartate (c), glycine (d), valine (e) and arginine (f). The * and ** indicate the C-terminal deamidated substrate and template, respectively, that remained after purification. The reactions with just the substrate R(FR) 3 are the black traces and those with the amyloid R(FR) 3 /(FE) 4 are the red ones. The R(FR) 3 concentration was 100 μM, the (FE) 4 concentration was 130 μM and amino acids were all at 100 μM Full size image

Thus, the reaction on the amyloid is not simply enhanced by an increase in the nucleophilicity of the N-terminal amine, but is influenced by the physicochemical properties of the incoming amino acid. Indeed, in the context of the R(FR) 3 /(FE) 4 amyloid, the addition of DL-phenylalanine is highly stereoselective for the L-enantiomer (Fig. 2). In the range of phenylalanine concentrations that we tested (25–200 μM), there is a distinct dependence of the product diastereomeric excess (d.e.) on the phenylalanine concentration. This would suggest that the reaction kinetics are not simply second order with distinct rate constants for L and D additions, but rather that preceding the condensation there is a binding event, perhaps with the N-[imidazolyl-(1)-carbonyl]-phenylalanine or N-carboxyanhydride intermediate, whose affinity is in the micromolar range. The stereoselectivity of the R(FR) 3 /(FE) 4 amyloid for five additional amino acids (A, V, L, Y and W) was similarly tested and found to be consistently L-selective, concentration dependent, and yielding higher product d.e. than the soluble R(FR) 3 peptide (Supplementary Fig. 3, Supplementary Table 2). As expected for the reaction between two chiral molecules, the addition of amino acids to soluble peptide displays some stereoselectivity albeit to a much lesser extent. In fact, soluble R(FR) 3 favors the D-enantiomers of leucine, phenylalanine, tyrosine and tryptophan, such that for these amino acids the R(FR) 3 /(FE) 4 amyloid has inverted and enhanced stereoselectivity relative to soluble R(FR) 3 . The largest increases in stereoselectivity for the amyloid relative to the soluble substrate occurred for phenylalanine and valine: for example, at 200 μM amino acid (100 μM each enantiomer) the d.e. of phenylalanine addition products increased from 0% to 71% and for valine from −3 to 76%. As expected, the stereoselectivity for L-additions produces an excess of D-enantiomers in the unreacted pool of amino acid (Supplementary Fig. 4).

Fig. 2 Stereospecificity of the single additions to R(FR) 3 /(FE) 4 amyloid. Reverse-phase HPLC chromatograms of the addition reactions of DL-phenylalanine with soluble substrate R(FR) 3 (a) and amyloid R(FR) 3 /(FE) 4 (b). The R(FR) 3 concentration was 100 μM, the (FE) 4 concentration was 130 μM and amino acids were at 25 μM (black), 50 μM (red), 100 μM (blue), 200 μM (green) of each enantiomer. The D-amino acid addition products are denoted with lower case letters. The single addiction product diastereomers are connected with a dashed line to illustrate differences in the yield of the products at different concentration of amino acid. The * indicates the C-terminal deamidated substrate that remained after purification. For a qualitative comparison of the specificity of addition of other amino acids, see Supplementary Fig. 3 and Supplementary Table 2 Full size image

Kinetics of amyloid-templated additions

For an initial assessment of the reaction kinetics, we measured the time dependence of product formation at our standard conditions (100 μM activated phenylalanine) over a period of 24 h. The results depicted in Supplementary Fig. 5 show that the majority of the (FR) 4 product is formed within 30 min, with the yield peaking around 3 h and then sinking about 20%, primarily due to multiple additions. In an effort to get a more detailed understanding of the reaction mechanism we measured the early kinetics of the reaction for both R(FR) 3 /(FE) 4 and the soluble R(FR) 3 at various L- and D-phenylalanine concentrations (Supplementary Figs. 6–8). The additions of L- or D-phenylalanine to soluble peptide appear to be first order with respect to phenylalanine (Supplementary Fig. 8c, d) as would be expected for a simple uncatalyzed addition. In contrast, the reaction kinetics of the L-additions to the (FR) 4 /(FE) 4 amyloid is much faster and of greater complexity, including a burst phase (Supplementary Figs. 7 and 8a). The data fit neither a first order reaction nor a Michaelis Menten-type mechanism (assuming a turn-over of 1). This complexity is specific for L-additions, since the additions of D-phenylalanine to (FR) 4 /(FE) 4 appear to be first order with respect to phenylalanine (Supplementary Fig. 8b) (for some further details we refer the reader to the Supplementary Discussion).

Consecutive additions to amyloid-templated substrates

To test the sequence specificity and consecutive templating capability of the amyloids, we designed the shorter substrate (FR) 3 (FRFRFR-NH 2 ), now two residues shorter than the template (FE) 4 . After demonstrating that the (FR) 3 /(FE) 4 mixture also formed amyloid aggregates (Supplementary Figs. 1 and 2) we performed stoichiometric addition reactions with phenylalanine, glycine, arginine, valine, or aspartate. Instead of selecting the hydrophobic amino acids phenylalanine and valine, this amyloid specifically enhances condensation with arginine. Four times more arginine addition occurred with the (FR) 3 /(FE) 4 co-aggregate than with the (FR) 3 soluble peptide, while the other amino acids reacted at least twice as much with the soluble peptide than with the co-aggregate (Fig. 3a). Even though the amyloid diminished their addition to the substrate, phenylalanine and valine actually yielded double addition products in reactions with the amyloid, consistent with the previous result (Fig. 1, Supplementary Table 1) that the template favors a hydrophobic amino acid for this position of the substrate. In fact, in a mixture of phenylalanine and arginine (100 μM each), (FR) 3 /(FE) 4 reacted to form the sequence-specific double addition product (FR) 4 (3.4%) as well as the double phenylalanine addition product FF(FR) 3 (3.9%) while the soluble (FR) 3 yielded only single additions of one or the other amino acid: arginine (1.9%), or phenylalanine (14%) (Fig. 3b).

Fig. 3 Sequence-specific, consecutive double addition reactions with the (FR) 3 /(FE) 4 amyloid. a Product yields in addition reactions with the individual amino acids phenylalanine, valine, aspartate, arginine and glycine. The yields for the reactions without template are in light gray, with template in gray and their ratio plotted on a different scale (right axis) is in black. The double addition products are outlined with the dotted lines. b Product yields in a mixture of arginine and phenylalanine. For all reactions, the (FR) 3 concentration was 100 μM, the (FE) 4 concentration was 130 μM and individual amino acids were all at 100 μM Full size image

With a yet shorter substrate, R(FR) 2 we wanted to test the amyloid templating for three consecutive residues, however, the R(FR) 2 /(FE) 4 mixture remained soluble with no obvious amyloid signatures (Supplementary Figs. 1 and 2). However, the fact that the (FR) 3 /(FE) 4 mixture forms an amyloid implies that the single phenylalanine addition product of R(FR) 2 would aggregate with (FE) 4 . To probe the combined sequence and stereoselectivity of the soluble R(FR) 2 /(FE) 4 mixture, we added both activated DL-phenylalanine and DL-arginine (each enantiomer at 200 μM). The precipitate from this reaction was collected at 100,000 g, solubilized in guanidine and analyzed by HPLC. Of the 64 possible triple addition products, only three could be detected in the insoluble fraction: two major products L-(FR) 4 and L-FF(FR) 3 and a minor product of undetermined stereochemistry, however most likely fFF RFRFR-NH 2 in which the final addition is a D-phenylalanine. (Fig. 4b). To help identify the numerous diastereomeric reaction products, the same reactions were carried out separately with D-amino acids or L-amino acids and also individually with arginine or phenylalanine. From these results, it was apparent that in the racemic reactions, the D-amino acids have little impact on the yield of the L-products. (Fig. 4a). The reaction with R(FR) 2 alone does not yield a precipitate and the HPLC chromatogram of its reaction products has no detectable (FR) 4 .

Fig. 4 Sequence-specific and stereospecific, consecutive triple addition reactions with the R(FR) 2 /(FE) 4 mixture. Reverse-phase HPLC chromatograms of the addition reactions of a mixture of arginine and phenylalanine (a), and a mixture of DL-arginine and DL-phenylalanine (b) with substrate R(FR) 2 , in presence (red) or absence (black) of template (FE) 4 . The maximum absorbance of the peaks that go off scale are listed above the peaks and the overlaid chromatograms have been scaled to represent the same amount of the total reaction. The substrate and template concentration was 100 μM, and amino acids were all at 200 μM of each enantiomer. The sequential additions of amino acids are indicated by the curved arrows: green for phenylalanine and blue for arginine, dotted lines are for D-enantiomers and lower case letters indicate the positions of D-residues. The isotactic and sequence-specific products are highlighted in gray. The labeled products were identified by mass spectrometry and control reactions with just D-enantiomers. The numbered products, identified only by mass spectrometry (sequence and stereochemistry not identified) have the following compositions: 1. R 4 F 3 , 2. R 4 F 3 , 3. R 3 F 4 , 4. R 3 F 5 5. R 3 F 4 Full size image

Regioselectivity in the additions to (OV) 4 templated by V(DV) 4

In order to address the relevance and general feasibility of this amyloid templating mechanism as a prebiotic reaction, we also designed an analogous substrate-template peptide pair from amino acids that are more likely to have been abundant in a prebiotic setting25. The valine/aspartate template V(DV) 4 (Ac-VDVDVDVDV-NH2) and valine/ornithine substrate (OV) 4 (OVOVOVOV-NH2) are, as with the FE/FR peptides, soluble in isolation and form an amyloid when mixed (Supplementary Fig. 9), yet they have an added complexity: the four ornithine residues and the N-terminus of (OV) 4 present a total of five addition-reactive sites. Indeed, in the standard conditions with two equivalents of activated valine, addition occurs equally well at all five sites of the soluble substrate, yielding the N-terminal addition product V(OV) 4 at 5%, constituting only 15% of the single addition products. With the (OV) 4 /V(DV) 4 amyloid, the yield of V(OV) 4 is increased to 14%, constituting 65% of the single addition products (Fig. 5a). Taking into account the five potential reactive sites, the increase in N-terminal specificity in the amyloid aggregate compared to soluble peptide is about 10-fold. Since all five valine addition products are not resolved by HPLC, the elution time for V(OV) 4 was identified with an authentic sample and each HPLC peak analyzed by NMR (Supplementary Fig. 10). The yield and regioselectivity is even higher for phenylalanine for which N-terminal additions comprise 85% of single addition products (or about 23-fold more than each sidechain) with a yield of 20% (Fig. 5b).

Fig. 5 Regio-specific and stereo-specific addition reactions with the (OV) 4 /V(DV) 4 amyloid. Reverse-phase HPLC chromatograms of the addition reactions with valine (a), phenylalanine (b), DL-valine (c) and DL- phenylalanine (d). Reactions with just substrate (OV) 4 are the black traces and those with the amyloid (OV) 4 /V(DV) 4 are the red ones. The ornithine sidechain addition products are indicated with the superscript S.C. The * indicates the C-terminal deamidated substrate that remained after purification. The (OV) 4 concentration was 100 μM, the V(DV) 4 concentration was 120 μM and amino acids were all at 200 μM of each enantiomer. The addition product to the N-terminus was identified via NMR and reverse-phase HPLC analysis of an authentic V(OV) 4 control peptide (see Supplementary Fig. 10) Full size image

Robustness of the amyloid templating reaction

The resilience of amyloids under harsh conditions is one of the characteristics that makes them interesting from a prebiotic perspective. The templating mechanism described above further supports the notion of the amyloid as a molecular scaffold that could have fulfilled the catalytic and genetic requirements of an early lifeform. In fact, the (OV) 4 /V(DV) 4 amyloid, with its putative prebiotic composition, retains enhanced N-terminal addition activity from pH 5.6–8.6, and in salt concentrations from 0 to 4 M NaCl. Within these ranges, the N-terminal specificity increases with decreasing pH or increasing NaCl concentration, both at a small expense to V(OV) 4 yield (Supplementary Tables 3 and 4). The stabilizing effect of the amyloid was very pronounced in a reaction at 90 °C for 6 h in which the amyloid both retained N-terminal addition specificity and resisted hydrolysis while the soluble peptide yielded dozens of products as a result of hydrolysis, ornithine sidechain additions and multiple additions (Fig. 6).

Fig. 6 Hydrolytic resistance of the (OV) 4 /V(DV) 4 amyloid. Reverse-phase HPLC chromatograms of the addition reactions with valine carried out at 90 °C for 6 h. The reactions with just the substrate (OV) 4 are the black traces and those with the amyloid (OV) 4 /V(DV) 4 are the red ones. The (OV) 4 concentration was 100 μM, the V(DV) 4 concentration was 120 μM and valine concentration was 200 μM Full size image

While the reaction outcomes are shown to be influenced by many factors, both yield (of sequence-specific products) and specificity are consistently higher for the amyloid substrates compared to the soluble substrates. Also, in the course of the study we found that phosphate, the buffer used in all experiments except the NaCl series, is moderately inhibitory to the CDI activated additions, reducing the yields 2–3-fold at 50 mM compared to yields at 20 mM NaPO 4 ; however, it has no effect on the specificity. Of course, the equilibrium between the amyloid and soluble peptides will also affect the yield and specificity of the reactions. Under our standard aggregation conditions (100 μM substrate, 120–130 μM template, pH 7.4) we quantitated the amount of substrate that remains soluble for the three different amyloids studied. We could not detect soluble substrate peptide for the (FR/FE) amyloids, however about 3% of (OV) 4 remained soluble in a mixture with its template V(DV) 4 .

In both the R(FR) 3 /(FE) 4 and (OV) 4 /V(DV) 4 amyloid systems, multiple addition products are either absent or significantly reduced compared to their non-templated reactions. Likewise, there are double but not triple additions to the (FR) 3 /(FE) 4 amyloid. Thus, it appears that the amyloids are inherently selective for the extension of substrates with a recessed N-terminus, that is, an N-terminus that does not extend to the end of the neighboring strands in the β-sheet. Although there is no high-resolution structural data on which to build a model, the FTIR spectra do indicate parallel β-sheets for the OV/DV system and anti-parallel β-sheets for the FR/FE system (Supplementary Figs. 1, 2 and 9). Therefore, we conclude that the inherent repetitive structures of the amyloid are generally well-suited to templating polymer addition reactions.

The concept of an amyloid as template for chemical reactions is not new. Nature has, in at least one known instance, taken advantage of the repetitive cross-β structure for the templated synthesis of the pigment melanin26. Just recently, an amyloid of the small peptide Ac-KLVFFAL-NH2 was designed that achieves template-directed polymerization of 6-amino-2-naphthaldehyde7. In terms, of self-replicative templating activities, amyloids have been shown to increase the rate of condensation of two halves of an amyloidogenic peptide via native chemical ligation14,15. The presented data, building upon these findings, greatly expands the prebiotic repertoire of amyloids by demonstrating sequence-selectivity, stereoselectivity and regioselectivity at the level of amino acids. Since the sequential addition of amino acids to a peptide in a self-replicative manner is an indisputable prerequisite for life as it developed on Earth, these results are of particular interest to studies on life’s molecular origins.