Peptide-based replicators

In the synthetic oligonucleotide-based synthetic replicators discussed in the previous section, transfer of information depends on a fairly specific, well-defined pattern of molecular recognition elements. Namely, recognition is afforded by hydrogen-bonding interactions between donor and acceptor elements, encoded within each oligonucleotide sequence. Formation of a self-replicating peptide, on the other hand, necessitates that the peptide template be able to associate with smaller peptide fragments in some form of a catalytically-active complex. Oligopeptides possess an extremely rich structural lexicon, arising from the increased number of building blocks used—20 amino acids compared to four nucleotides in replicating systems based on DNA. Inter peptide recognition is determined not only by the primary sequence of amino acids in each sequence, but also by the secondary and tertiary structures governed by the interactions between those amino acids, increasing the potential challenge of designing a self-replicating peptide. The first experimental demonstration of peptide replication was reported88 _{in 1996}

by Ghadiri and co-workers. The design of the 32-residue self-replicatinga-helical pep- tide (Figure 1.13) was inspired89by the leucine zipper domain of the yeast transcription factor GCN4. This peptide replicator design exploited a simple protein folding mo- tif—ana-helical coiled-coil—distinguished by peptide sequences composed of heptad repeats, resulting in two coiled-coils wrapped around each other with a slightly left- handed, superhelical twist. The sequence of the reported peptide replicator (Figure 1.13) implements six substitutions relative to the wild type GCN4. Of particular interest is the substitution of a neutral, hydrophilic asparagine residue (position 16 in the sequence), located within the core hydrophobic region, with a hydrophobic valine residue, which enabled90 _{equilibration between a dimeric and trimeric coiled-coil structure.}

R1 L5 Q4 E7 K3 _E 6 M2 K8 K15 a d e b f c g E22 L29 L12 L19 L26 V30 V23 V16 V9 K27 E20 L13 G31 A24 A17 Y10 S14 Y21 K28 E11 C18 R25 E32 R1 L5 Q4 E7 K3 E6 M2 K8 K15 a' d' e' b' f' c' g' E22 L29 L12 L19 L26 V30 V23 V16 V9 K27 E20 L13 G31 A24 A17 Y10 S14 Y21 K28 E11 C18 R25 E32

Figure 1.13 Design of ana-helical, coiled-coil peptide capable of self-replication, reported by Ghadiri and co-workers in 1996, featuring a heptad repeat (abcdefg). Recognition between peptides and their assembly into complexes is mediated by the recognition between the hydrophobic residues at position a and d (red), and electrostatic interactions between residues at position e and g (blue). Residues b, c and f (black) are exposed to the solvent and do not contribute to the recognition. Position of the two residues required for native chemical ligation, alanine (activated as thiobenzyl ester) and cysteine, is highlighted in yellow. Figure adapted from Ref. 88.

Monomeric coiled-coil peptides are generally present as random coils in aqueous solutions. However, these peptides can adopt a completelya-helical structure, providing that a suitable template framework for directing their assembly is present. As with other minimal replicating systems, an autocatalytic peptide system built from two smaller complementary peptides, each equipped with a reactive group, needs to assemble on a peptide sequence that positions these fragments in an orientation that facilitates their re- action. In a situation where these fragments are the constituent parts of a longer template sequence, the product formed by their reaction constitutes an identical copy—opening up the possibility of self-replication (Figure 1.14a). The Ghadiri peptide described here is capable of forming both dimeric and trimeric assemblies and the authors proposed that bothTand [T·T] could potentially serve as a template in the autocatalytic pathway.

HS H2N _N H O CH3 H N S O CH3 H N S O H2N O H N CH3 H N O N H H N O SH Peptide 1 Peptide 1 Peptide 1 Peptide 2 Peptide 1 Peptide 2 (a) (b) Spontaneous Rearrangement – BnSH Hydrophobic residue Solvent-exposed residue

Residue providing electrostatic interactions E

N

Template T

Template T _fragmentsPeptide

Ligation Template duplex Ternarycomplex [E·N·T] O SBn SH O SBn SH Autocatalytic channel

Figure 1.14 (a)Schematic representation of peptide self-replication, where an electrophilic component Eand nucleophilic fragmentNreact through a bimolecular reaction to give templateT. The peptide template can assemble the respective fragments in to a ternary, catalytically-active complex [E·N·T] which facilitates the ligation between the two reactive sites onEandN. The ligation produces a template duplex, the dissociation of which is necessary in order for efficient self-replication.(b)Mechanism of the native chemical ligation reaction that leads to the formation of template. The ligation occurs between the N-terminal preactivated thioester onE, and the C-terminal cysteine located on the nucleophilic fragmentN. Figure adapted from Ref. 88.

The recognition mediating the template-directed reaction in the Ghadiri peptide was afforded through the interactions of complementary hydrophobic and electrostatic peptide surfaces. Specifically, residues at position a and d (Figure 1.13, red) within the peptide sequence drive the inter-helical recognition through hydrophobic interac- tions, playing a pivotal role in determining the stability and orientation of coiled-coil peptides. Residues in position e and g (Figure 1.13, blue) within the heptad repeat are responsible for driving the intra-component recognition through electrostatic in- teractions. Residues b, c and f (Figure 1.13, black), on the other hand, are located on the solvent exposed surface, and, thus, do not contribute to the recognition. The ligation site (Figure 1.13, yellow) is positioned on the solvent-exposed surface, in order to avoid potential interference with the hydrophobic core responsible for recogni- tion. The peptide coupling strategy exploited by Ghadiri and co-workers employed a thioester-promoted native peptide ligation (Figure 1.14b), first described91_{by Kent and}

co-workers in 1994. Peptide templateTis formed through the reaction of an N-terminal, 17-residue electrophilic fragmentE, activated as a thiobenzyl ester and a 15-residue C-terminal nucleophilic fragmentN, bearing a free cysteine residue. The native ligation reaction proceeds through an intermediate thioester (Figure 1.14b), which undergoes

intramolecular rearrangement to produce the final, more thermodynamically stable amide bond at the ligation site.

The ability of the designed coiled-coil peptide to self-replicate was established unambiguously through template-instructed kinetic experiments, where the ligation reaction was examined in the presence of increasing quantities of preformed peptide templateT, added att= 0. The replication profile displayed parabolic growth, where the initial rate of ligation correlated with the square root of the concentration of the initial template added, suggesting that replication is limited by product inhibition despite the relatively high autocatalytic efficiency (e = 500). Through kinetic fitting and simulation of the experimental data, the authors were able to establish the reaction orderpas 0.63. This reaction order is higher than the reaction order observed generally in nucleotide-based self-replicating systems, and may potentially arise from catalysis through quaternary complexes, mediated by template duplex [T·T].

By exploring conservative substitutions of the residues at the key positions within the sequence of the peptide, a and d, responsible for molecular recognition, the authors were able to demonstrate that the efficiency of the replication mechanism is extremely sensitive to the identity of the residues within the peptide sequence. In particular, the authors analysed two conservative mutations, where an alanine residue was substituted for valine (residue 9) within the heptad repeat, and instead of leucine at position d (residue 26). Despite the conservative nature of the mutations in the residues responsible for the hydrophobic interactions, these altered peptides showed no significant template- assisted catalytic activity.

Reliance of the self-replication mechanism on molecular recognition was demon- strated through two carefully designed control experiments. The reaction betweenEand Nwas examined in the presence of guanidinium hydrochloride, a chaotropic reagent, which exerts a destabilising effect on complexes in the system, hindering the ability of the system to partake in recognition-mediated reactions. This experiment showed that the concentration-time profile in the presence of such chaotropic reagent closely mirrors the reaction profile determined for the background, uncatalysed reaction, with a concomitant loss of the sigmoidal reaction profile. Furthermore, no enhancement in the rate of formation ofTwas observed in the presence of added preformed template under these conditions. The second control experiment was specifically designed to probe whether the reactions of binary complexes with the individual smaller fragments,

i.e. [T·E] withNand [T·N] withE, contribute to the production of peptideT. These

experiments employed “crippled” peptide sequences, each containing a single mutation within the hydrophobic recognition-mediating core of both peptide fragments, namely substitution of a glutamic acid residue in place of valine (position 9) and leucine (posi-

tion 26). Kinetic analyses confirmed that the addition of the mutated templates (formed by the reaction of a “crippled” and native fragment), capable of associating withEorN in to binary complexes only, afforded no enhancement in the rate of formation of the native peptide. Taken together, the authors were able to establish unambiguously that a recognition-mediated enzyme-free peptide replication is possible in systems exploiting the coiled-coil structural motif.

The initial report of a self-replicating peptide by Ghadiri and co-workers was soon followed by several other reports of peptide replicators, exploiting similar design principles. Utilising the coiled-coil helical peptide platform, Chmielewski and co- workers have reported92,93two examples of peptide systems that could be modulated through environmental control, and, thus, allowing self-replication to be turned on and off selectively. Specifically, the Chmielewski laboratory showed that self-replication in a peptide system can be tuned by pH,92 _{as well as through ionic control.}93 _The

concept of environmental control can be illustrated on the pH modulated replicator E1E2 (Figure 1.15), for example, formed by reaction of two subunits, E1 and E2, incorporating two glutamate residues (at position e and g), protonated under acidic conditions. At physiological pH, however, these glutamate residues are negatively charged, resulting in destabilisation of the coiled-coil assembly. The random coil conformation adopted by the peptide at neutral pH is incapable of supporting self- replication. Therefore, the peptide can replicate successfully only at low pH (4.0), when the glutamate residues, essential for recognition, are protonated. Satisfyingly, the authors were able to establish through template-instructed experiments that under these conditions, the catalytic efficiency is similar to that observed in the Ghadiri system (e = 900). E1 E2 E1E2 E1 E2 E1E2 [E1·E2·E1E2] Self-replication pH 7.0 pH 4.0

Figure 1.15 Design of a self-replicating peptideE1E2modulated by pH, as described by Chmielewski and co-authors. The recognition-mediated reaction processes in the system, and, thus also the formation of catalytically-active complexes, are only effective at low pH (pH = 4), conditions at which the two glutamate residues are protonated. Figure adapted from Ref. 92.

As often observed with synthetic replicating systems based oligonucleotides, the Ghadiri and Chmielewski replicating peptides, while capable of templating their own formation, suffered from significant product inhibition. In the coming years, remarkable results in overcoming product inhibition in peptide-based replicating systems have been achieved by the Chmielewski laboratory in particular. Chmielewski and co-workers have explored two strategies for increasing the efficiency of replication. In 2002, Issac and Chmielewski exploited the findings reported94,95_{in the literature showing that the}

stability of coiled-coil assemblies can be modulated by altering the length of the peptide sequence. The authors adapted96_{the sequence of theE1E2peptide described previously,}

shortening it by one heptad repeat. The modification resulted in a self-replicating peptide with a dramatically icnreased catalytic efficiency (e = 500000) and reaction orderp= 0.91. In an alternative strategy, Li and Chmielewski achieved97exponential replication by introducing a proline residue at a strategic position within the peptide sequence, thus destabilising the product duplex. The replication efficiency of the proline-containing replicator increased toe = 320000, with a reaction order similar to that observed in the shorter-sequence peptide replicator.

Both Ghadiri and Chmielewski laboratories have been successful in developing these examples of individual self-replicating peptides into more complex networks where multiple catalytic pathways and replicators operate simultaneously. Chmielewski and co- workers have combined98 _{the two environmentally-responsive self-replicating peptides}

into a single system, where both auto- and crosscatalytic cycles are active. The expanded peptide network was assembled from four shorter peptide fragments,E1,E2,K1and K2, which permitted formation of the two native peptide templates,E1E2andK1K2, and two recombinant proteins,E1K2andK1E2. These mixed templates are capable of self-associatingviaanti-parallel coiled-coils and capable also of associating with each otherviaformation of parallel coiled-coils. Kinetic analyses of the various reaction pathways showed that under neutral pH conditions, the recombinantE1K2template is produced most rapidly. Despite this preference of the system towards production of E1K2, the authors were able to selectively amplifyE1E2 product by decreasing the pH of the reaction environment to 4. Similarly, the authors were able to direct the network towards enhanced production of K1K2, by undertaking the reaction under high salt conditions (at neutral pH). Using this framework, Chmielewski and co-authors demonstrated successfully that the production of a particular peptide replicator can be amplified selectively from a mixture of reactive components by modulation of the reaction environment, such as the pH or salt concentration, providing support for the potential role of proteins in the emergence of life.

The first multicyclic network explored by Ghadiri laboratory reported99 _{an example}

of a symbiotic, mutually auto- and crosscatalytic peptide network, where two replicators were capable of templating their own formation as well as the formation of each other. In fact, as a result of the higher catalytic efficiency of the crosscatalytic pathways relative to the autocatalytic ones, both replicators were able to coexist and enhance formation of each other within the network. In further work, Ghadiri and co-workers exploited100_{the sensitivity of the coiled-coil peptide replicator framework to changes in}

the residues required for recognition in designing a dynamic peptide network capable of error-correction (Figure 1.16). T E N [E·N·T] [T·T] T T9A E N E N [E·N·T9A] [E·N·T26A] T26A [T·T26A] [T·T9A]

Ⅰ

ⅠⅠ

ⅠⅠⅠ

Figure 1.16 Schematic representation of the recognition-mediated catalytic pathways active in an error- correcting, autocratic peptide network. A mixture of peptide fragments EandN, and their single-alanine mutants,E9A_andN26A_{, (simulating spontaneous generation of errors)} results in a wild type templateT(grey cylinder) and single mutation containing templates T9A_{(red cylinder) andT}26A_{(blue cylinder). The self-organised network amplifies the} templateTselectively by subjugation of the mutant templates for the production ofT. The double mutantT9A/26A_{is not shown as it was determined to be catalytically inactive.} Figure adapted from Ref. 100.

The authors achieved selective amplification of a single peptide replicator within this simultaneously auto- and crosscatalytic system by recruitment of the mutant peptides for the synthesis of the wild type peptide,T(Figure 1.16, grey). Slow spontaneous generation of errors/mutants, as observed traditionally in biological systems over time, was simulated by formation of structurally-related mutant peptides through bimolec- ular reaction of smaller fragments incorporating mutations. In addition to the native electrophilic and nucleophilic fragments,E and N(Figure 1.16, grey), the network included their single alanine mutants,E9A_{(Figure 1.16, red) andN}26A_{(Figure 1.16,}

T, T9A _{and T}26A _{with a single mutated residue and double mutant T}9A/26A_{. Under}

neutral conditions, the reaction system showed strong preference for the formation of the mutation-free speciesT. The double mutantT9A/26A_{was shown to be completely}

catalytically-inactive, whereas the two templates incorporating a single mutation were capable of crosscatalytic activity only, directed towards the enhanced formation of the native replicatorT. Interestingly, the error-free templateTwas found to be a selfish autocatalyst, which means that the autocatalytic cycle producing T worked in con- cert with the two crosscatalytic pathways to achieve selective production ofT. Within this peptide network, the authors have demonstrated an example of a peptide network capable of exhibiting two complex phenomena simultaneously, error-correction and sequence-specific replication, with potential significance in genotype stabilisation of self-replicating molecules.

The world as we know it is homochiral, yet, the origins of this biological homochi- rality have yet to be established and are a source101–104 _{of ongoing debate. Explor-}

ing the possible role of peptide replicators in this process, Ghadiri and co-workers have designed105 _{a replicating peptide network capable of stereospecific replication}

(Figure 1.17). TDD TLL TDL TLD ED EL ND NL [ED·ND_·TDD_] [TDD·TDD] [EL·NL·TLL] [TL·TLL_] TDD TLL

Figure 1.17 Schematic representation of stereospecific peptide replicators. The electrophilic fragments, EL_andED_{, and nucleophilic fragments,N}L_andND_{, combine to form four templates. The} homochiral templatesTLL_andTDD_{are capable of autocatalysis, while the heterochiral} templates can only be formed through uncatalysed bimolecular reactions. Black and orange cylinders represent peptide regions comprised of D- and L-amino acids, respectively. Figure adapted from Ref. 105.

This work extended their original peptide replicatorT to a system composed of two electrophilic fragments,EL_andED_{, and two nucleophilic components,N}L_andND_.

Reaction of fragments with the same stereochemistry produced homochiral templates TLL _{(Figure 1.17, orange) and T}DD _{(Figure 1.17, black), whereas reaction of mixed}

between the nucleophilic and electrophilic components showed that the homochiral products are formed efficiently and preferentially.

The heterochiral peptide templates, TLD _{and T}DL _{were shown to form through}

template-independent pathways only, and the authors suggest that this observation stems from the diminished ability of these two templates to form coiled-coil helical assemblies. Detailed kinetic analysis and template-instructed experiments revealed that TLL is capable of stereospecific self-replication, insensitive to addition of the homochiral TDD or the heterochiral templates. The chiroselective system exhibited strong sensitivity to mutations in even a single amino acid residue, which contributed to amplification of a single homochiral template, once formed. While the authors have, for the first time, demonstrated the feasibility of chiroselective replication, the ability of the homochiral templateTDD_{to self-replicate was not discussed in the study and, therefore,}

no conclusions can be drawn as to whether stereoselective replication is present in both homochiral replicators. Nevertheless, these results suggest that a peptide biopolymer