A Peptide-Based Oscillator.

Living organisms are replete with rhythmic and oscillatory behavior at all levels, to the extent that oscillations have been termed as a defining attribute of life. Recent studies of synthetic oscillators that mimic such functions have shown decayed cycles in batch-mode reactions or sustained oscillatory kinetics under flow conditions. Considering the hypothesized functionality of peptides in early chemical evolution and their central role in current bio-nanotechnology, we now reveal a peptide-based oscillator. Oscillatory behavior was achieved by coupling coiled-coil-based replication processes as positive feedback to controlled initiation and inhibition pathways in a continuously stirred tank reactor (CSTR). Our results stress that assembly into the supramolecular structure and specific interactions with the replication substrates are crucial for oscillations. The replication-inhibition processes were first studied in batch mode, which produced a single damped cycle. Thereafter, combined experimental and theoretical characterization of the replication process in a CSTR under different flow and environmental (pH, redox) conditions demonstrated reasonably sustained oscillations. We propose that studies in this direction might pave the way to the design of robust oscillation networks that mimic the autonomous behavior of proteins in cells (e.g., in the cyanobacterial circadian clock) and hence hint at feasible pathways that accelerated the transition from simple peptides to extant enzymes.

Dynamic Surface Layer Coiled Coil Proteins Processing Analog-to-Digital Information.

Surface layer proteins perform multiple functions in prokaryotic cells, including cellular defense, cell-shape maintenance, and regulation of import and export of materials. However, mimicking the complex and dynamic behavior of such two-dimensional biochemical systems is challenging, and hence research has so far focused mainly on the design and manipulation of the structure and functionality of protein assemblies in solution. Motivated by the new opportunities that dynamic surface layer proteins may offer for modern technology, we herein demonstrate that immobilization of coiled coil proteins onto an inorganic surface facilitates complex behavior, manifested by reversible chemical reactions that can be rapidly monitored as digital surface readouts. Using multiple chemical triggers as inputs and several surface characteristics as outputs, we can realize reversible switching and logic gate operations that are read in parallel. Moreover, using the same coiled coil protein monolayers for derivatization of nanopores drilled into silicon nitride membranes facilitates control over ion and mass transport through the pores, thereby expanding the applicability of the dynamic coiled coil system for contemporary stochastic biosensing applications.

Primitive selection of the fittest emerging through functional synergy in nucleopeptide networks.

Many fundamental cellular and viral functions, including replication and translation, involve complex ensembles hosting synergistic activity between nucleic acids and proteins/peptides. There is ample evidence indicating that the chemical precursors of both nucleic acids and peptides could be efficiently formed in the prebiotic environment. Yet, studies on nonenzymatic replication, a central mechanism driving early chemical evolution, have focused largely on the activity of each class of these molecules separately. We show here that short nucleopeptide chimeras can replicate through autocatalytic and cross-catalytic processes, governed synergistically by the hybridization of the nucleobase motifs and the assembly propensity of the peptide segments. Unequal assembly-dependent replication induces clear selectivity toward the formation of a certain species within small networks of complementary nucleopeptides. The selectivity pattern may be influenced and indeed maximized to the point of almost extinction of the weakest replicator when the system is studied far from equilibrium and manipulated through changes in the physical (flow) and chemical (template and inhibition) conditions. We postulate that similar processes may have led to the emergence of the first functional nucleic-acid-peptide assemblies prior to the origin of life. Furthermore, spontaneous formation of related replicating complexes could potentially mark the initiation point for information transfer and rapid progression in complexity within primitive environments, which would have facilitated the development of a variety of functions found in extant biological assemblies.

Signaling in Systems Chemistry: Programing Gold Nanoparticles Formation and Assembly Using a Dynamic Bistable Network.

Living cells exploit bistable and oscillatory behaviors as memory mechanisms, facilitating the integration of transient stimuli into sustained molecular responses that control downstream functions. Synthetic bistable networks have also been studied as memory entities, but have rarely been utilized to control orthogonal functions in coupled dynamic systems. We herein present a new cascade pathway, for which we have exploited a well-characterized switchable peptide-based replicating network, operating far from equilibrium, that yields two alternative steady-state outputs, which in turn serve as the input signals for consecutive processes that regulate various features of Au nanoparticle shape and assembly. This study further sheds light on how bridging together the fields of systems chemistry and nanotechnology may open up new opportunities for the dynamically controlled design of functional materials.

A chemically fueled non-enzymatic bistable network.

One of the grand challenges in contemporary systems chemistry research is to mimic life-like functions using simple synthetic molecular networks. This is particularly true for systems that are out of chemical equilibrium and show complex dynamic behaviour, such as multi-stability, oscillations and chaos. We report here on thiodepsipeptide-based non-enzymatic networks propelled by reversible replication processes out of equilibrium, displaying bistability. Accordingly, we present quantitative analyses of the bistable behaviour, featuring a phase transition from the simple equilibration processes taking place in reversible dynamic chemistry into the bistable region. This behaviour is observed only when the system is continuously fueled by a reducing agent that keeps it far from equilibrium, and only when operating within a specifically defined parameter space. We propose that the development of biomimetic bistable systems will pave the way towards the study of more elaborate functions, such as information transfer and signalling.

Open Prebiotic Environments Drive Emergent Phenomena and Complex Behavior.

We have been studying simple prebiotic catalytic replicating networks as prototypes for modeling replication, complexification and Systems Chemistry. While living systems are always open and function far from equilibrium, these prebiotic networks may be open or closed, dynamic or static, divergent or convergent to a steady state. In this paper we review the properties of these simple replicating networks, and show, via four working models, how even though closed systems exhibit a wide range of emergent phenomena, many of the more interesting phenomena leading to complexification and emergence indeed require open systems.

Emergence of native peptide sequences in prebiotic replication networks.

Biopolymer syntheses in living cells are perfected by an elaborate error correction machinery, which was not applicable during polymerization on early Earth. Scientists are consequently striving to identify mechanisms by which functional polymers were selected and further amplified from complex prebiotic mixtures. Here we show the instrumental role of non-enzymatic replication in the enrichment of certain product(s). To this end, we analyzed a complex web of reactions in ß-sheet peptide networks, focusing on the formation of specific intermediate compounds and template-assisted replication. Remarkably, we find that the formation of several products in a mixture is not critically harmful, since efficient and selective template-assisted reactions serve as a backbone correction mechanism, namely, for keeping the concentration of the peptide containing the native backbone equal to, or even higher than, the concentrations of the other products. We suggest that these findings may shed light on molecular evolution processes that led to current biology.The synthesis of biopolymers in living cells is perfected by complex machinery, however this was not the case on early Earth. Here the authors show the role of non-enzymatic replication in the enrichment of certain products within prebiotically relevant mixtures.

Bistability and Bifurcation in Minimal Self-Replication and Nonenzymatic Catalytic Networks.

Bistability and bifurcation, found in a wide range of biochemical networks, are central to the proper function of living systems. We investigate herein recent model systems that show bistable behavior based on nonenzymatic self-replication reactions. Such models were used before to investigate catalytic growth, chemical logic operations, and additional processes of self-organization leading to complexification. By solving for their steady-state solutions by using various analytical and numerical methods, we analyze how and when these systems yield bistability and bifurcation and discover specific cases and conditions producing bistability. We demonstrate that the onset of bistability requires at least second-order catalysis and results from a mismatch between the various forward and reverse processes. Our findings may have far-reaching implications in understanding early evolutionary processes of complexification, emergence, and potentially the origin of life.

Theoretical Models of Generalized Quasispecies.

Theoretical modeling of quasispecies has progressed in several directions. In this chapter, we review the works of Emmanuel Tannenbaum, who, together with Eugene Shakhnovich at Harvard University and later with colleagues and students at Ben-Gurion University in Beersheva, implemented one of the more useful approaches, by progressively setting up various formulations for the quasispecies model and solving them analytically. Our review will focus on these papers that have explored new models, assumed the relevant mathematical approximations, and proceeded to analytically solve for the steady-state solutions and run stochastic simulations . When applicable, these models were related to real-life problems and situations, including changing environments, presence of chemical mutagens, evolution of cancer and tumor cells , mutations in Escherichia coli, stem cells , chromosomal instability (CIN), propagation of antibiotic drug resistance , dynamics of bacteria with plasmids , DNA proofreading mechanisms, and more.

A bistable switch in dynamic thiodepsipeptide folding and template-directed ligation.

Bistable reaction networks provide living cells with chemically controlled mechanisms for long-term memory storage. Such networks are also often switchable and can be flipped from one state to the other. We target here a major challenge in systems chemistry research, namely developing synthetic, non-enzymatic, networks that mimic such a complex function. Therefore, we describe a dynamic network that depending on initial thiodepsipeptide concentrations leads to one of two distinct steady states. This bistable system is readily switched by applying the appropriate stimuli. The relationship between the reaction network topology and its capacity to invoke bistability is then analyzed by control experiments and theory. We suggest that demonstrating bistable behavior using synthetic networks further highlights their possible role in early evolution, and may shine light on potential utility for novel applications, such as chemical memories.

Coupled Oscillations and Circadian Rhythms in Molecular Replication Networks.

Living organisms often display rhythmic and oscillatory behavior. We investigate here a challenge in contemporary Systems Chemistry, that is, to construct "bottom-up" molecular networks that display such complex behavior. We first describe oscillations during self-replication by applying kinetic parameters relevant to peptide replication in an open environment. Small networks of coupled oscillators are then constructed in silico, producing various functions such as logic gates, integrators, counters, triggers, and detectors. These networks are finally utilized to simulate the connectivity and network topology of the Kai proteins circadian clocks from the S. elongatus cyanobacteria, thus producing rhythms whose constant frequency is independent of the input intake rate and robust toward concentration fluctuations. We suggest that this study helps further reveal the underlying principles of biological clocks and may provide clues into their emergence in early molecular evolution.

Robustness of synthetic circadian clocks to multiple environmental changes.

A molecular network that mimics circadian clocks from cyanobacteria is constructed in silico. Simulating its oscillatory behaviour under variable conditions reveals its robustness relative to networks of alternative topologies. The principles for synthetic chemical circadian networks to work properly are consequently highlighted.

Competition and cooperation in dynamic replication networks.

The simultaneous replication of six coiled-coil peptide mutants by reversible thiol-thioester exchange reactions is described. Experimental analysis of the time dependent evolution of networks formed by the peptides under different conditions reveals a complex web of molecular interactions and consequent mutant replication, governed by competition for resources and by autocatalytic and/or cross-catalytic template-assisted reactions. A kinetic model, first of its kind, is then introduced, allowing simulation of varied network behaviour as a consequence of changing competition and cooperation scenarios. We suggest that by clarifying the kinetic description of these relatively complex dynamic networks, both at early stages of the reaction far from equilibrium and at later stages approaching equilibrium, one lays the foundation for studying dynamic networks out-of-equilibrium in the near future.

Transient fibril structures facilitating nonenzymatic self-replication.

An emerging new direction of research focuses on developing "self-synthesizing materials", those supramolecular structures that can promote their own formation by accelerating the synthesis of building blocks and/or an entire assembly. It was postulated recently that practical design of such systems can benefit from the ability to control the assembly of amphiphilic molecules into nanostructures. We describe here the self-assembly pathway of short amphiphilic peptides into various forms of soluble ß-sheet structures--ß-plates, fibrils, and hollow nanotubes--and their consequent activity as autocatalysts for the synthesis of monomeric peptides from simpler building blocks. A detailed kinetic analysis of both the self-assembly and self-replication processes allows us to suggest a full model and simulate the replication process, revealing that only specific structures, primarily fibrils that are stable within the solution for a time shorter than a few hours, can be active as catalysts. Interestingly, we have found that such a process also induces fibril reproduction, in a mechanism very similar to the propagation of prion proteins by transmission of misfolded states.

Chemical and light triggering of peptide networks under partial thermodynamic control.

The kinetics of novel dynamic libraries that operate via reversible replication is described. In these systems, selective product formation is governed by peptides autocatalytic efficiency and by differences in their unfolding stability. We suggest ways to significantly alter the network behavior by chemical inputs (templates) or physical triggers (light).

ß-Sheet-induced chirogenesis in polymerization of oligopeptides.

The origin of long homochiral biopolymers in living systems has recently been the focus of intense research. In one particular research line, scientists studied, experimentally and theoretically, chiral amplification obtained during peptide formation by polymerization of amino acid building blocks. It was suggested that processes leading to temporal or spatial separation, and thus, to the growth of homochiral polymers at the expense of their heterochiral counterparts, can explain the chirality observed in larger molecules. We introduce a simple model and stochastic simulation for the polymerization of amino acids and ß-sheet formation, showing the crucial effects of the ß sheets on the distributions of peptide lengths. When chiral affinities are included, racemic ß sheets of alternating homochiral strands lead to the formation of chiral peptides, the isotacticity of which increases with length, consistent with previous experimental results in aqueous solutions. The tendency to form isotactic peptides is shown for both initially racemic and initially nonracemic systems, as well as for closed and open systems. We suggest that these or similar mechanisms may explain the origin of chiroselectivity in prebiotic environments.

Second order catalytic quasispecies yields discontinuous mean fitness at error threshold.

The quasispecies model describes processes related to the origin of life and viral evolutionary dynamics. We discuss how the error catastrophe that reflects the transition from localized to delocalized quasispecies population is affected by catalytic replication of different reaction orders. Specifically, we find that second order mechanisms lead to a discontinuity in the mean fitness of the population at the error threshold. This is in contrast to the behavior of the first order, autocatalytic replication mechanism considered in the standard quasispecies model. This suggests that quasispecies models with higher order replication mechanisms produce discontinuities in the mean fitness, and hence the viable population fraction as well, at the error threshold, while lower order replication mechanisms yield a continuous mean fitness function. We discuss potential implications for understanding replication in the RNA world and in virology.

Selection advantage of metabolic over non-metabolic replicators: a kinetic analysis.

A kinetic analysis and simulation of the replication reactions of two competing replicators-one non-metabolic (thermodynamic), the other metabolic, are presented. Our analysis indicates that in a rich resource environment the non-metabolic replicator is likely to be kinetically selected for over the metabolic replicator. However, in the more typical resource-poor environment it will be the metabolic replicator that is the kinetically more stable entity, and the one that will be kinetically selected for. Accordingly, a causal relationship between the emergence of a simple replicator and the emergence of a metabolic system is indicated. The results lend further support for the "replication first" school of thought in the origin of life problem by providing a mechanistic basis for the emergence of a metabolism, once a simple non-metabolic replicating system has itself been established. The study reaffirms our view that the roots of Darwinian theory may be found within standard chemical kinetic theory.