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).

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Logic operations can highlight information transfer within complex molecular networks. We describe here the design of a peptide-based replication system that can be detected by following its fluorescence quenching. This process is used to negate the signal of light-activated replication, and thus to prepare the first replication NAND gate.

Stable and reactive: A crystal structure at 1.35 Å of a thioester coiled-coil protein reveals high similarity to all-peptide-bond proteins. In these assemblies, the thioester bonds are kept reactive towards thiol molecules in the mixture. This enables efficient domain exchange between proteins in response to changes in folding conditions or introduction of external templates.

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.

Chemical synthesis and cell-based expression methods can afford incorporation of non-natural entities into proteins. New structures are obtained, and new functions gained, by attachment of amino acids with non-native side-chains, [1,2] modified backbones such as b-and g-amino acids, [3][4][5] or peptide bond analogues such as peptoids, esters, and thioesters. [6][7][8][9] Backbone modification with longer and flexible amino acids allows expansion of the conformational space occupied by proteins, while introduction of ester (depsipeptide) and thioester (thiodepsipeptide) bonds enhances their reactivity towards hydrolysis and other nucleophilic attacks. The durability of thioester bonds in neutral aqueous solutions and their reactivity in thiol-thioester exchange reactions make them a relevant choice for performing dynamic chemistry in water. [10][11][12][13][14][15] Particularly interesting is the possibility of utilizing such transformations for exchanging domains between different protein molecules, owing to sequence mutations or in response to chemical and physical changes.Self-organization of molecular networks has been extensively studied by scientists interested in systems chemistry. [16,17] When studying protein-based networks, it was demonstrated that the network connectivity and overall topology can be dictated by the sequence-specific information embedded in coiled-coil architectures, [18][19][20] and that careful design of the interhelical recognition interface can be used to affect the network in a predictable manner. [14,20,21] It is suggested here that the adaptive behavior of such networks, namely their rewiring in response to external triggers, can be greatly expanded if the coiled-coil proteins are formed within dynamic networks in which domain exchange readily takes place. Towards this end, we utilize coiled-coil protein analogues that contain thioester bonds within their sequences. We predict that to be mechanistically relevant for domain exchange, the thioester bond should be isostructural with the peptide bonds to maintain the 3D structure, and it should also be kept exposed and reactive towards small-molecule thiols and/or thiol-containing proteins. To highlight these characteristics, we provide here the first high-resolution structure (1.35 ) of a thioester coiled-coil protein, and compare it to the structure of the native and depsipeptide analogue proteins. The integrity of the thioester bonds and their accessibility to other molecules are revealed by analysis of the crystal structure, as well as from complementary thiolexchange assays. We then show using a set of mutants that the thioester stability can be correlated with the backbone regularity and the coiled-coil unfolding stability. Finally, a small library formed of these thioester mutants is screened for domain exchange in the absence and presence of an external template molecule, revealing significant template effect and exchange-product amplification.The sequence of the key thioester peptide (1 t ) was designed by replacing a glycin...

Peptide sequences modified with lanthanide-chelating groups at their N-termini, or at their lysine side chains, were synthesized, and new Ln(III) complexes were characterized. We show that partial folding of the conjugates to form trimer coiled coil structures induces coordination of lanthanides to the ligand, which in turn further stabilizes the 3D structure.

IntroductionBoolean algebra deals with the ''0'' and ''1'' values, utilized as presentations of false and true statements, respectively. The Boolean logic thus provides simple and concise means to describe the output of chemical processes that depend on more than one factor. Historically, the study of chemical logic gates has started with the design of small organic molecules that perform the desired functions when triggered by simple entities such as protons, hydroxyls, or metal ions [1,2]. Since the first demonstration by de Silva, many different chemical logic systems were developed, including metal-organic complexes, peptides, and DNA. The response of these systems to additional chemical triggers, as well as to electrochemical and light triggers, has been demonstrated quite frequently [3][4][5][6][7]. Interestingly, many of the recently described logic operations, and also the more complex arithmetic units, were designed based on pursuing dynamic processes instead of simple binding to one operating molecule [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24]. Beyond the basic demonstrations, scientists were able to exploit their new gates as smart devices that control various applications such as catalysis, sensing analytes, drug delivery, and even in vivo transcription. In a related line of research, modules of large biochemical systems have also been described to function as Boolean entities, in studies that used the ''top-down'' screening for such operations [25][26][27][28][29][30][31]. This chapter describes primarily our own research, in which we use synthetic networks made of small proteins as tools for performing chemical computations. We do so by practicing both the bottom-up approach, using individual molecules as gates, and the top-down approach, for which the entire molecular network is used to perform the desired functionality.Biology can serve as inspiration and can provide chemists with the design principles for engineering networks of interacting and replicating molecules. These can potentially be used as controllable tools for studying systems behavior. Toward this aim, different research groups have designed and characterized dynamic combinatorial libraries [32][33][34][35][36][37][38] and replication networks made of nucleic acids (DNA and RNA) [39][40][41][42], peptides [43][44][45][46], and small organic molecules [47][48][49][50].