A High‐Resolution Structure that Provides Insight into Coiled‐Coil Thiodepsipeptide Dynamic Chemistry

Abstract

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...

Self-Reproduction and Darwinian Evolution in Autocatalytic Chemical Reaction Systems

“…While the systems discussed above use irreversible native chemical ligation, Dadon et al employed a reversible trans-thioesterification chemistry to construct template-based self-reproducing peptides [ 142 , 143 , 144 ]. Here, similar coiled-coil architectures are used; however, the nucleophilic fragment is terminally modified to contain a thiol (using thiol glycolic acid) such that they can form reversible thioester bonds, since they are susceptible to free thiols, readily decomposing to starting material.…”

“…Here, similar coiled-coil architectures are used; however, the nucleophilic fragment is terminally modified to contain a thiol (using thiol glycolic acid) such that they can form reversible thioester bonds, since they are susceptible to free thiols, readily decomposing to starting material. This reversibility (decomposition) correlates well with the stability of the substrate-template complex, and therefore, the stable complexes result in efficient reproduction [ 143 ]. As discussed earlier in Section 2.4 , multiple stable steady states are critical for Darwinian evolution in self-reproducing networks [ 16 , 55 ].…”

From protein total synthesis to peptide transamidation and metathesis: playing with the reversibility of N,S-acyl or N,Se-acyl migration reactions

“…Though several chemical transformations fulfill these criteria, only few of them have been applied to peptide-based DCL approaches (Figure 2c-e). The most popular reversible chemistries in this field are the thiol-disulfide [9] exchange reaction ( Figure 2c), with significant applications in supramolecular assembly, the amine-imine [10] exchange reaction which includes the hydrazone bond (Figure 2d), and the thiol-thioester [11,12] exchange reaction (Figure 3e). …”

Thioesters Provide a Robust Path to Prebiotic Peptides

“…Previously, short thiol-terminated compounds and thioesters have been investigated in both aqueous and non-aqueous solutions as building block units for dynamic combinatorial libraries. [40][41][42][43][44] Based on the results presented here we propose that incorporation of even simple mercaptoacids into these systems might offer new avenues for dynamic combinatorial chemistry in water or under dry-down conditions. Of importance to origins research, mixtures of different building blocks, including amino acids, mercapto acids, and hydroxy acids are, arguably, more prebiotically plausible than pure amino acids as starting conditions for polymer evolution.…”