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Continuous self-assembly using enzyme mediated supramolecular switching

Periodic Reporting for period 1 - ASSEMZYME (Continuous self-assembly using enzyme mediated supramolecular switching)

Reporting period: 2015-03-24 to 2017-03-23

Over the past decades, chemists have extensively used molecular self-assembly as a bottom up approach to prepare well-ordered and truly sophisticated supramolecular architectures, many of which are able to exert specific functions. By exploiting the reversible nature of non-covalent interactions, great progress have been made in designing switchable, self-healing and self-replicating supramolecular systems that are able to change and adapt in response to external stimuli (e.g. light, pH, chemical modification, or enzymes). The latter have found exciting applications in catalysis, material science, and nanomedicine. Besides that, various kinetically trapped self-assembled structures have been obtained under kinetic control by rationally selecting the desired aggregation pathway. However, in spite of their remarkable structural complexity and responsiveness, artificial self-assembled systems lack the functional complexity that is the hallmark of living self-organized systems such as microtubules, ribosomes and biomembranes. This is because artificial self-assemblies evolve until a global (or local) free energy minimum is reached, after which they remain inactive. In other words, they reside either in thermodynamic equilibrium or kinetically trapped states. Living systems, on the other hand, continuously avoid equilibrium states, and are able to do so for extended periods of time, through a constant influx of energy. They reside in so-called dissipative non-equilibrium steady states, and continuously consume energy to keep their structure and function. The latter is at the bases of intricate biological functions, such as cell motility, muscle contraction, intracellular transport, and mitosis. For example, cells use fuel molecules like adenosine triphosphate (ATP) to control when and where supramolecular polymers such as actin should assemble and disassemble. Recently, artificial fuel-driven assemblies have been developed, in which the system can be pushed transiently out of equilibrium by addition of a chemical fuel, but then slowly relax towards its equilibrium states. Moreover, these systems can be refuelled, resulting in a new cycle of transient assembly, but the accumulation of waste from the fuel conversion, leads to a poisoning of the system, and to a limited number of possible cycles.
The aim of this project has been to realise non-equilibrium self-assembled steady states (NESS) of a supramolecular polymer, controlled by competitive enzymatic phosphorylation/dephosphorylation of the building blocks, in which the supramolecular assemblies are pushed and kept out of equilibrium by continuous influx of the chemical fuel ATP, and removal of waste through a membrane reactor.
Maintaining NESS conditions of a self-assembled system is a crucial advancement in the field of supramolecular chemistry, which will open the door to truly functional “living” systems.
At the end of the action, the proposed objectives were fully achieved, and we could successfully demonstrate that it is possible to keep supramolecular polymers in different NESS depending on the influx of chemical fuel supplied, and outflux of waste under continuous flow conditions.
As the first step, we prepared a not gelling supramolecular polymer based on a peptide-perylendiimide derivative that can be phosphorylated on a serine residue by a specific kinase enzyme (consuming the fuel ATP), and dephosphorylated back by a phosphatase enzyme (producing inorganic phosphate Pi as waste). We then studied in detail the kinetics of (de)phosphorylation, as well as the equilibrium self-assembly properties of the non-phosphorylated and phosphorylated polymers respectively. We found that phosphorylation, by changing the state of charge of the self-assembling molecules, results in increased thermodynamic stability and growth of polymer, as well as in switching of supramolecular chirality. Afterwards, successive dephosphorylation reverts the polymer completely back to its original structure. In other words, the supramolecular structure of the polymer can be reversibly controlled by enzymatic step-wise (de)phosphorylation, in analogy to stimuli-responsive materials.
We then worked out the experimental conditions at which both enzymes can work concurrently, and at commensurate speeds. The latter allowed us to obtain transient changes in the supramolecular structure of the polymer when adding ATP to a batch reactor containing the building blocks, and the two enzymes, in analogy with transient self-assembly, that is state-of-the-art in supramolecular chemistry. We could refuel our system, but waste products lead to phosphatase inhibition causing the reaction cycles to stop. The poisoning effect could be overcome by working in an open system under continuous flow conditions (see below). To shed light on the complex dynamics of the enzyme network, we developed a mathematical model based on mass action kinetics and performed numerical simulations.
Finally, we fabricated a membrane reactor, where different sustained NESS could be maintained depending on the level of fuel supplied and waste removed under continuous flow conditions. The result is that the assembly/disassembly process could be kept continuously working for days as long as the fuel is supplied.
These results, going beyond the state-of-the-art in the field, accomplish the objectives of the proposal, and have been reported in the paper “Non-equilibrium steady-states in supramolecular polymerization”, just accepted in NCOMM. In addition, they have been presented, so far, to four international conferences as poster and/or oral presentations (including Burgenstock and Gordon meetings).
In conclusion, our studies show that it is possible to obtain true non-equilibrium steady states of artificial self-assembled systems depending on the influx of chemical fuel supplied, and outflux of waste products. This is different from current switchable supramolecular systems that can reversibly interchange between two different non-dissipative states in response to stimuli, as well as from transient systems that are pushed temporarily out of equilibrium by adding aliquots of “fuel” periodically. Unlike commonly used batch or semi-batch conditions, where a fraction of the entire system is discarded due to outflow, we developed a membrane reactor that selectively exchanges fuel and waste, but leaves the self-assembling building blocks and costly enzymes in place. Our approach can be applied to currently existing stimuli-responsive or transient self-assembly systems, which could be maintained in this way out of equilibrium indefinitely.
In our case, the influx of ATP is the control parameter, which determines the nature of the steady-state, and the distance from the thermodynamic equilibrium. Exploring different steady-states depending on how hard the system is driven, can provide unique insight into the dynamics and structures of artificial self-assemblies far from equilibrium. In general, if supramolecular systems can be driven in this way, it could give rise to sustained supramolecular oscillations analogously to microtubules. To sum up, our work opens an avenue towards the development of more life-like self-assembled materials that can show true adaptability and eventually perform functions as complex as in living systems.