Chemical reaction cycles driven by phosphorylating agents

• Applicant: Professor Dr. Job Boekhoven
• Subject Area: Organic Molecular Chemistry - Synthesis and Characterisation
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 564321781

Biological processes are often regulated by the reversible phosphorylation of proteins. A demonstrative example is the Ca2+ ATPase, a transmembrane enzyme that pumps calcium ions from one side of the membrane to the other against a concentration gradient. Naturally, this process costs energy abstracted from ATP through hydrolysis. This energy is harvested by phosphorylating and dephosphorylating a site in the ATPase. That phosphorylation changes the protein structure, which pumps Calcium from one side to the other. Essentially, the chemical energy stored in the high-energy phosphorylation sources is converted into a transmembrane Ca2+ potential by transiently phosphorylating an amino acid. Given the importance of these reaction cycles, it begs the question: Can we create synthetic analogs of such cycles? With such analogs, we can attempt to build machinery like biology does. Those include pumps, motors, dynamic assemblies, or patterns. Besides, a synthetic analog could serve as a model for biological cycles and increase our understanding of the role of phosphorylation cycles in biology. For example, we can develop peptides whose folding is regulated by reaction cycles. Finally, the formation of structures out of equilibrium can be a model for studying protocells at the origin of life and a step toward synthetic life. Thus, the primary aim of this proposal is to create a chemical reaction cycle based on an amino acid's dynamic phosphorylation at the expense of a phosphorylating agent without the use of enzymes. We will use histidine phosphorylation as a starting point and mono-amidophosphate as fuel. When we quantitatively understand this cycle, we branch out to different amino acids and fuels. The second goal of this project is to design peptides that undergo a supramolecular step after phosphorylation. That way, the chemical potential can be used to drive supramolecular processes. Thus, we design peptides that become phosphorylated and a second class of peptides that form a complex with the phosphorylated state. Moreover, the second class of peptides will be catalytically active to dephosphorylate the activate peptide. Thus, the phosphorylated peptide has to form a complex with the catalyst to become dephosphorylated. This approach is critical to ensure optimal usage of the energy stored in the phosphorylating agent and also to perform work at the expense of the phosphorylating agent. Finally, we increase the functionality of the systems by introducing self-assembly. For example, we dock the dephosphorylating catalyst in a vesicle's membrane. Now, the activated peptide has to diffuse to the membrane before it can be deactivated. This project thus leads to very simplistic pumps that use the energy in phosphorylating agents to pump molecules. Future projects will build on these concepts such that transmembrane pumps can be developed.

• DFG Programme: Research Grants