CC-Trigger: toehold mediated protein coiled-coil displacement to introduce kinetic control of protein assemblies and advanced multi-state devices.

Proteins are nature’s most versatile workers, catalysing key reactions, processing signals, and moving cargo. Many of those functions occur through conformational changes or reconfigurations of protein complexes. Understanding the relationships between the sequence, 3D structure and function of proteins is not only of academic interest, but is also of great technological importance. The challenge in the field is creating larger dynamic protein assemblies that have several states and/or perform specific functions. Dynamic protein assemblies could be used to build drug delivery vehicles, smart responsive materials, biosensors and many other functions. DNA nanotechnology has produced amazing machines and multistate assemblies, including tweezers, switchable containers, molecular walkers and even cargo sorting robots. The common feature of those advances is the use the principle of toehold strand displacement, where a DNA strand can displace strongly bound strand from the complex using a single strand toehold. While DNA nanotechnology is easier to program, protein assemblies can perform more versatile range functions. The aim of this research project is to implement toehold mediated strand displacement principle using coiled coils (CCs) strands and demonstrate several exciting applications that this new technology would enable, for control of enzyme activity and localisation, reconfigurable multistate protein assemblies and logic gates. CC dimers are composed of a pair twisted alpha helices and share some similarities to DNA. Our group has a very successful track record in understanding and using short CCs. We have designed an orthogonal set of CCs (where each pair only interacts with each other), shown that orthogonality is maintained in vivo and used CCs to construct a new type of protein fold. Implementing the strand displacement principle into the protein world could help revolutionise the design of protein machines and devices that have strong potential in biomedicine applications. In DNA toeholds, the duplex strands that are being displaced must have very long dissociation half-live, therefore we hypothesise that long CCs (which also have slow dissociation) are need for the CC strand displacement to function. In WP1 we will design the first long artificial heterodimeric CCs, longer than 100 amino acid residues. In WP2 we will design and test several toehold constructs, systematically studying the effect of the length of the toehold and length of the duplex substrate produced in WP1 on the efficacy and kinetics of displacement. In WP 3 we will use advanced computational methods to design orthogonal sets of long CCs. The orthogonal set will enable several CC strand displacement reactions in the same system and construction of large CC protein origami. WP4 develops proof-of-principle applications that showcase the power of CC strand displacement. We plan to demonstrate two state switches, multistate reconfigurable CCPO assemblies and signal processing logic gates. The protein toehold displacement strategy, as proposed here, is a highly innovative approach, that could transform the field of CCPO and design of protein-based machines in general. Generating asset of orthogonal long designed heterodimeric CCs would be extremely useful to build larger CCPO structures that could encapsulate larger cargo and could be in different states visualised under the cryo-EM microscope. Our preliminary data attest that the design of long orthogonal heterodimeric CCs is indeed possible, which supports the feasibility of this project. CC strand displacement could have a huge impact on protein design and could generate a range of biomedical applications. DNA nanotechnology has already demonstrated that the strand displacement principle can be extremely powerful and versatile, we aim to bring the same versatility and programmability of applications to the more biocompatible and genetically encodable protein world.