OPTICAL CONTROL OVER MONOMERIC AND MULTIMERIC PROTEIN HYBRIDS
Living materials are based on proteins that adapt and change in structure and function continuously when in use; cellular microtubules, ATP synthases and ribosomes are but a few examples. Breathing life into man-made materials would allow improved understanding over protein regulation and dynamics, and how their integration into complex systems can lead to emergent functions across length scales. The focus of my research is to achieve optical control over functional proteins by connecting them to artificial molecular switches, with the aim to amplify molecular switching events across length scales and gain understanding over cooperative and systematic regulations of proteins.
We choose light because it offers spatiotemporal selectivity, is compatible with a wide range of phases and relatively non-destructive towards protein systems. Interfering with mechanisms such as allosteric communication or hierarchical self-assembly not only paves the way towards an improved understanding of cooperative or collective effects in living matter, but it is also associated with the generation of new classes of smart bio-hybrids. The works presented in this thesis involve diverse functional proteins, ranging from an allosteric transport protein, the human serum albumin (Chapter 3), to proteins forming cages (Chapter 4, 5, 6) all the way to chaperone proteins that are known to assist cellular protein folding (Chapter 6).
The work focuses on the use of spiropyran derivatives as photo-switches, because their irradiation induces not only a change in conformation, but also it yields a zwitterionic form that is bound to influence a protein environment. Chapter 3 and 5 demonstrate covalent incorporation of spiropyran switches into a cysteine and lysines that are present in the studied proteins. The covalent coupling involves reacting maleimide or succinimide moieties (attached to the photo-switchable units) with the proteins via thiol-Michael addition or amine-NHS ester reaction, respectively. Chapter 3 describes how such structural modification allows photo-controlling the natural allosteric regulation of the human serum albumin (HSA), and thus to modulate ligand binding by irradiation with light. On the other hand, modification of a bacterial encapsulin, a self-assembled protein cage that confines smaller proteins in its cavity (Chapter 4 and 5), yields local modifications only, and does not compromise the structural integrity and the behavior of the entire assembly.
Chapter 4 and 5 further confirm and demonstrate that bacterial encapsulins are robust, catalytically active, and biocompatible. They can also be labelled with light-switchable fluorophores on their lysines via reaction with succinimide-bearing spiropyran switches, and thus they are good candidates as smart nanoplatforms for in vitro and in vivo research purposes.
Chapter 6 demonstrates the light-fueled assembly of proteins by adding photo-switches into a medium containing the encapsulin and GroEL chaperone. This strategy enables addition of a large amount of photo-switches, which, in this context, results in the changes of the medium upon irradiation, i.e., pH and hydrophobicity. Mediated by a spiroyran-based photoacid, the described approach leads to an assembly of transient superstructures that depend on light irradiation to prevent disassembly. This assembly thus presents a dynamic and reversible order of a protein system that is sustained by a continuous input of energy, and this mechanism is reminiscent of the GTP-fueled assembly of cellular microtubules.
Overall, we have developed photo-responsive protein-based hybrids in which dynamic regulations such as allostery and self-assembly are prominent to protein functionality. Via incorporation of photo-switches, optical control has been demonstrated across length scales, from a monomeric structure to highly-defined multimeric architectures made of proteins. The presented research provides a means to interfere with dynamic regulations of proteins and supports strategies towards the development of biocompatible and smart materials.
