Supramolecular cucurbit[8]uril induced protein dimerization

Protein dimerization is a crucial biological process in which either two of the same proteins (homodimerization) or two different proteins (herodimerization) interact and form a functional assembly. In fact, proteins rarely show function and activity in their isolated form in a biological environment. The self assembly of proteins to form dimers or higher oligomeric aggregates is a common biophysical phenomenon which occurs in every cellular compartment such as cell membranes, the nuclear, and the cytosol. All cellular pathways such as enzyme activation, signal transduction, and even pathogenic pathways are significantly regulated via protein dimerization. Understanding and controlling the molecular mechanism that regulate protein dimerization is crucial for biomedical applications. Unfortunately, currently external control over these interactions is only possible in very limited cases. A robust and versatile supramolecular approach could therefore provide strong entries to explore and modulate the molecular mechanisms of protein dimerization. In this thesis it is shown that the supramolecular host molecule cucurbit[8]uril can efficiently be used to induce and reversibly control dimerization of proteins (fluorescent proteins, enzymes, and membrane proteins) having a genetically incorporated N-terminal phenylalanine-glycine-lycine (FGG) peptide motif. The proteins with an FGG-tag are easily generated by using autocleavage of an intein system under pH and temperature control. Cucurbit[8]uril selectively binds and dimerizes the FGG-tag in its hydrophobic cavity and via a key interaction between the N-terminal amine functionality of peptide and the carbonyl rim of cucurbit[8]uril. In order to proof the concept, we first studied the cucurbit[8]uril-induced dimerization of fluorescent proteins (CFP and YFP variants) with Forster resonance energy transfer (FRET), size exclusion chromatography, isothermal calorimetry (ITC), dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). The results showed that a very short, genetically encoded N-terminal FGG peptide motif can be used as a handle to control protein dimerization when the N-termini interact with the supramolecular host molecule cucurbit[8]uril in aqueous solution. The induced fluorescent protein dimers are significantly stable, they can be separated by size-exclusion chromatography, and the recognition is selective for the FGG motif over the classical N-terminal methionine residue. The SAXS study showed that cucurbit[8]uril induced YFP dimerization has a z-shaped structure of the two subunits, with a small tilt for some of the calculated structures and the two subunits are in very close proximity, though connected via a flexible linker. The studies on fluorescent proteins laid the foundation to explore the dimerization and subsequent activation of caspase enzymes, key regulators of apoptosis. Cucurbit[8]uril induced caspase-9 dimerization lead to strong enzymatic activation of the induced protein assembly. The supramolecular induced caspase-9 protein dimer is 50-fold more catalytically active than the caspase-9 monomer and can be fully reversed using a competitor ligand. Cucurbit[8]uril is a suparmolecular inducer of dimerization, which facilitates the formation of the caspase-9 dimer at low concentrations and simultaneously sustains the correct enzyme rearrangement upon dimerization. The final part of the investigations focused on the supramolecular induced dimerization of proteins in cells and of membrane proteins. These explorative studies show promising results that the above discovered concept can become amendable to proteins in a cellular environment, making cucurbit[8]uril induced protein dimerization become a strong molecular concept in biomedical research.