Necroptosis is a form of programmed cell death characterized by distinct processes, including cell volume increase, organelle swelling, loss of plasma membrane integrity, and the release of cellular contents into the extracellular environment. The key mediator of signaling during necroptosis is the protein MLKL (mixed-lineage kinase domain-like), which consists of a pseudokinase domain and a four-helix bundle (4HB) domain. Studies have demonstrated that the 4HB domain facilitates MLKL oligomerization and translocation to the plasma membrane, where it induces membrane permeabilization. As part of this Master’s thesis, a shorter peptide was designed using de novo protein design through the RFdiffusion software. The experimental objective was to evaluate whether the protein, designed with bioinformatics tools, can be expressed and bind to the target protein MLKLN-154 under laboratory conditions. To achieve this, both the target protein MLKLN-154 and the de novo designed protein GGNB-MLKL-BIND-015 were expressed, isolated, and purified. The expression of both proteins was carried out in bacterial cells, with affinity chromatography (IMAC and re-IMAC), serving as the critical purification step in both cases. Their potential interaction was assessed using size- exclusion chromatography (SEC), but the results showed no detectable interaction between the two proteins. Given that the molecular weight of the de novo binding protein is only 2.4 kDa, we also employed tricine-SDS-PAGE gels, which provide enhanced resolution for small proteins. Additionally, potential interaction was further investigated using tryptophan fluorescence spectroscopy, an intrinsic fluorescence method that detects shifts in the emission spectrum caused by environmental changes around tryptophan residues. The results provide insights into the capabilities of de novo protein design using modern bioinformatics tools and their translation into experimental work. This work lays the foundation for further research in bioinformatics and the practical application of theoretical models, while also highlighting the use of versatile analytical techniques, such as tryptophan fluorescence spectroscopy, which offers distinct perspective on protein- protein interactions at the molecular level.
|
