In nature, most proteins are found in an oligomeric, mainly homodimeric or homotetrameric form. This allows enzymes to have an increased local concentration of active sites, which speeds up the conversion of the substrate. This feature is evolutionarily favourable for most proteins and is conserved. However, there are still monomeric proteins that do not spontaneously associate into oligomeric complexes. One of these is cathepsin B, a cysteine protease with endo- and exonuclease activity. As part of the thesis, we wanted to use a rational design to find amino acid residues on the protein that could be mutated and thus allow oligomerisation of a naturally monomeric protein.
In the first part of the thesis, we used bioinformatics tools and theoretically known data to identify amino acid residues on the back surface that are reasonable to be modified by random saturation mutagenesis. We selected those that were facing out, i.e. from the interaction surface.
In the second part of the study, we wanted to in vitro test the in silico identified potential mutagenic sites. We introduced site-specific mutations by random saturation mutagenesis and checked the dimerization state with the BACTH system. Homodimerization of two molecules of procathepsin B occurred when the Glu9Val mutation was introduced. Heterodimerisation occurred when the mutation at site 15 was introduced, where one subunit carried the Pro15Gly mutation, and the other subunit was a wild-type protein. Finally, we in silico visualised the most probable dimer structure for both candidate dimers. The first one interacted via the bottom and the second one via the back surface of the protein.
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