Most cellular proteins are oligomers. Oligomeric proteins have a smaller solventaccessible surface area than monomers, which increases their stability. At the same time,
oligomerization can increase the local concentration of active sites and thus improve
enzyme activity. Despite the frequency of oligomeric proteins, cathepsin S, a member of
papain-like cysteine proteases, is a monomer. As a part of our thesis, we wanted to use
bioinformatic tools to determine potential interactive residues and introduce substitution
mutations on the surface of cathepsin S, which would consequently form a stable
homodimer.
In the first part of the task, using various publicly available programs, we determined the
interactive residues on the surface of cathepsin S and determined the surface amino acid
substitutions that would cause spontaneous homodimerization. Using the HADDOCK
2.4. program we determined the predicted homodimer structure, buried surface area, and
interaction energy. With three point mutations of amino acids (E15Y, P91W, K93W), we
managed to increase the buried surface area to 2478.8 Å2 and improve the interactive
energy of the homodimer, thereby stabilizing it. , K93W) labeled MutS5 initially forms a
stable dimer, which becomes less stable over time. We wanted to verify this in practice,
so we carried out an experimental part.
In the experimental work we used site-specific mutagenesis to introduce mutations in the
procathepsin S DNA transcript included in the plasmid pET-32/28b(+) and transformed
it into Rosetta Gami II [DE3] pLysS for the expression of the MutS5 protein. Using SDSPAGE, we proved that our mutated protein was mainly expressed in an insoluble form,
which means that it was most likely misfolded due to our mutations. With obtained form
of the protein, we cannot prove that properly folded »MutS5« forms a homodimer and
remains active
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