20.500.12556/RUL-140027 Racionalno načrtovanje in priprava dimernih variant človeškega katepsina S Rational design and preparation of dimeric variants of human cathepsin S Večina celičnih proteinov je oligomerov. Oligomerni proteini imajo namreč manjšo topilu dostopno površino kot monomeri, s tem pa se poveča njihova stabilnost. Hkrati lahko z oligomerizacijo zvišamo lokalno koncentracijo aktivnih mest in tako izboljšamo aktivnost encima. Kljub pogostosti oligomernih proteinov je katepsin S, pripadnik papainu podobnih cisteinskih proteaz, monomer. V sklopu naše diplomske naloge smo želeli z uporabo bioinformatskih orodij določiti potencialne interakcijske ostanke in uvesti substitucijske mutacije na površini katepsina S, ki bi posledično tvoril stabilen homodimer. V prvem delu naloge smo z uporabo različnih javno dostopnih programov določili interakcijske ostanke na površini katepsina S ter določili površinske substitucije aminokislin, ki bi povzročile spontano homodimerizacijo. S progrmom HADDOCK 2.4. smo določili predvideno strukturo homodimera, zakopano površino in interakcijsko energijo. S tremi točkovnimi mutacijami aminokislin (E15Y, P91W, K93W) smo uspeli povečali zakopano površino na 2478,8 Å 2 ter izboljšali interakcijsko energijo homodimera, s čimer smo ga stabilizirali Z računanjem molekulske dinamike smo dokazali, da teoretično mutiran katepsin S (E15Y, P91W, K93W) z oznako MutS5 na začetku tvori stabilen dimer, ki pa s časom postaja vse manj stabilen. To smo želeli preveriti v praksi, zato smo izvedli še eksperimentalni del. V ekperimentalnem delu smo z mestno-specifično mutagenezo uvedli mutacije v DNA zapis za prokatepsin S vključen v plazmid pET-32/28b(+) in tega transformirali v Rosetta Gami II [DE3] pLysS za ekspresijo proteina MutS5. Z NaDS-PAGE smo dokazali, da se naš mutirani protein izraža predvsem v netopni obliki, kar pomeni, da se je najverjetneje narobe zvil zaradi mutacij. V takšni obliki ne moremo dokazati, da pravilno zviti MutS5 tvori homodimer in pri tem ostane aktiven. 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 Cisteinski katepsini katepsin S proteinski inženiring Cysteine cathepsins cathepsin S protein engineering true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2022-09-09 13:55:00 2022-09-09 13:55:06 2022-11-17 13:06:32 0000-00-00 00:00:00 2022 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 22050 129798915 Sotosek_Timotej_-_Racionalno_nacrtovanje_in_priprava_dimernih_variant_cloveskega_katepsina_S.pdf Sotosek_Timotej_-_Racionalno_nacrtovanje_in_priprava_dimernih_variant_cloveskega_katepsina_S.pdf 1 AEDC88D13947E793B8E0C3BBDA58DEF0 01cae5aef9c13aff5ef7e820938e0fd57dc93b9efd14b3a67e1ec4888e85983d 30d6d53d-3036-11ed-92af-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=160686 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0