Racionalno načrtovanje in priprava dimernih variant človeškega katepsina B

Putar, Erik (author), Novinec, Marko (mentor)

Namen proteinskega inženiringa je priprava mutantnih proteinov, ki so v primerjavi z izvirnikom izboljšani ali kakorkoli modificirani v skladu z našimi potrebami. V okviru diplomske naloge smo z racionalnim pristopom proteinskega inženiringa želeli pripraviti homodimerno varianto človeškega katepsina B, ki je sicer monomerna papainu podobna cisteinska peptidaza. V naravi se proteini namreč nahajajo predvsem v homodimerni in homotetramerni obliki zaradi manjše topilu dostopne površine, ki poveča njihovo stabilnost. V primeru encimov lahko z oligomerizacijo potencialno izboljšamo njihovo afiniteto do substratov in hkrati dosežemo višjo lokalno koncentracijo aktivnih mest. Z uporabo prostodostopnih bioinformatskih orodij smo identificirali potencialne interakcijske ostanke na površini katepsina B ter z molekulskim umeščanjem in silico ustvarili model homodimera, ki je v stični površini med podenotama vseboval identificirane ostanke. Stično površino smo optimizirali z uvedbo treh hidrofobnih substitucij (H45W, N149F in K166W), s katerimi smo izboljšali stabilnost kompleksa ter povečali zakopano površino med podenotama. S simulacijami molekulske dinamike smo potrdili, da v vodnem topilu načrtan trojni mutant tvori stabilen homodimer. In silico ugotovitve smo nato želeli ovrednotiti še in vitro. Z mestno-specifično mutagenezo smo pripravili tri mutantne zapise za človeški prokatepsin B. Prvi je vseboval mutacijo za substitucijo K166W (enojni mutant), drugi K166W ter H45W (dvojni mutant) in tretji K166W, H45W ter N149F (trojni mutant). Vse tri mutante smo uspešno izolirali v topni obliki ter jim po aktivaciji določili njihove Michaelis-Mentenine konstante za sintetični substrat Z-FR-AMC (25 °C, 100 mM NaOAc, pH 5,5, 1mM EDTA, 5 mM DTT): 136 ± 10 µM za enojnega mutanta, 55 ± 6 µM za dvojnega mutanta in 45 ± 4 µM za trojnega mutanta. V zadnjem delu diplomske naloge smo z gelsko izključitveno kromatografijo poskusili določiti oligomerno stanje aktiviranih mutantov, kjer smo samo za enojnega mutanta nedvoumno lahko trdili, da se nahaja v monomerni obliki.

• Language: Slovenian
• Keywords: oligomerizacija, proteinski inženiring, katepsini
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-142887
• COBISS.SI-ID: 135080707
• Publication date in RUL: 30.11.2022

Secondary language

• Language: English
• Title: Rational design and preparation of dimeric variants of human cathepsin B

Abstract

The aim of protein engineering is to create mutant proteins which, in comparison to their wild type counterparts, are enhanced or in any way modified to fit our needs. In this thesis we attempted to construct a homodimeric variant of human cathepsin B through the application of basic rational protein engineering principles. Most proteins found in nature are homooligomers, namely homodimers and homotetramers. Oligomeric proteins have a smaller solvent-accessible surface than monomers and as a result manage to achieve greater overall stability. In an enzymatic context oligomerization could potentially enhance substrate affinity while simultaneosly increasing the local concentration of active sites. With the use of freely available bioinformatic tools we successfully identified potential interactive residues on the surface of cathepsin B. Using molecular docking we then predicted the homodimeric structure by engaging the determined interactive residues in the contact surface between the two subunits. In the next step we optimized the model's contact surface by introducing three hydrophobic substitutions (H45W, N149F and K166W), thereby increasing the overall stability and buried surface area of the complex. Lastly we performed molecular dynamics simulations in an aqueous solvent and confirmed that our in silico-designed triple mutant does indeed form a stable homodimer. We then attempted to assess our in silico findings experimentally. By applying PCR site-directed mutagenesis we successfully prepared three plasmid vectors each containing a mutated human procathepsin B sequence. The first one contained mutations resulting in a single substitution (K166W), the second two (H45W and K166W) and the third three (H45W, K166W and N149F). Succeding expression we successfully isolated all mutants in soluble form and determined their respective Michaelis-Menten constants for the synthetic substrate Z-FR-AMC (25 °C, 100 mM NaOAc, pH 5,5, 1mM EDTA, 5 mM DTT): 136 ± 10 µM for the single mutant, 55 ± 6 µM for the double mutant and 45 ± 4 µM for the triple mutant. Lastly we sought to determine the oligomeric state of our mutants through size-exclusion chromatography – here we were only able to determine that our single mutant is present as a monomer, while the oligomeric state of the other mutants remains ambiguous.

• Keywords: oligomerization, protein engineering, cathepsins