Raziskovanje vpliva ciklizacije peptida GSYWYNVWF na njegovo afiniteto do človeških imunoglobulinov G

Nabernik, Urša (avtor), Bratkovič, Tomaž (mentor), Bozovičar, Krištof (komentor)

Peptidi so skupina molekul, sestavljenih iz dveh ali več aminokislinskih ostankov, povezanih s peptidno vezjo. So vsestransko uporabni, med drugim igrajo pomembno vlogo afinitetnih ligandov imunoglobulinov (Ig) in so dobra alternativa naravnim Ig-vezavnim proteinom. Ker imajo bolj preprosto zgradbo v primerjavi s proteini, so bolj odporni na ostre pogoje čiščenja afinitetne kromatografske kolone, hkrati pa je njihovo pridobivanje cenejše. Peptidni afinitetni ligandi se selektivno in razmeroma močno vežejo na različne biološke molekule. Zlasti ciklični peptidi izkazujejo veliko selektivnost in afiniteto vezave, saj imajo manj dostopnih konformacij. Zaradi svoje togosti so lahko boljši ligandi od njihovih linearnih različic (če je vezavna konformacija podobna tisti, ki jo ciklični peptid zavzame v raztopini, pride namreč ob interakciji z vezavnim partnerjem do manjšega znižanja entropije, kar je termodinamsko ugodno). Vendar vsaka ciklizacija ne privede nujno do izboljšane afinitete. Nasprotno, lahko celo zelo poslabša afiniteto, če konformacija ob ciklizaciji postane neugodna. Zato je pri načrtovanju takih peptidnih različic smiselno postopno preverjati, kako določene spremembe v strukturi peptida vplivajo na afiniteto. Pri identifikaciji peptidov z visoko afiniteto večkrat uporabljamo tehniko predstavitve na bakteriofagu. To je robustna tehnika, ki olajša identifikacijo močnih in selektivnih peptidnih ligandov za ciljno tarčno makromolekulo. V magistrskem delu smo raziskali vpliv ciklizacije peptida GSYWYNVWF na njegovo afiniteto vezave. Na osnovi linearnega peptida, pridobljenega v pretekli razskavi, smo načrtovali nove ciklične peptidne različice, ki bi bile zmožne še močnejše vezave na regijo Fc IgG v primerjavi z linearnim peptidom. Zasnovane peptide smo predstavili na bakteriofagu ter ovrednotili njihovo vezavno afiniteto s testom ELISA. S takšnim pristopom smo uspešno odkrili peptid 6,11 Cys z aminokislinskim zaporedjem GSYWYCVWFGC (kjer sta cisteinska ostanka povezana z disulfidno vezjo), ki je izkazoval višjo afiniteto do IgG v primerjavi z njegovo linearno različico. Ugotovili smo, da sprememba mesta ciklizacije za samo eno mesto zelo vpliva na konformacijo peptida, ki postane neugodna in ima velik vpliv na vezavo. Ker je načrtovanje peptidov zamudno in ne obljublja želenih rezultatov, smo za lažje iskanje cikličnih peptidov z visoko afiniteto do IgG predlagali delno degeneriran oligonukleotid za pripravo kombinatorične knjižnice. S takim pristopom ohranjamo določene aminokisline na mestih, ki so pomembne za afiniteto peptida, vsa ostala mesta pa so popolnoma randomizirana. Na podlagi rezultatov smo za namen prihodnjih testiranj predlagali oligonukleotid, ki kodira peptidno zaporedje GXYWYCVWFXC, pri čemer X predstavlja randomizirani mesti, na katerih se lahko pojavi katerakoli od proteinogenih aminokislin.

• Jezik: Slovenski jezik
• Ključne besede: Ciklični peptidi, ligandi, afiniteta vezave, predstavitev na bakteriofagu, ELISA
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2022
• PID: 20.500.12556/RUL-140213
• Datum objave v RUL: 13.09.2022

NABERNIK, Urša, 2022, Raziskovanje vpliva ciklizacije peptida GSYWYNVWF na njegovo afiniteto do človeških imunoglobulinov G [na spletu]. Magistrsko delo. [Dostopano 3 julij 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=140213

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Investigating the effect of GSYWYNVWF peptide cyclization on its affinity for human immunoglobulins G

Izvleček

Peptides are a group of molecules consisting of two or more amino acid residues linked by a peptide bond. They are very useful, among other things they play an important role as affinity ligands of immunoglobulins (Ig) and represent a good alternative to natural Ig-binding proteins. Due to their simpler structure compared to proteins, they are more resistant to harsh cleaning conditions of the affinity chromatographic column. In addition, they can be produced cost-efficiently by chemical synthesis. Peptides as affinity ligands bind selectively and relatively strongly to different biological molecules. Specifically, cyclic peptides exhibit high selectivity and binding affinity as they have fewer accessible conformations. Due to their rigidity, they are often better ligands compared to their linear variants (if the binding conformation is similar to the one that the cyclic peptide occupies in the solution, there is only a small decrease in entropy upon target binding, which is thermodynamically favorable). However, not every cyclization necessarily leads to an improved affinity. On the contrary, it may even impair affinity if the conformation becomes unfavorable upon cyclization. Therefore, when designing such peptide variants, it makes sense to gradually examine how certain changes in the peptide structure affect affinity. The bacteriophage display technique is often used to identify high-affinity peptides. It is a robust technique that facilitates the identification of strong and selective peptide ligands for the target macromolecule. In this master's thesis, we investigated the influence of GSYWYNVWF peptide cyclization on its binding affinity. Based on the linear peptide obtained in the previous study, we designed several cyclic variants and assessed their binding affinity for IgG Fc region relative to that of the linear parent peptide. The designed peptides were presented on a bacteriophage and their binding affinity was evaluated by phage ELISA assay. With this approach, we found that the 6,11 Cys peptide with the amino acid sequence GSYWYCVWFGC (where cysteine residues are linked by a disulfide bond) showed a higher affinity for IgG compared to its linear variant. We noticed that changing the cyclization site for one site only greatly affects the conformation of the peptide, which becomes unfavorable and has a large negative effect on binding. Because peptide design is time-consuming and does not necessarily lead to ligands with desired properties, we proposed a partially degenerated oligonucleotide to prepare a phage-displayed combinatorial peptide library to ease the search for cyclic peptides displaying high affinity for IgG. With this approach, we preserve certain amino acids at sites that are important for peptide affinity and all other sites are completely randomized. Specifically, we proposed an oligonucleotide encoding the peptide sequence GXYWYCVWFXC for future testing, with X representing randomized sites where any of the proteinogenic amino acids may occur.

• Ključne besede: Cyclic peptides, ligands, binding affinity, phage display, ELISA