Načrtovanje in priprava samosestavljivih proteinskih polimerov na osnovi spektrinskih domen

Stell Pičman, Adrian Samuel (author), Jerala, Roman (mentor)

V sklopu raziskovalne naloge smo zasnovali modularne proteinske gradnike na osnovi spektrinskih ponovitev in ortogonalnih peptidov, ki tvorijo motive ovitih vijačnic (OV) z namenom, da se lahko samo-sestavijo v geometrijsko urejene visokomolekularne strukture. Izhajajoč iz para Dy3BB53:46, ki tvori filamente, smo zasnovali spremenjene različice proteinskih gradnikov, ki vsebujejo več peptidov OV. Z načrtno postavitvijo peptidov v proteinsko strukturo, smo predvideli samo-sestavljanje v premrežene oziroma razvejane strukture. Proteine smo proizvedli v E. coli in jih očistili z afinitetno kromatografijo Ni-NTA (NiNTA) ter z kromatografija z ločevanjem po velikosti (SEC). Nato smo jih karakterizirali z poliakrilamidno gelsko elektroforezo v prisotnosti natrijevega dodecilsulfata (NaDS-PAGE) in spektroskopijo cirkularnega dikroizma (CD). Polimerizacijo smo sprožili z dializo ali mešanjem predhodno dializiranih komponent. Dializo smo izvedli za zmanjšanje koncentracije soli in odstranitev glicerola. Kinetiko samo-sestavljanja in velikost struktur v raztopini smo spremljali z dinamičnim sipanjem svetlobe (DLS), poliakrilamidno gelsko elektroforezo v nativnih pogojih (nativna-PAGE) pa smo uporabili za potrditev tvorbe visokomolekularnih struktur. Z mikroskopijo na atomsko silo (AFM), interferenčno odbojno mikroskopijo (IRM) in fluorescenčno mikroskopijo s popolnim notranjim odbojem (TIRFM) smo razkrili raznolike in kompleksne samo-sestavljene strukture, vključno s filmi, razvejanimi filamenti in premreženji. Tako potrjujemo hipotezi, da se zasnovani proteini samo-sestavijo v visokomolekularne strukture in da so omenjene visokomolekularne strukture bistveno bolj razvejane in/ali premrežene od struktur, ki jih tvori izhodiščni proteinski par Dy3BB53:46. Z meritvijo AFM smo dokazali, da sušenje, kristalizacija soli in mehanske sile, povzročene zaradi spiranja, lahko vplivajo na končno obliko proteinskih struktur. Trenutno pomanjkljivo razumemo vpliv pogojev priprave vzorcev na končno morfologijo visokomolekularnih struktur. Kljub temu raziskava predstavlja obetaven korak k načrtovanju kompleksnih bioloških polimerov z načrtovano arhitekturo.

• Language: Slovenian
• Keywords: spektrinske ponovitve, distrofin, ovite vijačnice, samosestavljanje, proteinski polimeri
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [ A. S. Stell Pičman]
• Year: 2025
• PID: 20.500.12556/RUL-173119
• UDC: 544.431.7(043.2)
• COBISS.SI-ID: 248870915
• Publication date in RUL: 13.09.2025

Secondary language

• Language: English
• Title: Design and preparation of self-assembling protein polymers based on spectrin-like domains

Abstract

As part of our research project, we designed modular protein building blocks based on spectrin repeats and orthogonal coiled-coil (CC) peptides. The aim was to enable their self-assembly into geometrically ordered high-molecular-weight structures. Starting from the Dy3BB53:46 pair, which forms filaments, we designed modified versions of the protein building blocks containing multiple CC peptides. By strategically placing these peptides within the protein structure, we predicted self-assembly into cross-linked or branched structures. We produced the proteins in E. coli and purified them using Ni-NTA affinity chromatography and size-exclusion chromatography (SEC). They were then characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism (CD) spectroscopy. Polymerization was initiated by dialysis or by mixing previously dialyzed components. Dialysis was performed to reduce salt concentration and remove glycerol. The kinetics of self-assembly and the size of the structures in solution were monitored by dynamic light scattering (DLS), while native-PAGE was used to confirm the formation of high-molecular-weight structures. Atomic force microscopy (AFM), interference reflection microscopy (IRM), and total internal reflection fluorescence microscopy (TIRFM) revealed diverse and complex self-assembled structures, including films, branched filaments, and cross-links. This confirms our hypotheses that the designed proteins self-assemble into high-molecular-weight structures and that these high-molecular-weight structures are significantly more branched and/or cross-linked than the structures formed by the initial Dy3BB53:46 protein pair. AFM measurements demonstrated that drying, salt crystallization, and mechanical forces caused by rinsing can influence the final shape of the protein structures. We currently have an incomplete understanding of how sample preparation conditions affect the final morphology of the high-molecular-weight structures. Despite that, this research represents a promising step towards designing complex biological polymers with tailored architectures.

• Keywords: spectrin-like repeats, dystrofin, coiled-coils, self-assembly, protein polimers