Priprava in karakterizacija bipiramidalne proteinske kletke iz obvitih vijačnic za vezavo nanoteles

Satler, Tadej (author), Jerala, Roman (mentor), Plavec, Janez (comentor)

Proteini so makromolekule, ki sodelujejo pri skoraj vseh procesih v celici. Zaporedja aminokislin omogočajo tvorbo na milijone kompleksnih struktur proteinov z različnimi funkcijami. Področje oblikovanja de novo proteinov temelji na podlagi fizikalno-kemijskih zakonov ter proteinskih zaporedji, ki se razlikujejo od tistih v naravi. Pri enem izmed pristopov oblikovanja uporabljamo sekundarne strukture in njihove motive kot osnovne gradnike za modularno sestavo in tvorbo prostorske strukture. Primer takšnih gradnikov so α-vijačnice, ki ob interakciji tvorijo motiv obvite vijačnice. Povezovanje parov vijačnic je lahko paralelno ali antiparalelno, specifičnost povezovanja pa določajo hidrofobne in elektrostatske interakcije. Analogno sistemu združevanja komplementarnih verig DNA lahko oblikujemo kompleksne proteinske strukture z modularnim povezovanjem α-vijačnic v motiv obvite vijačnice. Peptidni gradniki so v tem primeru povezani znotraj ene polipeptidne verige, ki se ob združevanju ortogonalnih peptidnih parov zvije v proteinsko kletko iz obvitih vijačnic. Med doslej izvedene oblike proteinskih kletk spadajo tetraeder iz 12 peptidnih segmentov, kvadratna piramida iz 16 ter tristrana prizma in tristrana bipiramida iz 18 peptidnih segmentov. V magistrskem delu smo opisali pripravo in karakterizacijo bipiramidalne proteinske kletke sposobne vezave različnih nanoteles. Dve stranici oblikovane proteinske kletke tvorita peptidna para APH, za katera poznamo, da dobro vežeta različna nanotelesa. Bipiramidalno proteinsko kletko smo uspešno izolirali ter pokazali, da je v raztopini spontano zvita ter ima pričakovano velikost in obliko. Analiza vezave nanoteles je razkrila, da se na proteinsko kletko lahko specifično vežejo tako posamezna nanotelesa, kot tudi več različnih nanoteles hkrati. Tak protein zato predstavlja privlačno orodje za razvoj dostavnih sistemov. Glede na rezultate vezave smo pripravili različne komplekse nanoteles z bipiramidalno proteinsko kletko za kristalizacijo, žal pa nismo uspeli pridobiti proteinskih kristalov primernih za difrakcijsko analizo.

• Language: Slovenian
• Keywords: obvite vijačnice, proteinske kletke, nanotelesa
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2019
• PID: 20.500.12556/RUL-113186
• COBISS.SI-ID: 1538507203
• Publication date in RUL: 11.12.2019

SATLER, Tadej, 2019, Priprava in karakterizacija bipiramidalne proteinske kletke iz obvitih vijačnic za vezavo nanoteles [online]. Master’s thesis. [Accessed 8 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=113186

Secondary language

• Language: English
• Title: Preparation and characterization of bipyramidal protein cage from coiled coils for binding of nanobodies

Abstract

Proteins are the most abundant type of macromolecules that participate in almost every process in the cell. The sequences of simple amino acid monomers enable the formation of millions of complex protein structures with diverse functions. Field of de novo protein design is based on physical laws and protein sequences unrelated to those in nature. One of the design approaches is based on a modular noncovalent pairing of α-helices in coiled-coil secondary structures. Helices within the coiled-coil motif can be coupled in parallel or antiparallel fashion and the specificity of joining is determined by hydrophobic and electrostatic interactions. Analogous to the complementary pairing of DNA molecules, complex modular protein structures can be assembled with the intramolecular pairing of α-helices into coiled-coils. Orthogonal coiled-coil peptides are connected into a single polypeptide chain that upon pairing folds into polyhedron shaped protein cage. Known types of protein cages include a tetrahedron formed by 12 coiled-coil segments, square pyramid formed by 16 and a triangular prism and triangular bipyramid formed by 18 coiled-coil segments. In this thesis, we describe the preparation and characterization of bipyramidal protein cage for binding of nanobodies. Two edges of designed protein cage are formed by APH peptide segments, that were recognized to bind different nanobodies. The bipyramidal protein cage was successfully isolated and biophysically characterized. Protein is folded in solution and has expected size and shape. Binding analysis showed that protein cage could be targeted by individual nanobodies as well as multiple ones simultaneously, presenting an attractive tool for the development of delivery systems. According to the binding results, complexes of nanobodies with bipyramidal protein cage were prepared for crystallization trials, but unfortunately, we were unable to obtain quality protein crystals.

• Keywords: coiled-coil, protein cage, nanobody