IN VIVO SELF-ASSEMBLING COILED-COIL-BASED PROTEIN ORIGAMI

Lapenta, Fabio (author), Jerala, Roman (mentor)

Proteins are complex biopolymers that fold in a large array of nanostructures. Although resolved natural protein structures span over a large conformational landscape, a much wider sampling space, formed by polypeptides that nature has not sampled, is yet to be explored. The ability of designing novel protein structures from scratch, de novo protein design, offers a way to sample the unknown part of protein sequence-structure landscape and test new protein architectures. This thesis encompasses some of the recent advances concerning a modular approach to protein de novo design, coiled coil protein origami (CCPO) design, a strategy based on specific arrangements of coiled coil units within a polypeptide chain, which fold into polyhedral cages. Solubility and overall expression level of these cages were increased, which allowed the implementation of a purification protocol that did not require refolding passages and in turn permitted large-scale isolations and biophysical characterization. Variants of a soluble 12 coiled coil segment tetrahedral cage served as starting scaffold for initial characterization and for refining the design principles. Consequently, larger coiled coil cages such as a 16-segment coiled coil square-pyramid and an 18-segment trigonal prism were isolated, characterized and their conformation was analysed via Small Angle X-ray Scattering (SAXS) and Transmission Electron Microscopy (TEM) analysis. Ultimately, choosing to work on an 18-segment trigonal bipyramidal cage, we designed such fold both as a single chain polypeptide and as heterodimeric complex. Different strategies for oligomeric assembly were tested and a correctly folded heterodimeric bipyramidal complex was obtained upon self-assembly of different heterodimers. In addition, we devised a proteolysis-triggered conformational rearrangement of two subunits into a heterodimeric bipyramid. SAXS analysis confirmed the CCPO cages folding in accordance with our design. Overall, these implementations represented a leap forward in the increasingly expanding field of protein design, demonstrating the potential of a modular coiled coil based design.

• Language: English
• Keywords: Protein de novo design, coiled coil, protein cages
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2019
• PID: 20.500.12556/RUL-113428
• COBISS.SI-ID: 34550233
• Publication date in RUL: 03.01.2020

Secondary language

• Language: Slovenian
• Title: IN VIVO SAMOSESTAVLJANJE PROTEINSKEGA ORIGAMIJA NA OSNOVI OBVITIH VIJAČNIC

Abstract

Doslej znane proteinske strukture pokrivajo velik del konformacijskega prostora, ki je na voljo polipeptidni verigi. Kljub temu lahko slednja vsaj teoretično zavzame še veliko število drugačnih konformacij, ki jih narava ni vzorčila. Načrtovanje novih proteinskih struktur brez zanašanja na obstoječe strukture, tako imenovano proteinsko načrtovanje de novo, omogoča raziskovanje te, še ne popolnoma znane, povezave med aminokislinskim zaporedjem in proteinsko strukturo ter testiranje novih proteinskih arhitektur. Predloženo doktorsko delo predstavlja zadnje napredke na področju modularnega načrtovanja proteinov de novo na osnovi ovitih vijačnic. Razvita strategija, ki smo jo poimenovali proteinski origami, temelji na pravilnem povezovanju peptidov, ki tvorijo dimere ovite vijačnice, v polipeptidno verigo, ki se nato ob parjenju peptidov zvije v proteinsko kletko v obliki poliedra. Pomemben korak k posplošitvi metode proteinskega origamija za načrtovanje poljubnega poliedra je bilo povečanje topnosti in izražanja proteinov v bakterijskih celicah. Slednje je omogočilo razvoj postopka izolacije, ki ni temeljil na renaturaciji proteinov iz netopne frakcije in je tako omogočil izolacijo in biofizikalno karakterizacijo proteinov na večji skali. Priprava in karakterizacija več različic tetraedrične proteinske kletke, sestavljene iz 12 peptidnih gradnikov, sta nama omogočili oblikovanje pravil za načrtovanje proteinskih origamijev. Z upoštevanjem odkritih načel smo nato pripravili kvadratno piramido sestavljeno iz 16 peptidnih gradnikov in trigonalno prizmo z 18 peptidnimi gradniki. Obliko in velikost večjih nanostruktur smo potrdili z meritvami ozko kotnega sipanja rentgenskih žarkov (SAXS) in presevno elektronsko mikroskopijo (TEM). Poleg tega smo načrtovali tudi kletko v obliki trigonalne bipiramide, ki je vsebovala 18 peptidnih gradnikov. Tarčno zvitje smo skušali doseči z načrtovanje pravilnega zvitja ene same polipeptidne verige, kot tudi na osnovi pravilne interakcije dveh manjših podenot. Preizkusili smo več strategij oligomernega sestavljanja, pri čemer je le ena vodila do pravilno zvite heterodimerne bipiramide. Razvili smo tudi način za enostaven nadzor interakcije med manjšima podenotama na osnovi proteolitske cepitve. Z meritve SAXS smo potrdili, da so pripravljeni proteini zavzeli obliko trigonalne bipiramide. Vpeljane izboljšave metode proteinskega origamija predstavljajo pomemben napredek na področju načrtovanja proteinov in kažejo na velik potencial metode za razvoj kletk v medicinske in biotehnološke namene.

• Keywords: de novo načrtovanje proteinskih struktur, obvite vijačnice, proteinske kletke