Načrtovanje de novo proteinskega konformacijskega stikala na osnovi motiva ovite vijačnice

Aupič, Jana (author), Jerala, Roman (mentor)

Narava je tekom evolucije uporabila le del konformacijskega prostora, ki je na voljo polipeptidnim molekulam. Z načrtovanjem sintetičnih proteinov je mogoče ta nepreizkušen prostor načrtno preiskati in v njem odkriti popolnoma nova proteinska zvitja z novimi, v naravi neopaženimi lastnostmi. Eden izmed trenutnih izzivov na področju dizajniranja proteinov je načrtovanje proteinov, ki ob spremembi okoljskih pogojev spremenijo svoje konformacijsko stanje. Konformacijske spremembe strukture proteinov v odziv na kemijski ali fizikalni signal so osnovni del številnih regulatornih, transportnih in drugih mehanizmov v bioloških sistemih. Zmožnost načrtovanja proteinov, katerih konformacijsko stanje je mogoče natančno in reverzibilno nadzorovati, bi omogočila razvoj naprednih biomaterialov ali molekulskih strojev, prikrojenih za specifične aplikacije. V doktorskem delu smo z načrtovanjem vezavnih mest za kovinske ione razvili konformacijska stikala na osnovi motiva ovite vijačnice. Ovite vijačnice so pogost strukturni motiv v naravnih proteinih, z njimi pa je mogoče zgraditi tudi sintetične proteinske nanostrukture. V prvem delu smo načrtovali peptid poimenovan SwitCCh, ki je v prisotnosti Zn(II) ionov ali pri nizkem pH tvoril paralelen homodimer ovite vijačnice, drugače pa je v raztopini zavzel strukturo naključnega klobčiča. Dodatek Zn(II) ionov je povzročil tvorbo paralelnega homodimera, ob čemer se je temperaturna stabilnost peptida povišala za več kot 30 °C. Prehod med ovito vijačnico in razvitim stanjem je bil reverzibilen in ponovljiv. Peptid SwitCCh je bil ortogonalen glede na predhodno načrtovani set dimerov ovite vijačnice, kar pomeni, da bi ga bilo mogoče uporabiti kot kontrolni element za nadzorovanje zlaganja nanostruktur in materialov na osnovi ovite vijačnice. V drugem delu smo s pomočjo načrtovanja vezavnih mest za kovinske ione v predhodno načrtovani ortogonalen set razvili set Zn(II)-odzivnih ovitih vijačnic. Spektroskopija cirkularnega dikroizma in velikostno izključitvena kromatografija sklopljena s statičnim sipanjem svetlobe pri različnih kotih sta potrdili, da so se peptidi povezali v heterodimer ovite vijačnice le v prisotnosti Zn(II) ionov. Poleg tega so načrtovani peptidi delovali tudi kot pH stikala, saj so nizke vrednosti pH preprečile koordinacijo Zn(II) ionov, kar je vodilo do razvitja ovitih vijačnic. Na osnovi načrtovanega seta ovitih vijačnic smo uspeli pripraviti proteinski trikotnik, katerega zvitje je bilo pod kontrolo Zn (II) ionov. To kaže, da je načrtovani set Zn(II)-odzivnih ovitih vijačnic mogoče uporabiti za razvoj proteinskih kletk na osnovi ovitih vijačnic, katerih zvijanje in razvijanje je mogoče enostavno nadzorovati.

• Language: Slovenian
• Keywords: -
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2020
• PID: 20.500.12556/RUL-117406
• COBISS.SI-ID: 22790915
• Publication date in RUL: 09.07.2020

Secondary language

• Language: English
• Title: De novo design of coiled-coil protein switch

Abstract

De novo protein design represents an exciting opportunity to explore the conformational space unsampled by nature and develop novel protein folds and functionality. One of the current challenges in the protein design field is the design of proteins that change their conformation in response to environmental cues. Conformational change of proteins in response to chemical or physical signals is the underlying principle of many regulatory and transport mechanisms in biological systems. The ability to design proteins whose conformational state can be precisely and reversibly controlled would facilitate the development of smart bio-inspired materials or molecular machines tailored for specific applications. We explored metal-binding site design to engineer peptide-based conformational switches that assemble into a dimeric coiled-coil in response to the addition of Zn(II) ions. Coiled-coil dimers are present in many natural proteins and have been used to construct synthetic protein nanostructures. Firstly, we designed a peptide called SwitCCh that formed a parallel homodimeric coiled-coil in the presence of Zn(II) or low pH. The addition of Zn(II) promoted formation of a parallel homodimer with an increase in thermal stability by more than 30 °C. The peptide could be reversibly cycled between the coiled-coil and random conformation. Furthermore, the SwitCCh peptide was orthogonal to the previously developed coiled-coil dimer set, indicating it could be used for regulated self-assembly of coiled-coil based nanostructures and materials. We further advanced our work by utilizing metal-binding site design to render a previously designed orthogonal set of coiled-coil heterodimers Zn(II)-responsive. Circular dichroism spectroscopy and size exclusion chromatography coupled to multi-angle light scattering confirmed the designed peptides assembled into coiled-coil heterodimers only in the presence of Zn(II). Additionally, designed peptides also acted as pH switches, since low pH prevented coordination of Zn(II) and lead to disassembling of coiled-coils. Our results showed the incorporation of a metal binding site not only preserved orthogonality, but that it is also a viable strategy for increasing the size of orthogonal sets. The designed Zn(II)-responsive coiled-coils were used for the construction of a triangular fold, whose assembly and disassembly was under the control of Zn(II) ions, demonstrating the designed set could facilitate the development of coiled-coil protein cages with easily controllable folding and unfolding.

• Keywords: -