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ERC Advanced Grant MaCChines: Molecular machines based on coiled-coil protein origami; Principal investigator: prof. dr. Roman Jerala

Background

 

The aim of MaCChines is to advance design of modular proteins based on coiled-coil building modules (Coiled-coil protein origami CCPO) and take advantage of the unique properties of this platform. This type of de novo designed proteins are defined by the sequence of coiled-coil (CC) dimer-forming modules that are concatenated by flexible linkers into a single polypeptide chain that self-assembles into a polyhedral cage based on pairwise CC interactions (Gradišar et al., NatChemBiol 2013; Ljubetić, Lapenta et al., NatBiotech 2017).

These programmable molecules have many properties - they can be produced by cell factories in an energy- and resource-effective and sustainable manner, due to their structure defined at the nanoscale we can expect them to be efficient in recognition, delivery, and catalysis and to find applications in medicine, biotechnology and other fields.

The objective of MaCChines is to build foundations of a new branch of protein design strategy, develop and exploit unique features of de novo designed coiled-coil protein origami (CCPO), in order to enable new drug delivery technologies, construction of complex nanoscaffolds, new materials, sensors, rational design of molecular machines and new advanced therapeutics.

 

Results

Within the first half of the project we can report advances on several exciting directions along the proposed directions of the ERC AdG MaCChines project and also along some new ideas that have been conceived and realized during this project.

Several types of functionalized CC modules were developed and implemented:

  • New metal and pH dependent coiled—coil modules
    AUPIČ, Jana, LAPENTA, Fabio, JERALA, Roman. SwitCCh : metal-site design for controlling the assembly of a coiled-coil homodimer. ChemBioChem. 2018, vol. 19, 2453-2457
  • Fusion of CC modules with split proteases to generate fast responsive cellular logic
    FINK, Tina, LONZARIĆ, Jan, PRAZNIK, Arne, PLAPER, Tjaša, MERLJAK, Estera, LEBEN, Katja, JERALA, Nina, LEBAR, Tina, STRMŠEK, Žiga, LAPENTA, Fabio, BENČINA, Mojca, JERALA, Roman. Design of fast proteolysis-based signaling and logic circuits in mammalian cells. Nature chemical biology. 2019, vol. 15, 115-122
  • Application of CC modules for modulation of localization and regulation of biochemical/biological processes
    LEBAR, Tina, LAINŠČEK, Duško, MERLJAK, Estera, AUPIČ, Jana, JERALA, Roman. A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells. Nature chemical biology. 2020, vol. 16, 513-519

We introduced several new strategies of CCPO assembly:

  • Multichain CCPO assembly
    LAPENTA, Fabio, AUPIČ, Jana, VEZZOLI, Marco, STRMŠEK, Žiga, DA VELA, Stefano, SVERGUN, Dmitrii Ivanovich, CARAZO, José María, MELERO, Roberto, JERALA, Roman. Self-assembly and regulation of protein cages from pre-organised coiled-coil modules. Nature communications. 2021, vol. 12, 1-12
  • Design of the designed protein folding pathway and use of multiple copies of the same type of the CC peptide
    AUPIČ, Jana, STRMŠEK, Žiga, LAPENTA, Fabio, PAHOVNIK, David, PISANSKI, Tomaž, DROBNAK, Igor, LJUBETIČ, Ajasja, JERALA, Roman. Designed folding pathway of modular coiled-coil-based proteins. Nature communications. 2021, vol. 12, 1-12

Designed coiled-coil peptides were applied for tethering of Cas9 and exonuclease in a noncovalent manner, which significantly improved the efficiency of gene inactivation with potential applications in medicine, agriculture, biotechnology. We filed a patent application: Coiled-coil mediated tethering of CRISPR-CAS and exonucleases for enhanced genome editing: European Patent Application EP19192490.1, 2019-08-20. München: European Patent Office. This invention was the foundation of the ERC PoC project CCEdit, that started in 2020, within which we are further developing and translating this invention towards tangible applications.

 

Principal investigator: prof. dr. Roman Jerala

ERC MaCChines Team

dr. Helena Gradišar, dr. Ajasja Ljubetič, dr. Arvind Kumar Gupta, dr. Weijun Zhou, Žiga Strmšek

Maruša Ramšak, Sara Vidmar, Hana Esih, Jaka Snoj, Klemen Mezgec, Tadej Satler

 

Acronym: MaCChines

Project ID: 787115

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