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Self-assembling protein nanostructures
as the new platform for designed biomimetic smart materials Dr. Helena GRADIŠAR NIC, Department of synthetic biology and immunology EN-FIST, Centre of excellence CENTRE OF EXCELLENCE
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Department of synthetic biology and immunology
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Department of synthetic biology and immunology
Designed protein nanostructures DNA-based biosynthetic scaffolds Design of cellular logic circuits in mammalian cells Medical applications of synthetic biology Molecular mechanism of innate immunity Mechanism of signaling of Toll-like receptors Mechanism of activation of cytosolic inflammasomes MyD88 in cancer and autoimmunity Translational medicine: inhibitors, activators for vaccines and cancer therapy Nature Chemical Biology Nature Communications Nucleic Acid Research Molecular Therapy Nature Structural and Molecular Biology Nature Medicine Blood
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Protein nanostructures – rapid development
Over the last 15 years the field of protein assembly has undergone unexpected and rapid developments, and various inovative strategies have been proposed. Researchers used different buildig blocks, from fusions of different natural protein domains to synthetic molecules. They have been constructed and fabricated a variety of self-assembled nanomaterials, nanotubes, nanofibers, ring-like structures, spherically shaped structures, layers and crystals. They have been introduced a wide range of applications, such as nanodevices, biocatalysis and drug delivery, as well as therapy and diagnosis. Luo et al., Protein assembly: versatile approaches to construct highly ordered nanostructures, Chem Reviews, 2016
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Modular topological nano-cage – INNOVATIVE STRATEGY
We contributed to this exciting field with an innovative strategy. We developed the platforma for producing self-assembling structures from a single polypeptide chain chain. Luo et al., Protein assembly: versatile approaches to construct highly ordered nanostructures, Chem Reviews, 2016
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Programmable building blocks – Coiled-coils
Parallel dimer Antiparallel dimer We establish a set of orthogonal coiled coil- forming peptide pairs. First we had to constructed nano-cage we had to choose the building blocks. The coiled coils are very attractive from this point of view. We establish and tested a set of orthogonal pairs, which means that prticular peptide forms coiled-coil only with its designed partner. And now the question arises: how to build the tetrahedron from these building blocks?
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Self-assembling nanostructure
Design of tetrahedron- like cage from a single chain Sequential order of modules Toolbox of designed coiled coil-forming modules Deconstruction of a polyhedron into rigid modules Advantages: precise positioning of functional groups cavity bounded by coiled-coils adjustable shape and size Self-assembling nanostructure We reasoned that the polypeptide self-assembly problem could be circumvented by deconstructing the designed structural assembly into smaller independently-folded building elements whose interactions with other building elements are well understood and predictable Using Mathematical modeling and graph theory we came to topological solution We assumed that this type of structure posess many advantages:
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Production and isolation of polypeptide
Amino acid sequence MKQLEKELKQLEKELQAIEKQLAQLQWKAQARKKKLAQLKKKLQASGPGSPEDEIQQLEEEIAQLEQKNAALKEKNQALKYGSGPGDIEQELERAKASIRRLEQEVNQERSRMAYLQTLLAKSGPGQLEDKVEELLSKNYHLENEVARLKKLVGSGPGMKQLEKELKQLEKELQAIEKQLAQLQWKAQARKKKLAQLKKKLQASGPGSPEDEIQALEEKNAQLKQEIAALEEKNQALKYGSGPGQLEDKVEELLSKNYHLENEVARLKKLVGSGPGSPEDKIAQLKQKIQALKQENQQLEEENAALEYGSGPGSPEDENAALEEKIAQLKQKNAALKEEIQALEYGSGPGSPEDKIAQLKEENQQLEQKIQALKEENAALEYGSGPGDIEQELERAKASIRRLEQEVNQERSRMAYLQTLLAKSGPGSPEDKNAALKEEIQALEEENQALEEKIAQLKYGHHHHHHHH Production in E.coli Isolation Purity Single chain, concatenated modules. We produced and isolated pure polypeptide.
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Confirmation of tetrahedral
Characterization of structure Helical content Chemical stability Size determination Confirmation of tetrahedral nanostructure Gradišar et al.,Nature Chemical Biology, 2013 Comments in Nature, Science, Nature Biotechnology, C&E News… We characterized the obtained structures using different methods. High helical content. Higher stability in comparison to individual building modules. We Published the invention of new topological type of modular designed protein fold. The paper has been featured in comments in Nature...and many others. Visuatization by TEM and AFM
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Coiled-Coil Protein Origami Design platform (CoCoPOD)
Further we developed the computational platform that enables design of different polyhedra, computational simulations. The platform is roboust and flexible.
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Next generation of self-assembling polyhedral cages
(brez razlaganja, samo hiter prikaz) We designed different polyhedral structures. Produced them and characterized. Stability, folding kinetics, The structures with cavity, saxs (small angle Xray scattering) that contains the information about shape and size of particles.
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In vivo folding of nanocage – advantage for applications
BMDM Next step was to check if polyhedral structures can be formed in vivo. We have demonstrated that despite non-natural folds these protein structures are not perceived by mammalian cells as a foreign molecule and could be used safely. In vivo folding of the designed protein origami, not just in bacteria but also in mammalian cells and even in mice represents a major advantage for biomedical applications. Not stimulate the immune system. We showed that protein nanocages are biocompatible and suitable for biomedical applications. High biocompatibility – biomedical applications
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Functionalization of designed polyhedra smart materials
tethered assemblies shape and size of the scaffold and cavity regulated assembly/disassembly engineered binding and functional sites outside and/or inside of the cavity Introduction of functionalities leads to development of smart materials. There is a lot options how to functionalize the cages. Currently we are studying the functionalization of these structures. - opening/closing the cage by external conditions Ability to introduce functional residues, chemical groups and protein domains into selected positions Tethering protein assemblies into nanomaterials with selected geometry and stoiciometry
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Smart materials medical and biotechnological applications
Targeted delivery to selected tissue Designed vaccines Enzyme encapsulation Biocatalysis Smart functional materials Sensors Switches .... As smart materials the self-assembling nanocages have promising medical and biotechnological potential. They can be used for tergeted delivery Or designed for vaccines We can encapsulate the enzymes Biocatalysis improvement
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Conclusions Polyhedral protein nanostructures – advanced biomaterials with a great potential for apllications Production of is cost-effective and sustainable Current challenges are the size and functionalization of designed nanostructures Great expertise and infrastructure at NIC
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Projects SRA (ARRS) projects ERA SynBio (2014-2017)
BioMolecular Origami: establishing foundations in structural synthetic biology to engineer biomolcules for new routes to nanoscale objects and biomaterials Coordinator: prof. Jerala NIC; Partners: Oxford, Bristol, TUM, UW Seattle, UCLA M-Era.NET ( ) MediSURF: Designed nanostructured bioactive surfaces for precision medicines Partners: Leiden, Eindhoven, Bilbao, NIC On-going projects from the field of bionanomaterials
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Acknowledgement Prof. Dr. Roman JERALA, Head of Department
Dr. Ajasja Ljubetič Fabio Lapenta Dr. Igor Drobnak Jana Aupič Žiga Strmšek Dr. Andreja Majerle Nuša Krivec Mammalian cells: Dr. Mojca Benčina Dr. Iva Hafner Bratkovič Mice: Duško Lainšček Ex-colleagues: Dr. Vid Kočar Dr. Sabina Božič Tibor Doles Karen Butina Prof. Dr.Tomaž Pisanski Dr. Nino Bašič
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