2. 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

3. Department of synthetic biology and immunology

4. 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

5. 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

6. 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

7. 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?

8. 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:

9. 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.

10. 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

11. 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.

12. 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.

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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