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Modular protein nanostructures

DNA shows the way

Recently, DNA manipulation achieved spectacular results relying only on the base-pairing properties of DNA duplex and a huge amount of talent and imagination. Almost any type of 2D and 3D structure could be assembled relying only on synthetic DNA and self-assembly. Designed nanostructures, composed of intertwined complementary segments form extraordinary patterns and nano-objects ranging from tetrahedron, octahedron, cube, buckyball, dodecahedron to astonishing two dimensional lattices (He et al., 2008) and shaping different patterns by DNA origami. The design and preparation of such assemblies and conditions, under which these constructs form, can be relatively simple due to limited and easily predictable nucleotide interactions.

DNA has been thus tamed to form complex and well-defined designed structures but in fact in nature proteins and not nucleic acids are actually used as embodiments of function and as scaffold for structures.

Pros and cons of polypeptides in comparison to DNA for the embodiment of nanostructures

Polypeptides are composed of 20 different amino acids in comparison to 4 nucleotides in DNA. Those aminoacids confer to polypeptides a range of different properties, from charge, hydrophobicity to different interaction properties and chemical reactivity. Additionally polypeptide backbone allows polypeptides to assume much larger conformational variability or on the other hand restrict it more than nucleic acids. (Zhang, 2003). Pros and cons of using DNA and polypeptides as building material are presented in Table 2.

Approach to creation of modular protein nanostructures

Larger natural proteins are with exception of fibrils composed of folded domains. If we want to prepare the material at the macroscopic scale we have to order polypeptide building blocks into larger assemblies.

One approach is to rely on interactions between oligomerizing polypeptide domains, which can assemble noncovalently, but nevertheless specifically into larger superstructures. Attempts in this direction have already been made, where the oligomerizing domains were linked by rigid linkers as shown to assemble into small aggregates and fibrils (Padilla et al., 2001). This approach provides additional possibilities, such as creation of “smart materials” by regulating the assembly/disassembly by the presence of conditions which promote oligomerization of constituents (explored in the Results section).

The second approach towards building polypeptide nanostructures investigated so far was fibrils made of fragments of natural proteins or designed polypeptides. In these materials, usually small amyloidogenic peptides have been used and resulting structures (fibrils, hollow tubes etc.) did not offer much variety. An alternative building block for creating fibrils were coiled-coil forming segments, which have specificity for pairing with complementary coiled-coil forming segments into intertwined helices.

Coiled-coils

Coiled-coils are protein structural motifs where 2-7 α-helices wrap around each other typically in a left-handed orientation to form an intertwined superhelix. Coiled-coil segments are present in around 8 % of all proteins and are the main constituents of structural proteins keratin, myosin, tropomyosin but also dimeric transcription factors. Left-handed helices require 3.5 residues for each turn, therefore every seven residues or a heptad makes two helical turns, which span approximately 1 nm. This periodicity of sever residues leads to the characteristic requirements to stabilized this intertwined helix for particular residues in each heptad. Those residues are designated (a-b-c-d-e-f-g)n in one helix and (a'-b'-c'-d'-e'-f'-g')n in the other. Residues at positions a and d are usually occupied by nonpolar core residues found at the interface of two helices and are essential for the oligomerization, as they form a hydrophobic core, which contributes to overall stability and also define the oligomerization state of the coiled-coil segment. As leucine residues often occupied position d, this type of proteins is also called leucine zippers. Residue at positions e and g are solvent exposed polar residues at the edge of hydrophobic core that can interact with at positions e’ and g’ on the neighboring helix through electrostatic interactions. Those interactions are quite important for the design of artificial sequences as they can either stabilize or destabilize interactions and therefore also allow for the negative design. Residues b, c and f are typically hydrophilic and exposed to the solvent and may be available to introduce additional functional properties into the coiled-coil (Figure 1).

Figure 1: A) Helical wheel diagram showing interacting residues (a-d' and a'-d interactions between hydrophobic residues and e-e' and g-g' interactions between hydrophilic residues) in an antiparallel coiled-coil, B) 3-D structure of antiparallel coiled-coil determined by X-ray crystallography.

Coiled coils can assemble either in a parallel or antiparallel orientation, with respect to the orientation of polypeptide backbone. They can form either heterodimers or homodimers and all of those combinations have already been designed and verified experimentally for pairs of coiled-coil forming segments. Stability of coiled-coil is determined by the interactions between heptads, orientation and length of the segment. Coiled-coil interactions are probably some of the most well understood types of protein-protein interactions and we can predict the stability of interacting helices with relatively good accuracy (more detailed description in the modeling section) (Mason et al., 2007).

We come to the idea that coiled-coil segments could be used to assemble complex structures in a similar way as DNA assemblies by using coiled-coil segment specificity to govern pairing of the assembly. Coiled-coil segments would represent rigid rods that can form the edges between vertices of polyhedra or network connections of the lattice. All that is required is to prepare a set of coiled-coil forming segments that interact only with the designated coiled-coil forming partners (orthogonal set of coiled-coils). Coiled coil segments are delimited by flexible hinges, made of short peptide linkers between them to allow flexibility required for the assembly. At least three coiled-coil forming segments have to be present within a single polypeptide chain in order to form a 2D or 3D structure.

As an illustration, a tetrahedron (Figure 2) can be formed from a single type of polypeptide containing three coiled-coils which form antiparallel homodimers.

Figure 2: Tetrahedron formed from three antiparallel cioled-coil-forming segments

We developed this idea and discovered its exciting potentials as the complex structures can be formed already with very simple combinations. We have explored this idea in more detail in the Modeling section and proof of the principle of one specific design was experimentally confirmed, creating the first report of a coiled-coil based polypeptide lattice.

The idea of combining modular protein domain and/or coiled-coil forming segments into nanostructures was the background of our project to develop modular nanoBricks as the library that will allow to expand the universe of possible combinations of building nanostructures. In order to move beyond a mere proof of the principle we also developed a real world application based on formation of polypeptide membrane, which has been demonstrated to function for ultrafiltration of molecules and viruses. Application of the underlying idea of this project could be further developed by manufacturing numerous structures such as nano-cages, nano-networks and nano-wires. Additional advantage of such nanostructures built from polypeptide-forming coiled-coils are optionally added functional groups which can be introduced at solvent exposed residues b, c and f (e.g. cysteines for covalent modification of SH group or histidines for use as chelators of metal ions). These nanostructures can be used for diverse purposes such as drug delivery, chemical catalysis, metal binding, formation of conducting circuits and much, much more.