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What are E/K Coils?

E/K coils are synthetic coiled-coil domains designed specifically to bind to each other with high affinity and specificity (Litowski and Hodges, 2002) (Figure 1). They are composed of a heptad repeat that forms a coil structures that are able to interact with each other. These coils are able to interact with each other in an anti-parallel fashion that makes them useful for applications such as peptide capture, protein purification and in biosensors. For our project we decided to make use of the IAAL E3/K3 coils due to the balance they offer between affinity and specificity (Table 1).

How do these Coils Work?

These E3/K3 coils are able to form heterodimers due to the hydrophobic residues contained within the heptad repeat. In our case these are isoleucine and leucine residues. Designated by empty arrows in the helical wheel diagram below (Figure 2) these residues form the core of the binding domain of the coils. In order to prevent the homodimerization of these coils charged residues are included in the design. The electrostatic interactions between glutamic acid and lysine residues prevent an E-coil from binding with an E-coil for example. We selected the use of E3/K3 coiled-coils over other synthetic E/K coils as the isoleucine residue present shows a significant increase in the heterodimer over valine found in other coils. The alpha-helical propensity of the residues outside of the core interacting residues is also increased by utilizing an alanine residue instead of the serine residue found in ISAL and VSAL E/K coils. This selection maximizes the stability and specificity of the coils used in our system.

Ferritin Scaffold

One of the previously mentioned coils, the K-coil, will be attached to ferritin a ubiquitous 24-subunit iron storage protein. By attaching an E-coil to our protein of choice, TAL, we can fuse this protein to our ferritin scaffold. Why is this so significant..? why did we bind TALs to ferritin in the first place By using this method, we can bind multiple reporter proteins to the ferritin as well in order to amplify our output signal. In addition, the use of coils for fusions avoids the possibility of our large fusion protein causing problems in protein folding and nanoparticle assembly.

Upon further thought into this system, we determined the ferritin-coiled-coil system coudl be used for much more than the detectino of our one E.coli sequence. We can use this system to detect other sequences of DNA by exploiting the modifiability of the TAL system, along with the interchangeabillity of the coiled-coil system. Thus by attaching E-coils to TALs that target alternative sequences, we can use this system as a platform for a huge variety of DNA sequences. Furthermore, we can expand this system towards proteins and other molecules as well by replacing the TALs with antibodies or other binding proteins. This system shows the potential to bind a large number of ligands, only being restricted by the number of binding proteins that are currently known.

Another aspect of the ferritin nanoparticle that can be exploited is its composition, being composed of 24 subunits, as well as its potential as a novel reporter. Ferritin is typically composed of two chains, heavy and light. In our system, each one of these subunits can be bound to a reporter, effectively amplifying our catalytic activity by 24 due to the increased number of bound reporter proteins. But this activity can be scaled down through previously reporterd heavy-light chain fusions (Huh and Kim 2003). Rather than 24 subunits the nanoparticle would be reduced to 12 subunits, and thus the number of reporter proteins would decrease accordingly. We can scale this down even further due to our ability to transform the iron core of the protein into a catalytically active substance called Prussian Blue (Zhang 2012). Thus instead of multiple bound reporter proteins we can further scale down the output of our system to a system with essentialy one reporter. The ability of our system to scale up and down according to what is required means this system is applicable in areas ranging from low sensitivity to high sensitivity.