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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 DNA-binding protein, TAL, we can fuse proteins to our ferritin scaffold while avoiding the possibility of large fusion proteins interfering with protein folding and nano-particle assembly. But upon further thought into this system, we determined the ferritin-coiled-coil system is not only specific for detecting the E.coli sequence, but can expanded further as well. We can use this system to detect other sequences of DNA by swapping our E.coli TAL for a TAL that binds a different sequence, such as _________________-. Furthermore, we can expand this system towards proteins as well, by replacing the TALs with antibodies. Our system shows the potential to bind a limitless number of ligands, only being limited by the number of known binding-proteins.

Our scaffold protein, ferritin is typically composed of two subunits, the heavy and light chains. It has been previously shown that these two chains can be fused together to reduce the number of subunits involved in the nanoparticle, and thus reduce the number of proteins bound to it. Therefore depending on how we construct our ferritin, we can use this scaffold to scale the number of bound proteins from 24 to 12. We can one more step further and scale the reporter down aspect of the protein even more through the use of ferritin itself as a reporter. This unique property of our system means it can be used in a wide range of areas, ranging from low to high sensitivity applications.