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Team:Calgary/Project/OurSensor/Linker
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<p><b>Figure 3.</b> This basic qualitative assay was used to inform us whether certain elements of our system are able to bind to each other. Our TALE proteins were mounted to the membrane along with positive controls of three Prussian blue variants; two recombinant ferritins and one commercial protein. The membranes were then washed and blocked. Prussian blue ferritin with a coil was added to our TALE protein containing a coil. Prussian blue ferritin with a TALE that could bind to the DNA held by another TALE on the membrane was also added. A TMB substrate solution was added to cause a colourimetric change over 5 minutes. Positive results are indicated by dark rings of colour. Negative controls include a TALE with a coil on the same membrane and the same TALE and bovine serum albumin on separate membranes that were treated separately.</p> | <p><b>Figure 3.</b> This basic qualitative assay was used to inform us whether certain elements of our system are able to bind to each other. Our TALE proteins were mounted to the membrane along with positive controls of three Prussian blue variants; two recombinant ferritins and one commercial protein. The membranes were then washed and blocked. Prussian blue ferritin with a coil was added to our TALE protein containing a coil. Prussian blue ferritin with a TALE that could bind to the DNA held by another TALE on the membrane was also added. A TMB substrate solution was added to cause a colourimetric change over 5 minutes. Positive results are indicated by dark rings of colour. Negative controls include a TALE with a coil on the same membrane and the same TALE and bovine serum albumin on separate membranes that were treated separately.</p> | ||
Revision as of 09:08, 27 September 2013
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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).
Figure 1. Ribbon visualization of the E3/K3 IAAL coiled-coils.
| Coil Name | Peptide Sequence |
| IAAL E3 | NH2-EIAALEKEIAALEKEIAALEK-COOH |
| IAAL K3 | NH2-KIAALKEKIAALKEKIAALKE-COOH |
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.
Figure 2. A helical wheel representation of the IAAL E3/K3 coiled-coil heterodimer viewed as a cross-section based off of a similar figure created by Litowski and Hodges (2002). The peptide chain propagates into the page from the N to the C terminus. Hydrophobic interactions between the coils are indicated by the clear wide arrows. The intermolecular electrostatic interactions between the coils are displayed by the thin curved arrow (eg. Between Glu15 and Lys20)Letters a, b, c,and d designate the positions of IAAL repeat in the heptapeptide. THe e and g positions are occupied by the charged residues.
So do these Coils Actually Bind?
Figure 3. This basic qualitative assay was used to inform us whether certain elements of our system are able to bind to each other. Our TALE proteins were mounted to the membrane along with positive controls of three Prussian blue variants; two recombinant ferritins and one commercial protein. The membranes were then washed and blocked. Prussian blue ferritin with a coil was added to our TALE protein containing a coil. Prussian blue ferritin with a TALE that could bind to the DNA held by another TALE on the membrane was also added. A TMB substrate solution was added to cause a colourimetric change over 5 minutes. Positive results are indicated by dark rings of colour. Negative controls include a TALE with a coil on the same membrane and the same TALE and bovine serum albumin on separate membranes that were treated separately.
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. 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.
The result of this system is our DNA-binding protein, TAL, bound to ferritin, as well as multiple reporter proteins that can create an output for our system.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.
