Part:BBa_K2356001
14-3-3 tetramer with GFP
Main info
The sequence is designed by TU-Eindhoven 2017[http://2017.igem.org/Team:TU-Eindhoven] and starts with DNA coding for a His-tag, used for purification purposes, followed by a 14-3-3 dimer, a well researched and described protein. This is then followed by a linker with another 14-3-3 dimer, of which the last monomer is mutated, resulting in loss of binding capability. This domain is subsequently followed by a linker with GFP, a fluorophore. Expression of this protein should result in a trivalent scaffold that can fulfill multiple roles in protein-protein interaction (PPI) networks. The fluorophore allows localization and can be used to study the protein's behavior.
In short:
• 3978 DNA basepairs
• 1315aa protein (146 kDa)
• Binds to CT33 / CT52
• Binding possibilities to other molecules
• Green fluorescent
• His-tag purification
About 14-3-3
Proteins belonging to the 14-3-3 family are dimers, where each monomer consists out of nine anti-parallel alpha-helices. This causes the dimer to obtain a cup-like shape with two amphipathic binding grooves. The structure forms a rigid scaffold that is capable of anchoring proteins. 14-3-3 proteins are involved in multiple cellular processes and are mostly known to bind phosphorylated peptide motifs, especially those containing phosphoserine and phosphothreonine sequences. Most regions are conserved among different 14-3-3 isoforms, but the C-terminus appears to show more variability and is important in binding different target proteins.[1]
The 14-3-3 protein in this part is the specific tobacco isoform 14-3-3c and it is stripped of its last 18 C-terminal amino acids, called T14-3cΔC. This allows for higher affinity towards the CT33 peptide, more specifically towards the YDI tail, in the presence of small molecule fusicoccin.[2] Next to the shortening of 14-3-3, this part also connects two 14-3-3 dimers, forming a tetramer scaffold. Mutation of one or more monomers consecutively allows varying the amount of available binding pockets. Tunability of the number of binding pockets can be useful to create a valency that is ideal for phase separation or other Protein-Protein Interactions (PPIs). In this part, the 4th monomer is mutated, preventing it from binding CT33, yielding a trimeric scaffold. These three binding pockets can then be blocked using covalently attached ExoS domains, which can be cleaved by beforementioned MMPs. This would mean that PPIs will be induced only in the presence of these MMPs. ExoS is not yet included in this part, but could be added in a further stadium.
Connection to CT33
One motif that is known to bind to 14-3-3 is the phosphorylated C-terminus of H+-ATPase, an enzyme that catalyzes the hydrolysis of ATP to ADP.[3] The CT33 peptide comprises the final 33 amino acids of this C-terminus, which is referred. The binding of unphosphorylated CT33 and CT52 with YDI mutation to 14-3-3 family has extensively been researched and it was shown that this binding was particularly strong to the specific T14-3cΔC protein. This happened in the presence of a small molecule, called fusicoccin, which functions as stabilizer and resulted in a Kd of 0.85 nM.[2]
Due to this low Kd value and tunability of fusicoccin this binding is interesting for contributing to a PPI network based on 14-3-3 scaffolds, especially when the valency of 14-3-3 can be altered.
GFP
In many biological or chemical processes it is convenient to allow visualization of the behavior of molecules. One facile approach to such visualization is the attachment of a fluorophore, such as Green Fluorescent Protein (GFP). This protein is often used for this purpose, showing a major and a minor excitation peak at 395 and 475 nm, respectively, while the emission peak lies around 509 nm.[4] Using this domain in a protein network, where other proteins comprise different fluorophores, may yield significant information on interactions and localization.
His-tag
The very beginning of the part contains a sequence that encodes for the famous His-tag, comprising 6 consecutive Histidine amino acids, that binds to e.g. Nickel ions. This allows the protein to be purified through Immobilzed Metal Affinity Chromatography (IMAC).[5]
References
[1] Obsilova V, Kopecka M, Kosek D, Kacirova M, Kylarova S. Mechanisms of the 14-3-3 Protein Function : Regulation of Protein Function Through Conformational Modulation. 2014;63.
[2] Ottmann C, Marco S, Jaspert N, et al. Article Structure of a 14-3-3 Coordinated Hexamer of the Plant Plasma Membrane H + -ATPase by Combining X-Ray Crystallography and Electron Cryomicroscopy. 2007:427-440. doi:10.1016/j.molcel.2006.12.017.
[3] Morsomme P, Boutry M. The plant plasma membrane H+-ATPase;: structure , function and regulation. 2000;1465.
[4] Turoverov KK. NIH Public Access. 2010;9(4):338-369.
[5] https://www.thermofisher.com/nl/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/his-tagged-proteins-production-purification.html
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1306
Illegal EcoRI site found at 1461
Illegal PstI site found at 521
Illegal PstI site found at 3967 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1306
Illegal EcoRI site found at 1461
Illegal NheI site found at 1642
Illegal PstI site found at 521
Illegal PstI site found at 3967 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1306
Illegal EcoRI site found at 1461
Illegal BamHI site found at 3229 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1306
Illegal EcoRI site found at 1461
Illegal PstI site found at 521
Illegal PstI site found at 3967 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1306
Illegal EcoRI site found at 1461
Illegal PstI site found at 521
Illegal PstI site found at 3967
Illegal AgeI site found at 3958 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1009
Illegal SapI.rc site found at 2602
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