Part:BBa_K1965005
P3:cLuc
Introduction
This part is a genetic fusion of the synthetic nucleotide sequence encoding for the coiled coil peptide P3 (from Part BBa_K245120 of 2009 Slovenian iGEM team) and the C-terminal fragment of split firefly (Photinus pyralis (Common eastern firefly)) luciferase. P3 in combination with BBa_K1965006 (nLuc:AP4) form an antiparallel coiled coil, reconstituting luciferase activity.
The orientation of coiled coils is largely determined through interactions between amino acid residues in positions e and g [1], [2]. In coiled-coils with a parallel orientation, electrostatic interactions form between position g on the first and position e on the second alpha-helix (gn:en+1’ interaction). In coiled-coils with an antiparallel orientation, electrostatic interactions occur between g:g’ and e:e’ positions of the two helices [3]. The repeating and predicable nature of these interactions can be used for the rational design of coiled coils [4]. Antiparallel CC orientation allows for fusion of C-termini of N-part of split protein to N-termini of CC via a shorter linker, thereby likely resulting in more efficient reconstitution upon binding with appropriate CC partner. As represented in the wheel helical projection in 1, parallel CC are stabilized by electrostatic interactions g:e’ and e:g’, while interactions between g:g’ and e:e’ positions stabilize antiparallel CC. While CC orientation is mainly influenced by electrostatic interactions specific amino acid residues such as Asn inside CC core can contribute to the orientation as well. Due to polarity of the Asn residue two asparagines prefer interaction with each other rather than with other hydrophobic residues in vicinity such as Leu and Ile. These interactions stabilize the core of intended CC orientation and destabilize the core of CC in the opposite orientation.
Helical wheel projection of parallel (left) and antiparallel (right) coiled coil orientation. Interacting amino acid residues in positions e and g are connected with hatched line. The sequences entitled to each display are listed below.
Characterization
In order to compare the reconstitution efficiency of split protein dictated by parallel or antiparallel coiled coil interaction, we prepared fusion proteins with split firefly luciferase where we designed a new antiparallel peptide (AP4) and tested their activity in cells. Antiparallel coiled coils (AP4:P3) worked significantly better than parallel coiled coils (P4:P3) (2), thus demonstrating that a shorter linker between reporters and dimerizing units helps in the reconstitution of the split protein.
Reconstituted activity of the luciferase dictated by the parallel (left) and antiparallel coiled coil formation (right). HEK293-T cells were transfected with genetic fusions of coiled coil forming peptides and split luciferase. 24 h after transfection luciferase activity was measured. Coiled coil orientation is represented by coloring of each helix form blue (N-terminus) to red (C-terminus). N and C termini of split luciferase are represented by N or C, respectively.
Additionally, this part was characterized in combination with BBa_K1965021 (nLuc:AP4:TEVs:P3mS) and BBa_K1965022 (nLuc:AP4:TEVs:P3mS-2A). Upon activation of TEVp, the linker between AP4 and P3mS, which features the TEVp cleavage site it is cleaved, which enables P3mS to dissociate from AP4. After the dissociation of P3mS, AP4 is free to dimerize with P3:cLuc, which bring the nLuc part into close proximity of cLuc, enabling the split luciferase to regain its catalytic activity (3). The P3mS sequence is based on P3 coil sequence, which featured substitutions of Ala at the b and c position of the coil heptades with Ser and Gln, to increase its solubility, thereby easing its dissociation from AP4.
Upon addition of TEV protease the coil P3mS, which is now cleaved off from AP4, it is replaced by P3mS:cLuc, thereby promoting the reconstitution of firefly luciferase.
The construct was used in logic operations, whereby only in the presence of the protease the split luciferase was able to reconstitute and regain its activity (4).
Normalized luciferase activity was compared between samples with and without added TEVp to calculate the fold change of luciferase activity. Out of the four different constructs, the constructs which contained the inhibitory coils P3mS and P3mS-2A worked best, where we observed up to 15 times fold increase with the addition of TEVp.
References
[1]Woolfson, D. N. The Design of Coiled-Coil Structures and Assemblies. Adv Protein Chem 70, 79–112 (2005).[2] Oakley, M. G. & Kim, P. S. A buried polar interaction can direct the relative orientation of helices in a coiled coil. Biochemistry 37, 12603–12610 (1998).
[3] Litowski, J. R. & Hodges, R. S. Designing heterodimeric two-stranded α-helical coiled-coils: the effect of chain length on protein folding, stability and specificity. J. Pept. Res. 58, 477–492 (2001).
[4] Gradišar, H. & Jerala, R. De novo design of orthogonal peptide pairs forming parallel coiled-coil heterodimers. J. Pept. Sci. 17, 100–6 (2011).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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