Part:BBa_K1965006
nLuc:AP4
Introduction
This part is a genetic fusion of the synthetic nucleotide sequence encoding for the coiled coil peptide AP4 (implemented from part BBa_K245121 of 2009 Slovenian iGEM team) and the C-terminal fragment of split firefly (Photinus pyralis (Common eastern firefly)) luciferase. AP4 in combination with BBa_K1965005 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. 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.
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]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 103
Illegal NgoMIV site found at 1447
Illegal NgoMIV site found at 1468
Illegal AgeI site found at 1171 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1538
Illegal SapI.rc site found at 1353
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