Initial testing

The constructs B:nLuc, cLuc:A, A’:TEVs:B:nLuc and cLuc:A:PPVs:B’2A, which do not possess autoinhibitory segments, were tested for CC binding by measuring luciferase reconstitution. Constructs without protease cleavage sites (B:nLuc, cLuc:A ) were used as a control (4.12.1.). A’:TEVs:B:nLuc and cLuc:A:PPVs:B’2A were tested in the presence of TEVp and PPVp, which cleave off the autoinhibitory CC, resulting in split luciferase reconstitution. Additionally, different ratios of constructs were tested in order to obtain the best luciferase activity ((4.12.1.).

Results showed that very high amounts of the constructs based on same coiled coil sequences used by Shekhawat et al Shekhawat2009 (i.e. 50 ng of each) were needed to detect the firefly luciferase signal in mammalian cells (4.12.1). Therefore, we decided to engineer designed coiled coils from a toolbox, used by the ( 2009 Slovenian iGEM team ) Gradisar2011a. In order to design an antiparallel coiled coil-based system applicable for logic operation in living cells we took into consideration the rules that establish the orientation and strength of the affinity of the CCs and designed new coiled coils, expanding on the available collection of orthogonal CC and ( modeled ) the ratio of the affinities that are required to obtain the optimal response at low leakage.

Further explanation ...

Coiled coils

Alpha-helical segment interaction is a common feature in protein tertiary and quaternary structures, where helices form complexes of two or more coils (4.12.1.2). The most frequent interaction is between two alpha-helices, which form a dimeric coiled coil. Interactions can occur both in the two parallel or antiparallel orientation of the coil pairs Hadley2006. The interaction strength of different coiled coil pairs depends on their amino acid sequence and their structure, which determine the underlying noncovalent forces of attraction and repulsion the helices exert on each other. Understanding the rules that govern the interactions between coiled coils is thus inherently linked to understanding their amino acid sequences Woolfson2005.

Sequences of coiled coils that form interactions have a characteristic seven amino acid repeat, called heptad repeat. The position of each amino acid within a heptad is presented in a unified nomenclature (a,b,c,d,e,f,g). Interaction between two coils occurs on a continuous patch along the side of each alpha-helix with each patch facing the core of the dimer’s interface (4.12.1.2B). The amino acid residues which occupy this strip correspond to the a and d positions of the heptad; they are generally hydrophobic and represent the driving force behind dimerization Woolfson2005. Coiled coils are additionally stabilized by ionic interactions between polar amino acids (Asp, Glu, His, Lys, Asn, Gln, Arg, Ser or Thr) in positions e and g Woolfson2005, Gradisar2011a; while amino acids in positions b, c and f, which are less important to interactions between the helices; contribute to helix stability and solubility.

Two alpha-helices that form a coiled coil can interact either in a parallel or in an antiparallel orientation Oakley1998 (4.12.1.2 B and C). The orientation of coiled coils is largely determined through interactions between amino acid residues in positions e and g Woolfson2005, Oakley1998. 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 Litowski2001. The repeating and predicable nature of these interactions can be used for the rational design of coiled coils Gradisar2011a.
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 4.12.1.2g: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.

 Antiparallel coiled coils

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) (4.12.4), thus demonstrating that a shorter linker between reporters and dimerizing units helps in the reconstitution of the split protein.

To investigate whether the newly designed antiparallel CC is suited for implementation as logic unit into our system, the constructs nLuc:AP4 and P3:cLuc were compared to the coiled coil cLuc:A and B:nLuc from Shekhawat et al Shekhawat2009. Measurement of the reconstituted firefly luciferase activity showed that our designed coiled coils provided far higher (~50 fold) signal (4.12.5.), thus proposing the use of this new coiled coils for more sensitive logic gates that functions well in the cellular milieu.

The system presented by Shekhawat is able to process AND or OR logic functions but not those including negation (such as NOR, NAND etc.) We realized that this type of logic functions could be accomplished by introducing an additional cleavage site between the split reporter and coiled coil segment (4.12.6.1).

 Cleavable constructs

Constructs were therefore modified by the addition of TEVp cleavage site (TEVs) between nLuc and AP4 and PPVp cleavage site (PPVs) between P3 and cLuc. This represents a logic NOR gate based on the input signals, represented by TEVp and PPVp.

Indeed the system performed nicely (4.12.6.). Using this type of cleavage sites enabled us to design protease-based logic gates NOR, NOT A and NOT B (4.12.7.).

 Destabilized coiled coils

For implementation of the system with additional logic operations further modifications on our own CCs collection were needed. Analysis of the equilibrium model reveals that the affinity of the autoinhibitory segment should not be too strong, otherwise the inhibition will remain; but should also not be too weak, otherwise the system would be leaky and active already without cleavage. Stability of the coiled coil interaction can be tuned by introduction of non-favorable interactions e.g. by introducing Ala residues at a and d positions Acharya2002. We designed four different destabilized P3 coils by substituting b and c position with polar amino acids and a and d positions of different heptads with alanine residues (4.12.8.).

Those variably destabilized peptides were used as autoinhibitory coiled coil forming segments to test the difference in activity between the uncleaved and TEVp cleaved forms.

To test which one of our four destabilized CCs worked best, all constructs were tested in vivo with and without the presence of TEVp (4.12.9.). We concluded that P3mS and P3mS-2A demonstrated the highest fold increase in the luciferase activity upon the addition of TEVp. The other two constructs showed little to no increase in luciferase activity upon the addition of TEVp, suggesting that the peptides were destabilized too much leading to the leakage in the uninduced form.