Part:BBa_K245066

This part was designed to form a polypeptide chain, composed of three coiled-coil-forming segments. This polypeptide is able to self-assemble into a polyhedron, such as a box or form a two dimensional polypeptide lattice with dimension of each edge around 5 nm. This is the first example of self-assembled polygon composed of coiled-coil segments.

This part is composed of coding sequence for three polypeptide segments – two segments forming parallel heterodimeric coiled-coil (designed P1 and P2) and one parallel homodimeric coiled-coil-forming segment (GCN4-p1 (I-L)-from now on designated GCN). Between each coiled-coil-forming segment we introduced a dipeptide linker Gly-Ser using BioBrick standard BBF RFC 37, to allow the flexibility between coiled-coil-forming segments.

Coding sequences for GCN4, P1 and P2 are of synthetic origin. Each gene was cloned separately into our functionalized vector (BBa_K245005) in which T7 promoter, RBS and ATG are included in prefix and T7 terminator and STOP codon in suffix. Sequence coding for His tag is also inserted between ATG and multiple cloning site in prefix, which results in His-tag at the N-terminus of the polypeptide product of the part, inserted in this vector. Originating basic parts were BBa_K245118 (P1 inserted in functionalized vector BBa_K245005), BBa_K245119 (P2 inserted in functionalized vector BBa_K245005) and BBa_K245127 (GCN inserted in functionalized vector BBa_K245005).

Assemby of part BBa_K245066
There are four new inner restriction sites added in multiple-cloning site of the functionalized vector (BBa_K245005) (NgoMIV and AgeI in prefix; XmaI and BspEI in suffix). In this case vector containing P2 (BBa_K245119) was first cut, to obtain front vector and vector containing GCN (BBa_K245127) was cut to obtain front insert. Ligation of front insert into front vector resulted in a new intermediate part (BBa_K245052) where sequence coding for GCN is located at the 5’ and sequence coding for P2 is located at the 3’ of the part. After that GCN-P2 coding intermediate part (BBa_K245052) was cut as front vector, and BBa_K245118 was cut as front insert. After ligation of these front vector and front insert we gained new composite part P1-GCN-P2 (BBa_K245066). Because there are four new inner restriction sites added in multiple-cloning site of our functionalized vector (BBa_K245005) (NgoMIV and AgeI in prefix; XmaI and BspEI in suffix) this enabled us to create a friendly scar tccggc (SG) between basic parts (BBa_K245118, BBa_K245119 and BBa_K245127) during their ligation.
The resulting polypeptide product of a composite part (BBa_K245066) is also His-tagged due to design of our functionalized vector. Figure 1 shows the schematic representation of polypeptide product of this construct.

Figure 1: Scheme of the polypeptide product of a construct P1-GCN-P2. There is a methionine residue in front of a His-tag.

Composite part (BBa_K245066) in vector BBa_K245005, was transformed into a BL21(DE3) pLysS strain of E. coli where the protein expression was induced. Following cell lysis, supernatant (Figure 2A, lane 1) and insoluble fraction (inclusion bodies; Figure 2A, lane 2) were analyzed for the production of P1-GCN-P2 polypeptide. The protein was mainly produced in form of inclusion bodies (Figure 2A, lane 2), which were further purified (Figure 1A, lane 3). Purified protein (Figure 2A, lane 4) was prepared by solubilizing inclusion bodies in 6 M Gdn HCl and purification on nickel-NTA column under the denaturing conditions. Western blot revealed the presence of oligomers (Figure 2B) and mass spectrometry confirmed the identity of the polypeptide.

Figure 2: Production and analysis of K2 polypeptide. A) SDS PAGE of the soluble fraction of bacterial cell lysate (lane 1), insoluble fraction (lane 2), washed inclusion bodies (lane 3), protein purified by chelating chromatography (lane 4). B) Analysis of K2 after slow chemical annealing by Western blot (line 2). Additional bands showing oligomers of K2 are visible. Line 1 represents standard proteins.

Experiments done with P1-GCN-P2 polypeptide

Characterization of coiled-coil formation by circular dichroism

The shape of spectra of separate samples of constituent polypeptide segments P1 and P2 indicated that individual peptides are not structured, while the mixture of P1+P2 showed a high level of α-helical folding (Figure 3). The peptide concentration was 0.1 mg/ml in 10 mM HEPES, pH 7.5.

Figure 3: Far-UV CD spectra of peptides P1, P2 and mixture of P1 + P2 at 25° C. The shape of spectra indicated that individual peptides were not structured, while mixture of P1+P2 showed a high level of α-helical folding. The peptide concentration was 0.1 mg/ml in 10 mM HEPES, pH 7.5.

We analyzed the secondary structure of a polypeptide K2 under the native conditions (Figure 4). CD spectra showed strong helical signal, confirming that the coiled-coil interactions occur also in the context of a longer polypeptide and in the presence of linker sequences.

Figure 4: CD spectrum of K2 reveals a high fraction of α-helical secondary structure. K2 polypeptide forms a precipitate under the native conditions due to the formation of multiple coiled-coil interactions.
Slow chemical annealing of polypeptide self-assembly

We investigated the stability of P1-GCN-P2 from the CD analysis at different concentrations of the denaturing agent, which was around 4 M GdnHCl (Figure 5).

Figure 5: Secondary structure of the polypeptide at different concentrations of GdnHCl was determined by measuring the circular dichroism at 222nm on a CD spectrometer.

Analysis of self-assembled P1-GCN-P2 (K2) structures by microscopy

Self-assembled structures of polypeptide annealed at different concentrations were analyzed by AFM operating in acoustic alternative current mode. At low protein concentrations small aggregates with dimensions below 10 nm were observed, which is consistent with the expected size of the self-assembled box (Figure 6)

Figure 6: AFM scan of the assembly of nanostructure from P1-GCN-P2 (self-assembled over 20 hours from 5 to 1 M Gdn HCl at protein concentration of 0.5 µg/ml)

TEM was used to analyze the structure of self-assembled P1-GCN-P2 at 5 µg/ml. Results were very exciting as we can see the presence of a polygonal lattice with edges measuring below 10 nm. (Figure 7). This clearly shows that we can indeed form a two dimensional lattice made of polypeptides. There is clearly room for improvement, as the lattice contains a lot of defects, that could probably be solved by optimizing the polypeptide concentration, refolding conditions or modifications of the linker.

Figure 7: TEM image of the self-assembled nanostructure made of P1-GCN-P2.

P1-GCN-P2 was deposited on the grid for TEM analysis and stained with uranyl acetate. Image demonstrates formation of molecular network with edges at nanometer dimensions which is consistent with multiples of the designed edges consisting of P1-P2 and GCNp1-GCNp1 coiled-coils.

Potential applications

Polypeptide product of BBa_K245066 represents the first example of self-assembled polygon composed of coiled-coil segments. This opens exciting prospects for additional more complex assemblies and a whole new range of possible applications.

Assemblies based on interacting coiled-coil segments could be used as a scaffold for the attachment of different biological molecules, formation of biosensors, artificial enzymes and on the other hand the polypeptide could be biomineralized or prepared to conduct electricity, therefore representing an interface to the electronic circuits but could also be used themselves to build self-assembling electronic or optical circuits.