Team:Slovenia/Coiled-coil polyhedra Results.html
Polypeptide production and isolation
Plasmid, coding for K2 was assembled from BioBricks (BBa_K245118, BBa_K245119, BBa_K245127) and transferred into a BL21(DE3) pLysS strain of E. coli. Bacteria were grown according to the standard procedures, protein expression was induced with IPTG and bacteria were harvested after 4 hours. Following cell lysis supernatant (Figure 1A, lane 1) and insoluble fraction (inclusion bodies; Figure 1A, lane 2) were analyzed for the production of K2 polypeptide. The protein was mainly present in form of inclusion bodies (Figure 1A, lane 2), which were further purified (Figure 1A, lane 3). Purified protein (Figure 1A, lane 4) was prepared by solubilizing inclusion bodies in 6 M GdnHCl and purification on nickel-NTA column under the denaturing conditions. Western blot revealed the presence of oligomers (Figure 1B).
Figure 1: 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.
Characterization of coiled-coil formation by circular dichroism
We wanted to experimentally test the design of P1 and P2 coiled-coil-forming segments with respect to their lack of secondary structure in the isolated form and formation of a coiled-coil heterodimer. CD spectra of individual P1 or P2 peptides show that each of them is disordered in solution while their mixture (P1+P2) shows a high level of α-helical content (Figure 2). Although we did not strictly prove that P1 and P2 peptides form a parallel and not antiparallel coiled-coil dimer our results demonstrate that we designed an adequate pair of coiled-coil forming segments suitable for the formation of more complex assemblies.
Figure 2: 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 -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 3). 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 3: 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
Desired self-assembly includes interaction between several polypeptide chains. We presumed that the desired assemblies represent the energetic minimum, since all coiled-coil-forming potentials are satisfied. However with increasing number of polypeptide chains in the assembly the energetic differences between the correctly folded and misfolded structures decrease and kinetic limitations for formation of the most ordered structure may become important. In order to increase the fraction of correctly folded assemblies we wanted to remove the kinetic considerations by performing very slow annealing. In case of DNA origami this is usually done by a slow decrease of temperature over several hours or days. In our case temperature annealing did not give good results, presumably because the temperature-denatured state may not be completely unfolded and since both hydrophobic and electrostatic interactions take part in stabilization of coiled-coil assemblies, each with their different temperature dependence. Therefore we decided to use chemical annealing by decreasing the concentration of denaturing agent in solution towards the native conditions. For the efficient annealing we should keep the solution sufficiently long under the conditions, with equilibrium between the unfolded and folded structures. We determined the midpoint of transition of K2 from the CD analysis at different concentrations of the denaturing agent, which was around 4 M GdnHCl (Figure 4).
Figure 4: Secondary structure of the polypeptide at different concentrations of GdnHCl was determined by measuring the circular dichroism at 222nm on a CD spectrometer.
Usually slow refolding is performed by dialysis, which however may not be optimal if the difference in the concentration of both solutions is large, which may lead to the large concentration gradients at the edge of the dialysis tube. Therefore we invented a technique to perform chemical annealing over any desired length of time, by performing the dialysis against the continuously changing concentration of denaturing agent (Figure 5). From the analysis of the folding transition of K2 we determined that the optimal range should be between 5 M GdnHCl, where the large majority of the secondary structure is disrupted to 1 M GdnHCl, where almost all secondary structure is formed.
Figure 5: Scheme of the setup for slow chemical annealing of K2.
Buffer was slowly pumped into the beaker with mixing denaturant solution and protein samples in the dialysis bag, decreasing over 20 hours the conditions from the unfolding to the native conditions.
Continuous decrease of the denaturant in the dialysis solution was achieved by slowly pumping the buffer into the stirred dialysis solution, which initially contained 5 M GdnHCl, which was the same concentration as in the dialysis bag with K2 polypeptide. In this way the concentration difference between the solution inside and outside dialysis bag was minimal and programmable pump allowed setting the slope of the gradient to any desirable value, extending refolding from 12 hours towards potentially several days.
Concentration of the polypeptide represents an important factor in determining the type of self-assembled structures, either polygons, which require the assembly of eight chains or lattice, which requires over hundreds of molecules. At low polypeptide concentrations formation of polygons should be favored since they should form oligomers that are big enough to form a closed structure, satisfying all coiled-coil-forming potentials. Refolding procedure allowed us to perform the self-assembly in parallel using several different concentrations at the same time.
K2 dissolved in 6 M GdnHCl at 10 mg/ml was diluted to 0.5 µg/ml, 5 µml and 50 µml in 5 M GdnHCl in 20 mM HEPES buffer pH 7. 100 ml of the solution was placed in a dialysis bag with membrane cutoff of 3.5 kDa, and placed in 2 l beaker containing a 200 ml of the 5 M GdnHCl in 20 mM HEPES buffer pH 7 (Figure 5A). Solution was stirred at room temperature with magnetic mixer at 1000 rpm. To the bottom of the dialysis solution in the vicinity of rotating magnet a solution with 20 mM HEPES pH 7 was added by a pump set to the flow rate of 1 ml/min. Within 20 hours the concentration of denaturing agent in the solution decreased to 1 M (Figure 5B). At this point, when the self-assembly was already completed the dialysis bag with protein solution was transferred to the solution containing 2 l or 20 mM HEPES pH 7 and dialyzed for 3 hours and the procedure was repeated twice, to remove the denaturant (Figure 5C).
Sensitivity of DLS is rather low as it requires sample at concentrations above 0.2 mg/ml, therefore we had to concentrate the annealed polypeptide samples. DLS results (Figure 6) showed that solution contained two populations of K2 aggregates and both were present at appreciable amount. Hydrodynamic radius (RH) of one population was 8 nm, and for the other 88 nm, indicating the presence of initial steps of larger assemblies in addition to small aggregates, compatible with the expected box.
Figure 6: DLS correlation curve for 50 µM K2 solution. When data were transformed to RH distribution two populations one with RH of 8 nm and another with RH of 88 nm, were observed. Note that the scattering power of larger aggregates dominates the correlation curve and the observable presence of smaller aggregates indicates that their number is significant in the solution.
Polypeptide production and isolation
Self-assembled structures of K2 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 7).
Figure 7: AFM scan of the assembly of nanostructure from K2 (self-assembled over 20 hours with slow gradient of Gdn HCl from 5 to 1 M at protein concentration of 0.5 µg/mL).
TEM was used to analyze the structure of self-assembled K2 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 8). 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, which could probably be solved by optimizing the polypeptide concentration, refolding conditions or modifications of the linker.
Figure 8: TEM image of the self-assembled nanostructure made of K2. K2 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.
K2 thus 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.
One can imagine that assemblies based on interacting coiled-coil segments can be used as a scaffold for 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.