CONCLUSIONS
Evaluation of the dissociation of 3D printed models of polyleucine coiled-coils provides scant evidence for steric entanglement of the interface, with measurable entanglement occurring only with antiparallel coiled-coils. Parallel coiled-coils are more common than antiparallel coiled-coils and formation of antiparallel coiled-coils has tended to rely upon designed cysteine mutations and enforced disulfide bonds, or specific charged/polar interactions in antiparallel vs. parallel coiled-coil orientations 18. The analysis of 3D printed coiled-coils supports the geometric analysis of Richmond and Richards 3 in that steric interactions between adjacent side chains oppose shear and torsional movement of packed helices, but separation normal to the helical axis is unimpeded by any steric consideration (it would be opposed principally by non-covalent attractive forces). Thus, the “zipper”, “jigsaw”, and “Velcro” descriptors (implying steric entanglement, Fig. 2) for the polyleucine coiled-coil interface are unsupported by these results.
Our initial expectation in evaluating potential entanglement associated with alternative domain-swapped definitions of the trefoil motif trimer was that the wild-type Monofoil definition of N- and C-termini would yield the greatest entanglement. This was based upon the hypothesized role of enhanced stability afforded by domain swapped entanglement in symmetric protein architecture 14,19-21. However, the wild-type Monofoil definition yielded the least entanglement. Expression and characterization of the stability and folding properties of Monofoil and the domain-swapped polypeptides, described herein, have previously been reported 16. We note there is a general decrease in the level of protein expression upon increased steric entanglement (Table 2). The inability of the permutant #2 3D model to disassemble despite input of substantial kinetic energy indicates a high energy barrier to unfolding, and therefore, the potential for kinetic trapping of both unfolding and folding (Fig. 6). The inhibition of efficient expression in response to increased entanglement is consistent with this interpretation.
The general avoidance of interface entanglement with 3D printed models of polyleucine helices GCN4 and 14-3-3 coiled-coils, and the lowest entanglement being exhibited by the natural termini definition of the Monofoil trimeric oligomer (compared to all domain-swapped alternatives), indicates that entanglement at oligomeric interfaces is likely to be selected against in protein evolution. Thus, the present work suggests that the de novo design of oligomeric interfaces should avoid steric entanglement as a strategy to enhance stability due to the potential negative consequences of kinetic trapping of the folding/unfolding pathway.