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.