Heptad insertion extends the E. coli ROP protein by 50

Antibody function involves conformational variability, yet their extreme flexibility complicates measurement of their structure and properties. They also have numerous ligands, suggesting that a rigid bivalent ligand construct of appropriate length could interact with an antibody to reduce its flexibility for imaging, measurement or functional effect. Such a construct would ideally have an inter-ligand spacer that is fairly rigid and with length between 6 and 10 nm. Coiled-coil bundles of alpha helices represent a common motif in protein structure whose relatively simple parallel geometry makes them suitable for rational modification including applications in metrology. In this study, we describe a heptad-insertion heuristic for extending bundles and apply it to the E. coli ROP/ROM protein, which is a 13 kDa, thermostable RNA-binding unit that is naturally a 4-helix dimer and has been engineered to self-associate in various ways to form larger assemblies. We first introduced a tryptophan residue into the core (wild-type lacks tryptophan) to support precise quantitation, and then extended the protein to 150 % of its native length by inserting four helical heptads. We report the engineering process and crystal structures of the tryptophan mutant and the lengthened protein, which also contains a new phenylalanine in the core. Where wild-type has a length of 4.3 nm, the extended variant has length 6.5 nm and may serve as a rigid module for higher order constructs.