@article{Karageorgos2026, 
author = {Ioannis Karageorgos and D. Travis Gallagher},
title = {Heptad insertion extends the E. coli ROP protein by 50 %},
year = {2026},
journal = {BioDesign Research},
volume = {8},
number = {1},
keywords = {Coiled coil, Crystal structure, Helical bundle, Heptad repeat, Protein engineering},
url = {https://www.sciopen.com/article/10.1016/j.bidere.2025.100061},
doi = {10.1016/j.bidere.2025.100061},
abstract = {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.}
}