摘要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.