By extending a naturally rigid bacterial protein by 50% while preserving geometry and stability, the study introduces protein-based “molecular rulers” that could probe antibody shape, limit unwanted motion, and support safer, more effective antibody drug design.
Antibodies are essential modern biomedicines used to treat cancer, autoimmune disorders, and infectious diseases, but their effectiveness depends not only on binding chemistry but also on physical shape. Each antibody carries two antigen-binding sites separated by a variable distance, typically 6–14 nanometers, which is crucial for bivalent binding and signaling. However, antibodies are highly flexible in solution, making their true conformational range difficult to measure. Existing methods often yield averaged or incomplete information, limiting predictive models. A promising solution is to use rigid molecular spacers of known length to bridge both binding sites simultaneously. Protein coiled-coil bundles, with regular geometry and tunable design, offer an attractive platform for such nanometer-scale tools.
A study (DOI: 10.1016/j.bidere.2025.100061) published in BioDesign Research on 8 December 2025 by Ioannis Karageorgos and David Travis Gallagher, Material Measurement Laboratory of the National Institute of Standards and Technology, demonstrates that rigid protein coiled-coil bundles can be rationally and precisely extended while preserving stability and geometry, providing a foundation for nanometer-scale protein tools to measure and control the conformational behavior of antibodies and other flexible biomolecules.
Using a structure-guided protein engineering strategy combined with high-resolution X-ray crystallography, the researchers first introduced a single tryptophan residue into the rigid four-helix bundle of the bacterial ROP protein to enable accurate quantitation without perturbing its fold, and then extended the bundle by systematically inserting additional coiled-coil heptad repeats while iteratively optimizing sequence composition to preserve correct dimer assembly and stability. Structural analysis of the intermediate variant, ROP+Trp (PDB 7KAE), revealed that the engineered tryptophan packs cleanly into the protein core, stacking against Glu5 while projecting its indole nitrogen toward solvent, and that this substitution leaves the backbone essentially unchanged, with an overall Cα RMSD of only 0.9 Å relative to wild type and solution behavior indistinguishable from the native protein. Building on this validated scaffold, the team lengthened the protein from 56 to 85 residues by duplicating four heptads at multiple positions, followed by iterative sequence diversification to avoid symmetry-related misassembly. Crystallographic data showed that the resulting extended construct forms a well-ordered, left-handed coiled-coil bundle in which each helix wraps approximately 120 degrees around the central axis, closely matching AlphaFold predictions with an RMSD of 0.8 Å over 170 Cα atoms. Despite substantial sequence remodeling—affecting roughly a quarter of all positions and enriching helix-favoring residues such as alanine, glutamate, glutamine, and lysine—the core remained tightly packed, with disorder confined to a small number of surface side chains. To further demonstrate design flexibility, the researchers reintroduced a single phenylalanine at a new core position, Leu69, guided by structural modeling and validation tools; crystallography confirmed that the aromatic ring fits snugly into the hydrophobic core without distorting the backbone and preserves a conserved, water-coordinated packing motif seen in wild-type ROP. Throughout the design process, clusters of compensatory mutations emerged around splice points and the new aromatic site, resolving steric clashes over successive iterations and highlighting how precise backbone knowledge and helix-biased residue selection can enable rational, large-scale extension of rigid protein bundles.
In conclusion, the extended protein acts as a rigid, nanometer-scale module that can serve as a scaffold or spacer to engage both antibody binding sites simultaneously. It may enable measurement of inter-site distances, restrict flexibility, and stabilize defined antibody conformations, while also illustrating a general strategy for creating protein-based metrological tools at the macromolecular scale.
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References
DOI
10.1016/j.bidere.2025.100061
Original Source URL
https://doi.org/10.1016/j.bidere.2025.100061
Funding information
CBMS is primarily supported by the National Institutes of Health, National Institute of General Medical Sciences (NIGMS) through Grant # P30GM133893, and by the DOE Office of Biological and Environmental Research FWP # BO070.
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