Abstract
Abstract
Long, non‐contractile tails composed of helical hexameric protein repeats that assemble into continuous tubular structures characterize siphoviruses. The siphovirus tail tube protein gp39 contains two cysteine residues per monomer. Cryo‐electron microscopy revealed that these cysteines are oriented toward the interface between the rings, which facilitates the formation of inter‐ring disulfide bonds. Phylogenetic analysis revealed that in the phage tail tube protein‐3 (PF08813) family, only a single branch, representing approximately 14% of its members, contains disulfide bonds within the tubular structure, indicating a clear evolutionary adaptation to stabilize the structure. Structural characterization of gp39 alongside its homolog gp13, which naturally lacks disulfide crosslinks, provided insight into this stabilization strategy. Both gp39 and gp13 can self‐assemble into tubular structures independently of disulfide bond formation. When cysteine residues were inserted into gp13 at the same positions as in gp39, disulfide bonds were formed, as confirmed by Raman spectroscopy. Differential scanning fluorimetry further demonstrated that variants containing disulfide bonds exhibit enhanced thermal stability. These results reveal the evolutionary and structural basis by which disulfide bonds ensure the stability of phage tail structures and establish the fundamental principles for the design of robust, thermally stable protein nanotubes.