Resolving Orsay Virus δ Protein Architecture Using Molecular Rulers in Single-Molecule Force Spectroscopy

The Orsay virus δ protein lacks repeat structures, making traditional force spectroscopy analysis difficult. By engineering a construct with titin (I27)4 domains as a molecular ruler, the team could calibrate force-extension curves and resolve domain architecture despite weak unfolding signals. Single-molecule force spectroscopy (SMFS) revealed distinct mechanical unfolding steps corresponding to δ protein domains. The method offers a general strategy for studying multidomain proteins with low mechanical stability. These findings may inform understanding of viral capsid assembly and infectivity.