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

Understanding the mechanical stability and architecture of viral proteins can provide valuable information about their biological function, but it remains a significant biophysical challenge. This study employs single-molecule force spectroscopy (SMFS) to investigate the multi-domain architecture of the Orsay virus δ protein, which lacks repeat structures and exhibits weak unfolding peaks. We engineered a construct using titin (I27) ₄ domains as an internal molecular ruler, enabling us to bracket the δ protein peaks to determine domain length and identify unfolding forces with an atomic force microscope (AFM). To address limitations of one-dimensional (1D) force distributions in resolving overlapping structural states, we created a two-dimensional (2D) mechano-structural signature map. By plotting kinetic stability (unfolding force F ) against physical structural footprint (domain length L ), we distinguished distinct unfolding domains, successfully separating degenerate 1D data into two statistically distinct populations corresponding to the δ protein’s internal domain (I) and C-terminal domain (C). This label-free method provides the first mechanical evidence of the δ protein’s multi-domain architecture. It establishes a robust, multi-dimensional framework for decoding the mechanics of complex biomolecular assemblies in their native state.