Calpain small subunit homodimerization is robust and calcium‐independent

Calpains dimerize via penta-EF-hand (PEF) domains, but their mechanical stability and kinetics remain incompletely characterized. Here, we used single-molecule force spectroscopy on an EGFP-tagged calpain small subunit PEF (CAPNS1-PEF) homodimer to quantify dissociation mechanics. Across retraction speeds of 0.2-10 μm/s, homodimer rupture required large forces (> 300 pN). Rupture forces and force-induced unfolding did not change when Ca²⁺ was removed, suggesting Ca²⁺-independent mechanics, consistent with crystal structures showing minimal Ca²⁺-induced conformational changes in the PEF domain. Together, these results indicate that EF-hands in the CAPNS1 homodimer function primarily as structural elements within a mechanically robust, Ca²⁺-insensitive dimerization scaffold.