A pathological isoleucine to asparagine missense mutation disrupts the structure and function of the C2B domain of otoferlin. (A) The fluorescence spectra of endogenous aromatic residues of the isolated WT (left) and I318N (right) mutant C2B domain differ in their spectral profiles. Although a notable difference in the fluorescence intensity (FI) of WT C2B is observed in 0.1 mM EGTA versus 1 mM calcium conditions, the spectra of the I318N mutant shows no discernable change in fluorescence in response to calcium. (B) The membrane aggregation activity of C2B is disrupted by the I318N mutation. After injection of protein at t = −5 min, calcium was added at t = 0 min to a final concentration of 1 mM. OD₄₀₀ measurements for the WT C2B (left) and I318N (right) reveal that the point mutation abrogates calcium-triggered C2B-mediated liposome aggregation. The OD₄₀₀ values after addition of calcium were 0.11 ± 0.02 for WT and 0.02 ± 0.01 for the mutant C2B (n = 3). (C) Reconstituted fusion assays performed in the presence of WT or I318N mutant C2B. After the addition of 1 mM free calcium, WT C2B accelerated SNARE-mediated fusion, whereas the I318N failed to enhance the rate or extent of fusion.