Fig. 6

Mapping the position of the regulatory domain on the surface of PLS3 core. a Positions of single-cysteine mutations in the loops harboring OI-associated mutations (A250C in the 240–268 and K452C in the 446–456 loops) and in the ABD1–ABD2-connecting linker (I388C and T391C in the 377–394 linker) are shown in red on a homology-based model of PLS3 (Phyre2⁸⁶; color scheme as in Fig. 1). The proposed position of RD is encircled by a dashed line. b–e Acrylodan labeling of the cysteine residues described in (a) introduced as the only cysteines into the full-length PLS3 (FL) and PLS3 lacking the Ca²⁺-binding regulatory domain (ΔRD). Labeling was assessed for each construct in the presence of either 1 mmol·L⁻¹ EGTA or CaCl₂. The rates were calculated by fitting the data to a pseudo-first order kinetic model and plotted as means ± SE (n = 4 for b, n = 5 for c, n = 3 for d, e); individual data points are plotted as circles. Statistical significance was determined by two-sample (equal variances) Student’s t test with two-tailed distribution