Fig 1. Kinetics of brominated PC exchange between mixed DDM/lipid micelles and SERCA1a solubilized at the same detergent/lipid ratio.

(A) Trace a1, to 2 mL of SR vesicles suspended at 0.04 mg protein/mL (together with 0.02 mg endogenous lipid/mL) in buffer A at 20°C, 0.6 mM EGTA (EG) followed by 0.6 mM CaCl₂ (Ca) were first added as controls, to illustrate the classical Ca²⁺-dependent changes in SERCA1a intrinsic fluorescence as well as the usual down-drift in fluorescence intensity accompanying such measurements (see Materials and Methods). SERCA1a was then solubilized by adding mixed DDM/egg PC micelles (D/L, containing the unbrominated lipid at a ratio of 5 g detergent/g lipid, i.e. ~7 mol/mol) at final concentrations of 0.4 and 0.08 mg/mL for DDM and egg PC, respectively. Mixed micelles with brominated lipids (D/BrL) were then added and this was followed by addition of excess DDM (to reach a final total DDM concentration of 4 mg/mL). For Trace a2, additions were performed first with brominated (D/BrL) and then with unbrominated (D/L) lipid. Traces a3 and a4 correspond to experiments similar to those in a1 and a2, respectively, but now measuring light scattering at 290 nm. (B) In Traces b1 and b2, unbrominated lipid was used but DDM was replaced by 5,6-brominated DDM (BrD) at the same molar concentration. Traces here have not been corrected for dilution effects. This dilution effect only becomes somewhat significant when adding excess DDM at 3.2 mg/mL (32 μL of a 200 mg/mL stock solution resulting in a 1.6% dilution). Numbers indicate the concentrations of detergent and lipid added at each step to the cuvette, in mg/mL. The cartoon on top depicts the principle of the experiment. Lipids are represented with a grey headgroup, detergent molecules are represented with a white headgroup and bromine ions are displayed as black dots. Each trace corresponds to one experiment representative of three independent experiments.