Disulfide cross-linking of CLC-ec1-Cy5 at R230C and L249C. (A) Top view of the CLC-ec1 homodimer showing R230C (orange) and L249C (yellow) positions. (B) SDS-PAGE of R230C/L249C (on the C85A/H234C background) and with the addition of DTT. Red circle, monomer; gray circles, dimer; white circles, tetramer. (C) Size exclusion chromatography profiles of WT and R230C/L249C. (D) Functional Cl⁻ transport of WT-Cy5 and R230C/L249C-Cy5, reconstituted at χ* = 10⁻⁵ subunits/lipid (1 µg/mg); red line indicates F_0,Cl. (E) F_0,Cl and k of R230C/L249C-Cy5 (RC-LC-Cy5) show no significant difference compared with WT-Cy5. Data are presented as mean ± SEM. (F) Photobleaching data of R230C/L249C-Cy5 (red, P_Cy5 = 0.72 ± 0.02) compared with the saturating range of WT-Cy5 (black) and monomer (dashed) versus dimer (solid) models using the 2:1 POPE/POPG liposome size distribution (P_Cy5 = 0.72, P_non-specific = 0.14, bias = 4 and A_lipid = 0.6). Data are presented as mean ± SEM; n = 5, incubated at room temperature for 1–2 d in MLVs before extrusion.