Figure 3. Glutaraldehyde cross-linking... | Rockefeller University Press

Figure 3.

$Figure 3. Glutaraldehyde cross-linking of WT-Cy5. (A) Side view of CLC-ec1 homodimer, with subunits colored light and dark gray. Residues with primary amine groups that are potential glutaraldehyde cross-linking sites are highlighted as follows: lysines are turquoise, with those within 5 Å of another lysine colored dark blue; the N terminus is represented by a circle. (B) SDS-PAGE of WT (on the C85A/H234C background), WT + glutaraldehyde, and WT + 2% SDS followed by addition of glutaraldehyde. Red circle, monomer; gray circles, dimer; white circles, tetramer. (C) Size exclusion chromatography profiles of WT and glutaraldehyde cross-linked WT. (D) Functional Cl− transport of WT-Cy5 and WT-Cy5 + glutaraldehyde, reconstituted at χ* = 10−5 subunits/lipid (1 µg/mg). The red line indicates the fraction of chloride trapped in empty liposomes (F0,Cl). (E) F0,Cl and chloride transport rate (k) show a reduction of activity in the presence of glutaraldehyde (****, P ≤ 0.0001; and ***, P ≤ 0.001). Data are presented as mean ± SEM. (F) Photobleaching data of WT-Cy5 + glutaraldehyde (red, PCy5 = 0.70 ± 0.04) compared with the saturating range of WT-Cy5 (black) and monomer (dashed) versus dimer (solid) models using the 2:1 POPE/POPG liposome size distribution (PCy5 = 0.72, Pnon-specific = 0.14, bias = 4, and Alipid = 0.6). Data are presented as mean ± SEM; n = 4–6, incubated at room temperature for 7–31 d in MLVs before extrusion.$

Glutaraldehyde cross-linking of WT-Cy5. (A) Side view of CLC-ec1 homodimer, with subunits colored light and dark gray. Residues with primary amine groups that are potential glutaraldehyde cross-linking sites are highlighted as follows: lysines are turquoise, with those within 5 Å of another lysine colored dark blue; the N terminus is represented by a circle. (B) SDS-PAGE of WT (on the C85A/H234C background), WT + glutaraldehyde, and WT + 2% SDS followed by addition of glutaraldehyde. Red circle, monomer; gray circles, dimer; white circles, tetramer. (C) Size exclusion chromatography profiles of WT and glutaraldehyde cross-linked WT. (D) Functional Cl⁻ transport of WT-Cy5 and WT-Cy5 + glutaraldehyde, reconstituted at χ* = 10⁻⁵ subunits/lipid (1 µg/mg). The red line indicates the fraction of chloride trapped in empty liposomes (F_0,Cl). (E) F_0,Cl and chloride transport rate (k) show a reduction of activity in the presence of glutaraldehyde (****, P ≤ 0.0001; and ***, P ≤ 0.001). Data are presented as mean ± SEM. (F) Photobleaching data of WT-Cy5 + glutaraldehyde (red, P_Cy5 = 0.70 ± 0.04) 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 = 4–6, incubated at room temperature for 7–31 d in MLVs before extrusion.