Figure 4

Titration of atTic20 in POPC liposomes with SDS. (A) Far-UV CD spectra of reconstituted atTic20 in POPC liposomes titrated with SDS (0 – 84 mM). (A-i) shows the low [SDS] region of the spectra in greater detail (0-1.3 mM), where higher SDS concentrations enhanced negative ellipticities at both 208 and 222 nm. (A-ii) shows the high [SDS] region (1.3-84 mM) in greater detail, where increased SDS concentrations resulted in only small changes in the molar ellipticity of atTic20. (B) Plot of R_θvs. [SDS] reveals a cooperative dissociation of associated atTic20 in the presence of SDS. The R_θ was normalized and K_1/2 ([SDS] at 50% protein dissociation) was calculated using the Hill fitting (K_1/2 = 0.5 ± 0.05 mM). (C) Semi-native PAGE (12%) of atTic20 and atTic20ΔN20 in POPC liposomes in the presence of increasing concentrations of SDS added to the sample buffer (the electrophoretic running buffer contained 2 mM SDS in all cases). The gels were stained with Coomassie Brilliant Blue. Monomeric (M), dimeric (D), trimeric (Tr) and tetrameric (T) forms of atTic20 and atTic20ΔN20 are indicated. (D) Relative proportion of the different associated states of atTic20 and atTic20ΔN20 measured during the SDS titration as determined by measuring band intensity on the semi-native PAGE gels shown in C. Band intensity was quantified using Quantiy One (Bio-Rad) as explained in the Methods.