Fig. 6. Superimposed 900-MHz TROSY spectra from a cholesterol titration of the Notch-TMD demonstrate an absence of specific association of cholesterol with the Notch-TMD.

NMR samples were prepared with increasing concentrations of cholesterol (0, 2.5, 5, 10, and 15 mol %). Samples contained ~0.35 mM protein in 15% (w/v) DMPC/DHPC bicelles. There are no significant chemical shift perturbations that suggest direct cholesterol binding [compare to results for C99 (30)]. Moreover, even the modest shifts seen for some residues vary linearly with cholesterol concentration rather than showing any sign of saturation of binding.