Fig. 3.

Intramembrane kinetics of γ-secretase within a proteoliposome. (A) Fluorescence quenching of N43 when incorporated into vesicles is alleviated after dissolving the proteoliposome with 0.25% Nonidet P-40 detergent. (B) Circular dichroism spectrum of N43 incorporated into a proteoliposome reveals an α-helical structure. (C) Labeling of a notch peptide with a Cys on its N terminus with a membrane impermeable dye in the presence and absence of melittin to allow dye passage into the interior of the proteoliposome (mean ± SD, n = 2). (D) After reconstitution of γ-secretase and N43 substrate into proteoliposomes, the reaction was initiated by resuspending the proteoliposomes in 50 mM Hepes pH 7.0 and 150 mM NaCl. Product was formed linearly with time. (E) Kinetics of N43 cleavage by γ-secretase within a proteoliposome (mean ± SD, n = 4).