Fig. 1
Noxa BH3 peptide and membrane-restricted obatoclax (OBX) directly antagonize the ability of Mcl-1 to inhibit Bak-mediated calcein release from proteoliposomes. a Left. Model for the regulation of liposome bilayer permeabilization by the tBid-Bak-Mcl-1 axis. Membrane anchoring of Mcl-1 and Bak is achieved by replacing their C-terminal TM segments with chemical functionalities (blue circle) that interact with modified head groups (red circle) of liposome phospholipids. Bilayer permeabilization is assayed by the acquisition of calcein (Cn) fluorescence upon its release from liposomes induced by tBid. Right. Chemical structures of obatoclax and des-methoxy obatoclax. b Obatoclax binds avidly to lipid vesicles. Addition of lipid vesicles (0–40 μM) to obatoclax (0.15 μM) in buffer at 37 °C leads to rapid obatoclax partitioning into vesicle bilayers and enhancement of obatoclax fluorescence (λex/λem = 540/575 nm, slitwidths = 10/10 nm); obatoclax half-maximally associates with bilayers at 13 ± 1 μM lipid (mean/half-range of two experiments). c Obatoclax transfers rapidly between distinct bilayers. Obatoclax (0.3 μM) added to lipid vesicles (10 μM) incorporating NBD-PE causes rapid energy transfer-mediated quenching of NBD-PE fluorescence (λex/λem = 470/538 nm, slitwidths = 10/10 nm) as obatoclax partitions into the vesicle bilayers (first arrow). On subsequent addition of sonicated vesicles lacking NBD-PE (20 μM; second arrow), obatoclax transfers from NBD-incorporating to NBD-PE-free vesicles, partially restoring NBD-PE fluorescence, over a time scale of seconds. d Proteolipsomes harboring membrane-anchored Mcl-1 and/or Bak derivatives (BakΔC*; Mcl-1ΔC*) were challenged with tBid in the presence or absence of obatoclax or Noxa BH3 peptide. Shown are representative fluorimetric assays from 3 independent experiments of calcein release from liposome in response to 40 nM tBid over time (right panel). Concentrations of assay constituents are given in the left panel