Figure 1.

Bax activation scheme, spin labeling and activity assays. (a) Sketch of conformational changes of Bax during activation. The protein exists at least in seven conformational states (a-g). Soluble monomeric Bax (state “a”) can transiently interact with the mitochondrial outer membrane MOM (“b”). Upon interaction with an activator-type BH3-only protein membrane insertion is induced (“c”)^20,22 followed by the formation of homo-dimers (“d”) that assemble into higher order oligomers^10,18,22,24 (“e”). The oligomers form a toroidal pore in the MOM allowing the release of Cyt c^39,59 (“f”). These pores can grow to “mega-pores” able to release mitochondrial DNA^14,17. (b) The four spin labels used in this study, targeting cysteine residues. (c) Room temperature cw spectra of the nitroxide-labeled proteins, with the degree of labeling (spin/cysteine ratio) indicated in %. Asterisks indicate residual free label. For Gd-labeled Bax see Supp. Fig. 2. (d) Structural models of monomeric¹² and dimeric active Bax¹⁸ with one MTSL (R1) label attached per site using the software MMM⁵⁵. Intra- and inter- monomer distances are highlighted by dotted lines (black for the wild type variants, red for the C87 variants). (e) Graphical scheme of the assay used to address Bax-induced pore formation. (f) Kinetic curves of pore formation (left panels) and data points after 30 min (right panels) for the Bax_WT variants (upper panels) and for the Bax_C87 variants (lower panels), both compared to unlabeled Bax_WT. Dotted lines: Bax alone (50 nM) with vesicles; solid lines: 50 nM Bax with 50 nM cBid and vesicles. Note that the maximally reachable fraction of permeabilized vesicles varies for different experiments, depending on the lipid preparation (100% permeabilization was detected by adding detergent).