FIGURE 4.

Domain 3 conformational changes are blocked in PFO^W165T. Membrane binding and the D3 structural changes normally associated with assembly and formation of the oligomeric β-barrel pore were detected by placing fluorophores at specific locations and comparing the emission intensities of native PFO and PFO^W165T derivatives in the absence (solid line) and presence (dashed line) of liposomes. A, membrane binding of PFO and PFO^W165T was detected by comparing the emission intensities of undecapeptide tryptophans of wild-type PFO (WT) and PFO^W165T in the absence and presence of liposomal membranes (4). B, disruption of the domain D2-D3 interface was detected by monitoring the aqueous exposure (and resulting decrease in the emission intensity) of the buried NBD, located at cysteine-substituted Asn-197, as D3 swings away from D2 (9). C, disengagement of β5 from β4 was detected by monitoring the decrease in the emission of NBD positioned at cysteine-substituted Val-322, which is buried beneath β5. As β4 and β5 separate, NBD moves from a nonpolar to polar environment, and its emission intensity decreases (7). D, intermolecular association of β1 and β4 from adjacent membrane-bound monomers is shown by the π-stacking of pyrene dyes attached to Cys-substituted Thr-179 (located in β1) and Val-322 (located in β4) and the resultant broad excimer emission near 470 nm (7). Pyrene emission spectra are shown for an equimolar mixture of pyrene-derivatized PFO^T179C and PFO^V322C in wild-type (WT) and PFO^W165T backgrounds in the absence (solid line) and presence (dashed line) of liposomes.