This project is aimed at the understanding of the physico-chemical mechanisms of membrane remodeling during physiological and pathogenic events. A frame-shifted region of the influenza A virus PB1 gene encodes a novel protein, termed PB1-F2, a mitochondrial protein that can induces cell death. Many pro-apoptotic proteins are believed to act at the mitochondrial outer membrane to form an apoptotic pore with lipids. We studied the interaction of isolated, synthetic (s) PB1-F2 peptide with planar phospholipid bilayer membranes. The presence of nanomolar concentrations of peptide in the bathing solution induced a trans-membrane conductance that increased in a potential-dependent manner. Positive potential on the side of protein addition resulted in a several fold increase in the rate of change of membrane conductance. sPB1-F2 treated membranes became permeable to monovalent cations, chloride, and to a lesser extent, divalent ions. Despite varying experimental conditions, we failed to detect distinctive conductance levels typical for large, stable pores, protein channels, or even pores that are partially proteinaceous. Rather, membrane conductance induced by sPB1-F2 fluctuated and visited almost all conductance values. sPB1-F2 also dramatically decreased bilayer stability in an electric field, consistent with a decrease in the line tension of a lipidic pore. Since similar membrane destabilizing profiles are seen with pro-apoptotic proteins (e.g. Bax) and the cytoplasmic helix of HIV gp41, we suggest that the basis for sPB1-F2 induced cell death may be the permeabilization and destabilization of mitochondrial membranes, leading to macromolecular leakage and apoptosis. Neuronal death is often preceded by functional alterations at nerve terminals. The BCL-2 family protein BCL-xL is an anti-apoptotic protein found in the adult nervous system, including the giant presynaptic terminal of the squid stellate ganglion, where it is known to form ion channels in the outer mitochondrial membrane. Its naturally occurring N-truncated protease cleavage product delta-N BCL-xL is pro-apoptotic. Application of delta-N Bcl-xL to mitochondria inside the squid presynaptic terminal rapidly induced the opening of large multi-conductance channels in mitochondrial membranes. The maximal conductance of these channels was significantly larger than that of control recordings or that with full length BCL-xL. Mutants of delta-N Bcl-xL lacking a C-terminal mitochondrial anchor region, or in which the death producing BH3 domain was mutated, failed to induce channel activity. Moreover, delta-N Bcl-xL failed to produce any channel activity when applied to plasma membranes, suggesting that a component of mitochondrial membranes was necessary for its actions. The occurrence of the large conductance openings was reduced by NADH, an inhibitor of the outer mitochondrial membrane channel VDAC. Pharmacological and genetic experiments on yeast mitochondria lacking VDAC indicated that the NADH-sensitive DN Bcl-xL ?induced channel activity results from a functional interaction between DN Bcl-xL and VDAC. Furthermore, exposure of squid ganglia to hypoxia, a death stimulus to neurons, rapidly induced proteolysis of full length BCL-xL and this was blocked by the protease inhibitor zVAD, suggesting that proteolysis of BCL-2 family proteins may contribute to the induction of large conductance activity in the outer mitochondrial membrane. Mixtures of cationic and anionic surfactants crystallized at various ratios in the absence of added salt, form micron-sized colloids. Here we propose and test a general mechanism explaining how this ratio controls the shape of the resulting colloidal structure, which can vary from nanodiscs to punctured planes: during co-crystallization, excess (non-stoichiometric) surfactant accumulates on edges or pores rather than being incorporated into crystalline bilayers. Molecular segregation then produces a sequence of shapes controlled by the initial mole ratio only. Using freeze-fracture electron microscopy, we identified three of these states and their corresponding coexistence regimes. Fluorescence confocal microscopy directly showed the segregation of anionic and cationic components within the aggregate. The observed shapes are consistently reproduced upon thermal cycling, demonstrating that the icosahedral shape corresponds to the existence of a local minimum of bending energy for facetted icosahedra when the optimal amount of excess segregated material is present.
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