No abstract

The mechanism of pore formation of lytic peptides, such as melittin from bee venom, is thought to involve binding to the membrane surface, followed by insertion at threshold levels of bound peptide. We show that in membranes composed of zwitterionic lipids, i.e. phosphatidylcholine, melittin not only forms pores but also inhibits pore formation. We propose that these two modes of action are the result of two competing reactions: direct insertion into the membrane and binding parallel to the membrane surface. The direct insertion of melittin leads to pore formation, whereas the parallel conformation is inactive and prevents other melittin molecules from inserting, hence preventing pore formation.Lytic or antimicrobial peptides are small proteins (12-50 amino acids) that affect cells by disrupting the barrier function of lipid membranes (1). There is much interest in these peptides because of their potential pharmaceutical applications, e.g. as cancer drugs and antibiotics (2). Because of their small size and high stability, they can be obtained in large quantities. Of all lytic peptides, the 26-residue melittin is the best studied to date (for review see Ref.3). Melittin is the major constituent of the venom of the European honeybee Apis mellifera. Melittin has been reported to have anticancer effects and is already being used to treat pain and arthritis in Asia (4).Melittin can bind within milliseconds to lipid membranes and adopts an amphipatic ␣-helical conformation, oriented either parallel or perpendicular to the plane of the membrane. The perpendicular conformation is embedded in the membrane and is needed for pore formation, whereas the parallel conformation is inactive (5-10). These observations led to the proposal of a two-step model for pore formation, where melittin at low concentrations binds parallel to the membrane (Fig. 1A, step 1) and at higher concentrations shifts toward the perpendicular orientation (step 2), causing pore formation (10 -12). The transition from parallel to perpendicular is still poorly understood, especially because the cationic (5ϩ at neutral pH) melittin interacts strongly with the lipid headgroups (partitioning coefficient of 10 4 -10 5 M Ϫ1 for phosphatidylcholine (PC) 2 (12-17)), and the energy of the transition must be very high (3,11). Based on the high affinity of melittin for PC headgroups, it has been widely accepted that the concentration of melittin needed for pore formation is not dependent on the absolute melittin concentration but rather on the ratio of melittin to lipid molecules. However, this important implication has never been directly tested by varying the lipid concentration and determining the fractions of bound and free melittin. In this study we analyzed the lipid concentration dependence of melittin action and studied the reversibility of melittin binding, thereby resolving pore formation from binding events. MATERIALS AND METHODSMelittin was purchased from Genscript (Piscataway, NJ), and lipids were from Avanti Polar Lipids (Albaster, AL). Liposomes ...

SummaryWe determined the diffusion coefficients (D) of (macro)molecules of different sizes (from~0.5 to 600 kDa) in the cytoplasm of live Escherichia coli cells under normal osmotic conditions and osmotic upshift. D values decreased with increasing molecular weight of the molecules. Upon osmotic upshift, the decrease in D of NBD-glucose was much smaller than that of macromolecules. Barriers for diffusion were found in osmotically challenged cells only for GFP and larger proteins. These barriers are likely formed by the nucleoid and crowding of the cytoplasm. The cytoplasm of E. coli appears as a meshwork allowing the free passage of small molecules while restricting the diffusion of bigger ones.

Molecular self-assembly is the basis for the formation of numerous artificial nanostructures. The self-organization of peptides, amphiphilic molecules composed of fused benzene rings and other functional molecules into nanotubes is of particular interest. However, the design of dynamic, complex self-organized systems that are responsive to external stimuli remains a significant challenge. Here, we report self-assembled, vesicle-capped nanotubes that can be selectively disassembled by irradiation. The walls of the nanotubes are 3-nm-thick bilayers and are made from amphiphilic molecules with two hydrophobic legs that interdigitate when the molecules self-assemble into bilayers. In the presence of phospholipids, a phase separation between the phospholipids and the amphiphilic molecules creates nanotubes, which are end-capped by vesicles that can be chemically altered or removed and reattached without affecting the nanotubes. The presence of a photoswitchable and fluorescent core in the amphiphilic molecules allows fast and highly controlled disassembly of the nanotubes on irradiation, and distinct disassembly processes can be observed in real time using fluorescence microscopy.

Bacterial populations harbor a small fraction of cells that display transient multidrug tolerance. These so-called persister cells are extremely difficult to eradicate and contribute to the recalcitrance of chronic infections. Several signaling pathways leading to persistence have been identified. However, it is poorly understood how the effectors of these pathways function at the molecular level. In a previous study, we reported that the conserved GTPase Obg induces persistence in Escherichia coli via transcriptional upregulation of the toxin HokB. In the present study, we demonstrate that HokB inserts in the cytoplasmic membrane where it forms pores. The pore-forming capacity of the HokB peptide is demonstrated by in vitro conductance measurements on synthetic and natural lipid bilayers, revealing an asymmetrical conductance profile. Pore formation is directly linked to persistence and results in leakage of intracellular ATP. HokB-induced persistence is strongly impeded in the presence of a channel blocker, thereby providing a direct link between pore functioning and persistence. Furthermore, the activity of HokB pores is sensitive to the membrane potential. This sensitivity presumably results from the formation of either intermediate or mature pore types depending on the membrane potential. Taken together, these results provide a detailed view on the mechanistic basis of persister formation through the effector HokB.