We have previously demonstrated that spontaneous phospholipid transfer between bilayer vesicles at higher vesicle concentrations is characterized not only by a first-order desorption rate but also by a second-order process dependent on vesicle concentration (Jones & Thompson, 1989b). We have extended our studies to examine the mechanism of this second-order process by investigating transfer as a function of lipid type, temperature, aqueous medium composition, and vesicle size. The results suggest a mechanism of concentration-dependent transfer in which the rate of lipid monomer desorption from vesicle bilayers is enhanced in transient vesicle-vesicle complexes.

The transfer kinetics of [3H]-1-palmitoyl-2-oleoylphosphatidylcholine ([3H]POPC) and 1-palmitoyl-2-(pyrenyldecanoyl)phosphatidylcholine (PyrPC) from POPC small unilamellar vesicles were examined at 37 degrees C with lipid concentrations ranging from 0.1 to 40 mM. The rate of [3H]POPC transfer was determined by analyzing the movement of this lipid from charged donor to neutral acceptor vesicles. The rate of decay of the ratio of the intensity of pyrene excimer fluorescence to that from the pyrene monomer (E/M) upon addition of an unlabeled vesicle population to a population containing PyrPC was used to evaluate PyrPC transfer. For both lipids, the kinetic data are best described by a model which assumes that transfer occurs by vesicle collisions as well as by desorption from the bilayer. For [3H]POPC, the off-rate constant is 0.014 h-1 while the collisional rate constant is 0.0016 mM-1 h-1. PyrPC has an off-rate constant of 0.023 h-1 and a collisional constant of 0.0015 mM-1 h-1. These numbers were calculated by assuming the rate of interbilayer transfer to be negligible relative to that of intervesicular transfer. The large transfer fluxes in the high vesicle concentration range where the collisional process dominates suggest that spontaneous transfer may be of importance in membrane biogenesis.

We have determined the conformation of an analogue of the Escherichia coli LamB signal peptide inserted into a model membrane using the transferred nuclear Overhauser effect (trNOE) NMR technique. In order to make NMR analysis feasible, a water-soluble LamB signal peptide analogue was designed by inserting three basic residues (KRR) into the N-terminal region of the wild-type sequence (with a Val-->Trp mutation for fluorescence measurements), viz., MMITLRKRRKLPLAVAVAAGWMSAQAMA-NH2. For the purpose of the trNOE study, the binding affinity of the peptide for phospholipid vesicles was tuned by adjusting the proportion of acidic lipid in the vesicle. Circular dichroism and fluorescence measurements showed that the KRR-LamB signal peptide spontaneously inserted into the lipid bilayer with a conformational transition from a mostly random coil to a predominantly alpha-helical structure. The trNOE analysis revealed that the alpha-helix extended from approximately the beginning of the hydrophobic core (residue Leu8) to the C-terminus. The continuity of the helix was somewhat disrupted at the end of the hydrophobic core (around residue Gly17). Furthermore, the topological arrangement of the peptide within the lipid bilayer was explored by NMR line broadening induced by a paramagnetic nitroxide-labeled lipid. The line-broadening results demonstrated that the residues in the helical region are well integrated into the acyl chain region of the bilayer. The N-terminal part of the peptide showed many trNOEs, but without any indication of a helical conformation. The line-broadening analysis indicates that this part of the peptide primarily interacts with the membrane surface.

The lack of methods for isolating Borrelia burgdorferi outer membranes (OMs) has hindered efforts to characterize borrelial surface-exposed proteins. Here we isolated OMs by immersion of motile spirochetes in hypertonic sucrose followed by isopycnic ultracentrifugation of the plasmolyzed cells. The unilamellar vesicles thus obtained were shown to be OMs by the following criteria: (i) they contained OspA and OspB; (ii) they did not contain flagellin, NADH oxidase activity, or the 60-kDa heat shock protein; and (iii) their morphology by freeze-fracture electron microscopy was identical to that of OMs of intact organisms. Consistent with previous studies which employed immunoelectron microscopy and detergent-based solubilization of B. burgdorferi OMs, only small proportions of the total cellular content of OspA or OspB were OM associated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fluorography of OMs from spirochetes metabolically radiolabeled with [ 3 H]palmitate or 35 S-amino acids demonstrated that the OMs contained both nonlipidated and lipidated proteins. This fractionation procedure was also used to isolate OMs from virulent and avirulent isolates of the well-characterized B. burgdorferi N40 strain. SDS-PAGE fluorography revealed that OMs from the two isolates differed with respect to both nonlipoprotein and lipoprotein constituents. When whole cells, protoplasmic cylinders, and OMs were immunoblotted against sera from mice persistently infected with B. burgdorferi N40, the majority of antibody reactivity was directed against intracellular proteins. The availability of isolated OMs should facilitate efforts to elucidate the complex relationship(s) between B. burgdorferi membrane composition and Lyme disease pathogenesis.