Nisin is a cationic polycyclic bacteriocin secreted by some lactic acid bacteria. Nisin has previously been shown to permeabilize liposomes. The interaction of nisin was analyzed with liposomes prepared of the zwitterionic phosphatidylcholine (PC) and the anionic phosphatidylglycerol (PG). Nisin induces the release of 6-carboxyfluorescein and other small anionic fluorescent dyes from PC liposomes in a delta psi-stimulated manner, and not that of neutral and cationic fluorescent dyes. This activity is blocked in PG liposomes. Nisin, however, efficiently dissipates the delta psi in cytochrome c oxidase proteoliposomes reconstituted with PG, with a threshold delta psi requirement of about -100 mV. Nisin associates with the anionic surface of PG liposomes and disturbs the lipid dynamics near the phospholipid polar head group-water interface. Further studies with a novel cationic lantibiotic, epilancin K7, indicate that this molecule penetrates into the hydrophobic carbon region of the lipid bilayer upon the imposition of a delta psi. It is concluded that nisin acts as an anion-selective carrier in the absence of anionic phospholipids. In vivo, however, this activity is likely to be prevented by electrostatic interactions with anionic lipids of the target membrane. It is suggested that pore formation by cationic (type A) lantibiotics involves the local perturbation of the bilayer structure and a delta psi-dependent reorientation of these molecules from a surface-bound into a membrane-inserted configuration.

The major coat protein (gVIIIp) of bacteriophage M13 solubilized in sodium dodecyl sulfate (SDS) detergent micelles was used as a model system to study this protein in the lipid-bound form. In order to probe the position of gVIIIp relative to the SDS micelles, stearate was added, spin-labeled at the 5- or 16-position with a doxyl group containing a stable nitroxide radical. The average position of the spin-labels in the micelles was derived from the line broadening of the resonances in the 13C spectrum of SDS. Subsequently, we derived a model of the relative position of gVIIIp in the SDS micelle from the effect of the spin-labels on the gVIIIp resonances, monitored via 1H-15N HSQC and TOCSY experiments. The results are consistent with the structure of gVIIIp having two helical strands. One strand is a long hydrophobic helix that spans the micelle, and the other is a shorter amphipathic helix on the surface of the micelle. These results are in good agreement with the structure of gVIIIp in membranes proposed by McDonnell et al. on the basis of solid state NMR data [McDonnell, P. A., Shon, K., Kim, Y., & Opella, S. J. (1993) J. Mol. Biol. 233, 447-463]. This study indicates that high-resolution NMR on this membrane protein, solubilized in detergent micelles, is a very suitable technique for mimicking these proteins in their natural environment. Furthermore, the data indicate that the structure of the micelle near the C-terminus of the major coat protein is distorted.(ABSTRACT TRUNCATED AT 250 WORDS)

The unusual base composition of the genome of the human malaria parasite Plasmodium falciparum prompted us to systematically investigate the occurrence of homopolymeric DNA tracts in the P. falciparum genome and, for comparison, in the genomes of Homo sapiens , Saccharomyces cerevisiae , Caenorhabditis elegans , Arabidopsis thaliana , Escherichia coli and Mycobacterium tuberculosis. Comparison of theobserved frequencies with the frequencies as expected for random DNA revealed that homopolymeric (dA:dT) tracts occur well above chance in the eukaryotic genome. In the majority of these genomes, (dA:dT) tract overrepresentation proved to be an exponential function of the tract length. (dG:dC) tract overrepresentation was absent or less pronounced in both prokaryotic and eukaryotic genomes. On the basis of our results, we propose that homopolymeric (dA:dT) tracts are expanded via replication slippage. This slippage-mediated expansion does not operate on tracts with lengths below a critical threshold of 7-10 bp.

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The crystal structure of the dimeric gene V protein of bacteriophage fl was determined using multiwavelength anomalous diffraction on the selenomethioninecontaining wild-type and isoleucine-47 methionine mutant proteins with x-ray diffraction data phased to 2.5 A resolution.The structure of the wild-type protein has been refined to an R factor of 19.2% using native data to 1.8 A resolution. The structure of the gene V protein was used to obtain a model for the protein portion of the gene V protein-single-stranded DNA complex.Gene V protein of bacteriophage fltt is a member of a class of proteins involved in DNA replication that bind to singlestranded nucleic acids with high affinity and cooperativity but little sequence specificity (1-4). Gene V protein coats the single-stranded DNA (ssDNA) intermediate in bacteriophage fl DNA replication, forming an ordered superhelical protein-DNA complex (1,(5)(6)(7)(8). This protein-DNA complex facilitates packaging of the ssDNA into new phage particles. Gene V protein also binds with some specificity to a translational operator sequence on phage fl gene II mRNA (9-12).The gene V protein provides a general model for proteinssDNA interactions that are strong, yet not sequencespecific. Gene V protein binding to single-stranded nucleic acids and to oligonucleotides has been studied using chemical modification, spectroscopic techniques, and mutagenesis (13-24). The structure of the protein-ssDNA complex has been studied using electron microscopy and solution scattering methods and is found to consist of a regular left-handed superhelix in which the gene V protein dimers are arrayed on the outside of the superhelix and the ssDNA strands are inside (8,25). The structure of the gene V protein is also of interest because its small size and the large number of mutants available have made it a useful model for determining effects ofamino acid substitutions on protein stability and function (23,26,27).A model for the crystal structure of the wild-type (WT) gene V protein has been reported (28), but recent NMR studies have demonstrated that the positions of amino acids involved in the segments of antiparallel (-structure are not compatible with those in the model (21), and a new determination of the structure was necessary. The multiwavelength anomalous diffraction (MAD) technique (29) was ideally suited for this purpose, as the WT gene V protein contains two methionine residues (Met-1 and Met-77) that might be substituted in vivo in Escherichia coli by selenomethionine. Here we report the determination of the gene V protein structure using the MAD technique, the refinement of the structure using x-ray diffraction data on the WT gene V proteinAt and a model for protein-protein contacts in the gene V protein-ssDNA superhelical complex. MATERIALS AND METHODSModeling of the Protein Portion of Gene V Protein-ssDNA Complex. The gene V protein dimer was placed with its internal two-fold axis of symmetry perpendicular to the axis of the superhelix to be generated and Phe-73 pointing ei...