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  • Biochimica et Biophysica Acta (BBA) - Biomembranes 09/2014; 1838(9):2139. DOI:10.1016/j.bbamem.2014.06.001
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    ABSTRACT: We previously performed a lipid vesicle-based, high-throughput screen on a 26-residue combinatorial peptide library that was designed de novo to yield membrane permeabilizing peptides that fold into β-sheets. The most active and soluble library members that were identified permeabilized lipid vesicles detectably, but not with high potency. Nonetheless, they were broad-spectrum, membrane-permeabilizing antibiotics with minimum sterilizing activity at low μM concentrations. In an expansion of that work, we recently performed an iterative screen in which an active consensus sequence from that first generation library was used as a template to design a second generation library which was then screened against lipid vesicles at very high stringency. Compared to the consensus sequence from the first library, the most active second generation peptides are highly potent, equilibrium pore-formers in synthetic lipid vesicles. Here we use these first and second generation families of peptides to test the hypothesis that a large increase in potency in bacteria-like lipid vesicles will correlate with a large improvement in antimicrobial activity. The results do not support the hypothesis. Despite a 20-fold increase in potency against bacteria-like lipid vesicles, the second generation peptides are only slightly more active against bacteria, and at the same time, are also more toxic against mammalian cells. The results suggest that a "pipeline" strategy towards the optimization of antimicrobial peptides could begin with a vesicle-based screen for identifying families with broad-spectrum activity, but will also need to include screening or optimization steps that are done under conditions that are more directly relevant to possible therapeutic applications. © 2013 Wiley Periodicals, Inc. Biopolymers, 2013.
    Biopolymers 01/2014; 102(1). DOI:10.1002/bip.22281
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    ABSTRACT: We previously reported the development of a highly accurate statistical algorithm for identifying β-barrel outer membrane proteins or transmembrane β-barrels (TMBBs), from genomic sequence data of Gram-negative bacteria (Freeman,T.C. and Wimley,W.C. (2010) Bioinformatics, 26, 1965-1974). We have now applied this identification algorithm to all available Gram-negative bacterial genomes (over 600 chromosomes) and have constructed a publicly available, searchable, up-to-date, database of all proteins in these genomes. For each protein in the database, there is information on (i) β-barrel membrane protein probability for identification of β-barrels, (ii) β-strand and β-hairpin propensity for structure and topology prediction, (iii) signal sequence score because most TMBBs are secreted through the inner membrane translocon and, thus, have a signal sequence, and (iv) transmembrane α-helix predictions, for reducing false positive predictions. This information is sufficient for the accurate identification of most β-barrel membrane proteins in these genomes. In the database there are nearly 50 000 predicted TMBBs (out of 1.9 million total putative proteins). Of those, more than 15 000 are 'hypothetical' or 'putative' proteins, not previously identified as TMBBs. This wealth of genomic information is not available anywhere else. The TMBB genomic database is available at http://beta-barrel.tulane.edu/. wwimley@tulane.edu.
    Bioinformatics 07/2012; 28(19):2425-30. DOI:10.1093/bioinformatics/bts478
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    ABSTRACT: Moving freely: A recent model for voltage gating of potassium channels proposed that the four arginine residues of the voltage-sensing S4 helix (left) are in direct contact with the membrane lipids and move into the hydrocarbon core of the membrane during gating. It is demonstrated that the physical properties of the isolated S4 sequence (right) are sufficient to allow it to freely translocate across synthetic membranes.
    Angewandte Chemie International Edition 07/2012; 51(29):7150-3. DOI:10.1002/anie.201202741
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    ABSTRACT: We recently developed an orthogonal, high-throughput assay to identify peptides that self-assemble into potent, equilibrium pores in synthetic lipid bilayers. Here, we use this assay as a high-throughput screen to select highly potent pore-forming peptides from a 7776-member rational combinatorial peptide library based on the sequence of the natural pore-forming peptide toxin melittin. In the library we varied ten critical residues in the melittin sequence, chosen to test specific structural hypotheses about the mechanism of pore formation. Using the new high-throughput assay, we screened the library for gain-of-function sequences at a peptide to lipid ratio of 1:1000 where native melittin is not active. More than 99% of the library sequences were also inactive under these conditions. A small number of library members (0.1%) were highly active. From these we identified 14 potent, gain-of-function, pore-forming sequences. These sequences differed from melittin in only 2-6 amino acids out of 26. Some native residues were highly conserved and others were consistently changed. The two factors that were essential for gain-of-function were the preservation of melittin's proline-dependent break in the middle of the helix and the improvement and extension the amphipathic nature of the α-helix. In particular the highly cationic carboxyl-terminal sequence of melittin, is consistently changed in the gain-of-function variants to a sequence that it is capable of participating in an extended amphipathic α-helix. The most potent variants reside in a membrane-spanning orientation, in contrast to the parent melittin, which is predominantly surface bound. This structural information, taken together with the high-throughput tools developed for this work, enable the identification, refinement and optimization of pore-forming peptides for many potential applications.
    Journal of the American Chemical Society 06/2012; 134(30):12732-41. DOI:10.1021/ja3042004
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    ABSTRACT: To enable selection and characterization of highly potent pore-forming peptides, we developed a set of novel assays to probe 1) the potency of peptide pores at very low peptide concentration; 2) the presence or absence of pores in membranes after equilibration; 3) the interbilayer exchangeability of pore-forming peptides; and 4) the degree to which pore-forming peptides disrupt the bilayer organization at equilibrium. Here, we use these assays to characterize, in parallel, six membrane-permeabilizing peptides belonging to multiple classes. We tested the antimicrobial peptides LL37 and dermaseptin S1, the well-known natural lytic peptides melittin and alamethicin, and the very potent lentivirus lytic peptides LLP1 and LLP2 from the cytoplasmic domain of HIV GP41. The assays verified that that the antimicrobial peptides are not potent pore formers, and form only transient permeabilization pathways in bilayers which are not detectable at equilibrium. The other peptides are far more potent and form pores that are still detectable in vesicles after many hours. Among the peptides studies, alamethicin is unique in that it is very potent, readily exchanges between vesicles, and disturbs the local bilayer structure even at very low concentration. The equally potent LLP peptides do not exchange readily and do not perturb the bilayer at equilibrium. Comparison of these classes of pore forming peptides in parallel using the set of assays we developed demonstrates our ability to detect differences in their mechanism of action. Importantly, these assays will be very useful in high-throughput screening where highly potent pore-forming peptides can be selected based on their mechanism of action.
    Biochimica et Biophysica Acta 02/2012; 1818(7):1625-1632. DOI:10.1016/j.bbamem.2012.02.009
  • Neurochemical Research 01/2012; 37(6):1150-3. DOI:10.1007/s11064-011-0699-4
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    ABSTRACT: Our laboratory has previously reported an antibody-based assay for hexavalent uranium (UO(2)(2+)) that could be used on-site to rapidly assess uranium contamination in environmental water samples (Melton, S. J.; et al. Environ. Sci. Technol. 2009, 43, 6703-6709). To extend the utility of this assay to less-characterized sites of uranium contamination, we required a uranium-specific adsorbent that would rapidly remove the uranium from groundwater samples, while leaving the concentrations of other ions in the groundwater relatively unaltered. This study describes the development of hydrogel particles containing amidoxime groups that can rapidly and selectively facilitate the uptake of uranyl ions. A miniemulsion polymerization technique using SDS micelles was employed for the preparation of the hydrogel as linked submicrometer particles. In polymerization, acrylonitrile was used as the initial monomer, ethylene glycol dimethacrylate as the crosslinker and 2-hydroxymethacrylate, 1-vinyl-2-pyrrolidone, acrylic acid, or methacrylic acid were added as co-monomers after the initial seed polymerization of acrylonitrle. The particles were characterized by transmission electron spectroscopy, scanning electron microscopy (SEM) and cryo-SEM. The amidoximated particles were superior to a commercially available resin in their ability to rapidly remove dissolved UO(2)(2+) from spiked groundwater samples.
    ACS Applied Materials & Interfaces 11/2011; 4(1):163-70. DOI:10.1021/am201204r
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    ABSTRACT: Here we test the hypothesis that membrane-spanning β-sheets can exhibit structural plasticity in membranes due to their ability to shift hydrogen-bonding patterns. Transmembrane β-sheet forming peptides of the sequence AcWL(n), where n = 5, 6, or 7, which range from 21 to 27 Å in maximum length, were incorporated into bilayers made of phosphatidylcholine lipids with saturated acyl chains containing 14, 16, or 18 carbons, which are 36-50 Å in thickness. The effect of the peptide β-sheets on fluid- and gel-phase bilayers were studied with differential scanning calorimetry and circular dichroism spectroscopy. We show that AcWL₅ forms a stable, peptide-rich gel phase in all three lipids. The whole family of AcWL(n) peptides appears to form similarly stable, nonmembrane-disrupting β-sheets in all bilayer phases and thicknesses. Bilayers containing up to 20 mol % peptide, which is the maximum concentration tested, formed gel phases with melting temperatures that were equal to, or slightly higher than, the pure lipid transitions. Given the range of peptide lengths and bilayer thicknesses tested, these experiments show that the AcWL(n) family of membrane-inserted β-sheets exhibit remarkable structural plasticity in membranes.
    Biophysical Journal 08/2011; 101(4):828-36. DOI:10.1016/j.bpj.2011.06.059
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    ABSTRACT: Combinatorial peptide chemistry and orthogonal high-throughput screening were used to select peptides that spontaneously translocate across synthetic lipid bilayer membranes without permeabilization. A conserved sequence motif was identified that contains several cationic residues in conserved positions in an otherwise hydrophobic sequence. This 9-residue motif rapidly translocates across synthetic multibilayer vesicles and into cells while carrying a large polar dye as a "cargo" moiety. The extraordinary ability of this family of peptides to spontaneously translocate across bilayers without an energy source of any kind is distinctly different from the behavior of the well-known, highly cationic cell-penetrating peptides, such as the HIV tat peptide, which do not translocate across synthetic bilayers, and enter cells mostly by active endocytosis. Peptides that translocate spontaneously across membranes have the potential to transform the field of drug design by enabling the delivery of otherwise membrane-impermeant polar drugs into cells and tissues. Here we describe the chemical tools needed to rapidly identify spontaneous membrane translocating peptides.
    Journal of the American Chemical Society 06/2011; 133(23):8995-9004. DOI:10.1021/ja2017416
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