No description available.

Antimicrobial peptides (AMPs) are not only fast microbe-killing molecules deployed in the host defense of living organisms but also offer valuable lessons for developing new therapeutic agents. While the mode of action of AMPs is not clearly understood yet, membrane perturbation has been recognized as a crucial step in the microbial killing mechanism of many AMPs. In this thesis, we first present a physical basis for the selective membrane-disrupting activity of cationic AMPs. To this end, we present a coarse-grained physical model that approximately captures essential molecular details such as peptide amphiphilicity and lipid composition (e.g., anionic lipids). In particular, we calculate the surface coverage of peptides embedded in the lipid headgroup-tail interface and the resulting membrane-area change, in terms of peptide and membrane parameters for varying salt concentrations. We show that the threshold peptide coverage on the membrane surface required for disruption can easily be reached for microbes, but not for the host cell -- large peptide charge (≳4) is shown to be the key ingredient for the optimal activity-selectivity of AMPs (in an ambient-salt dependent way). Intriguingly, we find that in a higher-salt environment, larger charge is required for optimal activity. Inspired by membrane softening by AMPs, we also study electrostatic modification of lipid headgroups and its effects on membrane curvature. Despite its relevance, a full theoretical description of membrane electrostatics is still lacking -- in the past, membrane bending has often been considered under a few assumptions about how bending modifies lipid arrangements and surface charges. Here, we present a unified theoretical approach to spontaneous membrane curvature, C₀, in which lipid properties (e.g., packing shape) and electrostatic effects are self-consistently integrated. Our results show that C₀ is sensitive to the way lipid rearrangements and divalent counterions are modeled. Interestingly, it can change its sign in the presence of divalent counterions, thus stabilizing reverse hexagonal (H_II) phases.

A method was developed which allowed for more consistent liposome quality, reducing the standard error of initial rates for Zn2+ diffusion by 30%. Introducing low concentration of platelet-activating-factor (PAF), 1-palmitoyl-L-lyso-3-phosphocholine (Lyso-PC), or 1-oleoyl-2-acetyl-sn-glycero-3-phosphocholine (Acyl-PAF) to 1palmitoyl.-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes did not have any noticeable impact on zinc diffusion. Since diffusion is dependent on membrane composition and properties, it can be concluded that PAF, Lyso-PC, and Acyl-PAF did not alter POPC liposome properties significantly. Zn2+ and Cd2+ kinetic experiments showed binding to the liposome surface prior to diffusion and a mutual diffusing species, the monohydroxo complex. Although Mn 2+ did not diffuse to any measurable extent, binding to the liposome surface was also observed. Cue+ and Pb 2+ on the other hand follow a more complex diffusion mechanism, which requires further investigation. / Department of Chemistry

Membranes are essential components of all living organisms forming the borders of cells and their organelles. Planar lipid membranes deposited on solid substrates (solid supported membranes) provide models to study the functions of membrane proteins and are used as biosensing platforms. However, despite remarkable progress, solid supported membranes are not stable to harsh conditions such as dehydration, high temperature and pressure, and mechanical stress. In addition, the direct deposition of membranes onto a solid substrate often causes restricted mobility and denaturation of reconstituted membrane proteins. Membrane stability can be addressed by altering the structure of the component lipids. Bolalipids are an interesting class of bipolar lipids that have been proposed for biosensing applications. Membranes formed from mixtures of a bolalipid, C20BAS, and dioleoylphosphaphatidylcholine, POPC, were characterized by atomic force spectroscopy (AFM). The lipid mixtures produced a phase separated membrane consisting of thinner bolalipid-rich and thicker monopolar-rich POPC regions, with a height difference of approximately 1-2 nm. This confirmed an earlier prediction that some bolalipid/PC membranes would phase separate due to the hydrophobic mismatch between the two lipids. Interestingly, the surface coverage of the two phases was inconsistent with what one would expect from the initial starting lipid ratios. The complex membrane morphologies observed were accredited to the interplay of several factors, including a compositionally heterogeneous vesicle population, exchange of lipid between the vesicle solution and solid substrate during formation of the supported membrane, and slow equilibration of domains due to pinning of the lipids to the solid support. Decoupling the membrane from its underlying surface is one strategy to maintain the structure and mobility of membrane proteins. This decoupling can be achieved by depositing the membrane on a soft cushion composed of a water swelling hydrophilic polymer. A polyelectrolyte multilayer (PEM) and a tethered poly(ethylene) glycol (PEG) polymer are the two types of polymer cushions used in this study. The PEMs consist of the charged polysaccharides, chitosan (CHI) and hyaluronic acid (HA) which offer the advantage of biocompatibility over synthetic PEMs. DOPC lipid bilayers were formed at pH 4 and 6.5 on (CHI/HA)5 films. At higher pH adsorbed lipids had low mobility and large immobile lipid fractions; fluorescence and AFM showed that this was accredited to the formation of poor quality membranes with defects and pinned lipids rather than to a layer of surface-adsorbed vesicles. However, more uniform bilayers with mobile lipids were produced at pH 4. Measured diffusion coefficients were similar to those for bilayers on PEG cushions and considerably higher than those measured on other polyelectrolyte films. The results suggest that the polymer surface charge is more important than the surface roughness in controlling formation of mobile supported bilayers. The suitability of polymer supported membranes for the incorporation of integral membrane proteins was also assessed. The integral membrane protein Ste14p, a 26 kDa methyltransferase enzyme, was reconstituted into POPC membranes on PEM and PEG supports. A combination of fluorescence microscopy, FRAP, AFM and an in situ methyltransferase activity assay were utilized to characterize the protein incorporated polymer supported membranes. Fluorescence measurements showed that more protein was incorporated in model membranes formed on the PEG support, compared to either glass or PEM cushions. However, the protein activity on a PEG support was comparable to that of the protein in a membrane on glass. FRAP measurements showed that the lipid mobilities of the POPC:Ste14p bilayers on the various supports were also comparable. Lastly, as a new platform for manipulating and handling membrane proteins, nanodiscs containing reconstituted Ste14p were studied. Nanodiscs are small, soluble and stable bilayer discs that permit the study of membrane proteins in a uniform phospholipid bilayer environment. Empty and protein containing nanodiscs were deposited on a mica surface and imaged by AFM. AFM showed that protein containing samples possessed two subpopulations of nanodiscs with a height difference of ~1 nm. The taller discs, ~20% of the population, contained protein. Other experiments showed that the packing of the nanodisc samples was influenced by their initial stock concentration and that both imaging force and the addition of Mg2+ caused formation of larger bilayer patches.

No description available.

Off-resonance unenhanced total internal reflection (TIR) Raman Spectroscopy was explored to investigate supported single lipid bilayers with incorporated membrane peptides/proteins at water/solid interface. A model membrane was formed on a planar supported lipid layer (pslb) by the fusion of the reconstituted small unilamellar vesicles (SUVs), and the intensity of bilayer was confirmed by a comparison of Raman spectral intensity in the C-H stretching modes with C₁₆TAB. With prominent Raman sensitivity attained, we studied the 2-D phase transition of DMPC and DPPC pslbs and the temperature-dependent polarised spectra revealed a broad transition range of ca. 10 °C commencing at the calorimetric phase transition temperature. We applied polarised TIR-Raman Spectroscopy to pslbs formed by DMPC SUVs reconstituted with a model membrane-spanning peptide gramicidin D. A preferential channel structure formed by dissolution of trifluoroethanol could be probed by polarised Raman Spectroscopy qualitatively showing an antiparallel β-sheet conformation (different from "standard" one) and our Raman spectra by correlation with NMR and CD data confirmed single-stranded π^6.3 β-helical channel structure in the single bilayer. We also studied the membrane-penetrating peptide indolicidin in the presence of DMPC pslb over the chain melting temperature and a β-turn structure was dominantly observed concomitant with membrane perturbation. Dynamic adsorption of DPPC to form pslb from a micellar solution of n-dodecyl-β- _D-maltoside could be examined with high sensitivity of every 1-min acquisition. Finally we used polarised TIR-Raman scattering to porcine pancreatic phospholipase A₂ hydrolytic activity on DPPC pslbs and revealed lipid-active conformation different from that of the enzyme alone.

Thesis (M.S.)--University of Texas at El Paso, 2008. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

Thesis (Ph.D.)--Cleveland State University, 2008. / Abstract. Title from PDF t.p. (viewed on Oct. 8, 2008). Includes bibliographical references. Available online via the OhioLINK ETD Center. Also available in print.

Thesis (M.S.)--University of North Carolina at Wilmington, 2005. / Includes bibliographical references (leaves: 53-55)

Thesis (Ph.D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xxii, 352 p.; also includes graphics. Includes bibliographical references (p. 346-352). Available online via OhioLINK's ETD Center