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

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.

The first step in the synthesis of steroid hormones occurs in the mitochondria where cholesterol is converted to pregnenolone by cytochrome P450scc (CYP11A1). Cholesterol is insoluble in water and is supplied to the CYP11A1 directly from the inner mitochondrial membrane to which the enzyme is bound. The aim of this study was to characterise the interaction of bovine CYP11A1 with the phospholipid membrane. The effect of osmotic stress provided by glycerol on the spin-state, activity and degree of hydration of CYP11A1 was also investigated. Multiple sequence alignment of mitochondrial P450s revealed that there are 46 absolutely conserved residues, with the highest conservation in the heme-binding region at the C-terminal. The greatest variablility between subfamilies is in the regions believed to be involved in substrate binding (SRSs), as defined for the CYP2B family. The secondary structure prediction for CYP11A1 suggests that there is strong similarity in secondary structure to P450s of known structure. A model structure of CYP11A1 was built from primary sequence alignment to template P450 structures using the SwissModel automated server. From the model and other bioinformatic analyses, the distal face of the P450 which includes the A’ helix, F-G loop and beta sheet 1 regions, were predicted to interact with the membrane. Tryptic digests of CYP11A1 were performed with the aim of identifying membrane bound peptides that may be protected from protease activity. HPLC tryptic maps were similar in profile between soluble and vesicle-bound P450 which suggests that there is not a large region of CYP11A1 protected from protease digestion when the enzyme is attached to a membrane. Mass spectrometric analysis of peptides resulting from tryptic digestion revealed a number of peptides in the soluble digest that were not present in the digest of vesicle-bound P450. These peptides were located at the N-terminal and the J to J’ helix and interestingly, there was an absence of C-terminal peptides for both digests. This C-terminal peptide could be detected in digests of vesicle-bound P450 but not in digests of soluble P450 by tricine SDS polyacrylamide gel electrophoresis, Western transfer and N-terminal sequence analysis. Based upon the bioinfomatic and tryptic digestion data, a set of N- and C-terminal deletion mutants of CYP11A1 were expressed in E. coli and fractionated based on their association with the soluble or membrane fraction of the cells. The N-terminal deletion of the A’ helix resulted in an increase in the proportion of CYP11A1 in the soluble fraction while the C-terminal deletion did not alter membrane localisation. There are eight tryptophan residues in mature CYP11A1. The accessibility of these tryptophans to a water-soluble fluorescence quencher was determined for soluble and vesicle-bound enzyme. When CYP11A1 was associated with the vesicle membrane an average of 2 tryptophan residues were protected from quenching compared to soluble CYP11A1. This suggests that these tryptophan residues become buried within the membrane following association of CYP11A1 with the vesicles and are no longer accessible to quencher. The only free cysteine (C265S) of bovine CYP11A1 was removed by site directed mutagenesis and new cysteine residues introduced at selected sites based upon earlier results and the modelled CYP11A1 structure. The cysteine mutants were expressed, purified and labelled with the environmentally sensitive fluorescent probe, N-(7-nitrobenz-2-oxal-3-diazol-4-yl)ethylenediamine (NBD). There was an increase in the hydrophobicity of the NBD environment following the association of CYP11A1 with vesicles for the labeled mutants V212C and L219C. This indicates that these residues which are in the F-G loop, become localized to a more hydrophobic environment following membrane binding. Labeled cysteine residues introduced into the A’, B’ and G helices and β4-2 did not show major changes in hydrophobicity following membrane integration of CYP11A1. Osmotic stress of CYP11A1 induced by glycerol resulted in a low-spin spectral response and inhibition of activity. The change to low spin correlated with the dissociation of five or six water molecules from CYP11A1 and the inhibition of activity with cholesterol as substrate correlated with the dissociation of two molecules of water. In conclusion, this study shows that CYP11A1 is held to the membrane, at least in part, by the F-G loop region, and that the removal of water from the active site of CYP11A1 by osmotic stress causes a low spin spectral response and inhibition of activity.

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

Honors Project--Smith College, Northampton, Mass., 2010. / Includes bibliographical references (p. 102-105).

Thesis (M.Sc.)--Memorial University of Newfoundland, 2003. / Bibliography: leaves 60-66.