Thesis advisor: Jianmin Gao / My graduate research focuses on engineering and characterizing gramicidin A (gA), a natural fifteen-residue transmembrane channel peptide. It consists of D- and L- amino acids at alternate positions. gA is believed to fold into a β-helix in membranes, and two folded monomers at each leaflet of the lipid bilayer dimerize to form a transmembrane channel. gA shares the common features of other known membrane channels: a well defined structure that only allows the passage of specific ions, a gating mechanism, and a high abundance of aromatic residues. This dissertation includes two subprojects: I. Understanding Channel Formation: Aromatic Modifications of Gramicidin A Channel Ion channels are key elements in signaling and molecule transport, and therefore crucial for normal function of cells. Defective ion channels are known to be responsible for a number of diseases. Although hundreds of crystallographic structures of membrane proteins have been deposited into the PDB in the past few decades, our knowledge on this large family of proteins is still limited and mostly descriptive. Study of small peptides in model membranes is a good simplification of the more complex biological systems. In chapter 1, I will introduce my research using gA as a model system to understand the significant role of aromatic residues in membrane channel structure formation. Channel activities of these gA-Ar mutants were evaluated by ion leakage assays. The structure activity relationship of a library of gA mutants was discussed. The alternating chirality of amino acids was proven to be essential for gA channel activity. Several additional interesting observations are discussed. II. Towards Bacterium Specific Ion Channels: Solublized Gramicidin A as Potential Systemic Antibiotics The rapid development of multidrug resistance by pathogenic bacteria poses a serious threat to society and demands new antibiotics with different mechanisms. Often considered as a model transmembrane channel, gA also has proven antibiotic activities. The gA channel facilitates passive diffusion of water and monovalent cations (e.g. H+, Na+, K+) thus killing bacteria by disrupting the ion gradient across the cell membrane. However because of its poor solubility and high toxicity, its medicinal application as an antibiotic has been limited to topical reagents. A detailed understanding of gA allows rational optimization of the gA-WT to potential systemic antibiotics. Bacterial membranes are composed of a large fraction of anionic species, therefore, we hypothesize that strategic incorporation of cationic residues into gA will afford bacterium-specific toxicities. In addition, the charged residues will greatly improve the water solubility of gA. In chapter 2, I will introduce my research on developing soluble and bacterium specific gA as a potential systemic antibiotic. We firstly incorporated D-Lys at the C-terminus to obtain our first generation of gA based antibiotics. The best candidate (D-Leu10,12,14D-Lys gA) shows significantly increased water solubility (~ 1, 000 times) and therapeutic index (˃ 50 times). Modifications on the Lys side chain were then carried out to fine tune the antibiotic activities of these cationic gA. My research has pointed out a possible strategy to convert hydrophobic membrane channel peptides into potential systemic antibiotics. In addition to targeting the negative charges of bacterial membranes with cationic gA mutants, we proposed a novel strategy in which boronic acid is used to chase after the 1,2-diol substructure in the PG headgroup through boronate ester formation. Polyvalent display of boronic acids on a peptide scaffold results in enhanced binding with diols, showing promise of the boronate approach in the development of bacterium specific reagents. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Marburg, Universiẗat, Diss., 2005.

Lettuce and tomato field trials were performed in an unheated polytunnel in Aberdeenshire to investigate the efficacy and mode of activity of <i>Brevibacillus brevis</i> against grey mould disease (<i>Botrytis cinerea</i>).  This was achieved by means of treating plants with whole cultures of <i>B. brevis</i> WT (containing gramicidin S and biosurfactant) and <i>B. brevis</i> E1 (containing biosurfactant only) and also with supernatant and spore fractions of such cultures so that the effects of treatments containing one, both or neither of the potentially active components, gramicidin S (bound to the bacterial spore) and biosurfactant (released into the culture medium) could be assessed.  In winter lettuce, WT and E1 reduced grey mould by up to 79% (p=0.05) with no significant disease (p=0.05) between efficacy of these treatments.  WT reduced disease by 59% in spring lettuce but at low significance (p=0.2) although marketable yields were significantly improved (p=0.05).  The biocontrol treatments WT, E1, SWT and SE1 reduced (p=0.05) grey mould by up to 48% in tomato leaves and 73% of WT treated plants had no stem lesions, significantly more (p=0.05) than lesion-free control plants (17%).  These results suggest that both the biosurfactant and gramicidin S play a role in disease reduction.  The biosurfactant appears to be responsible for disease reduction on aerial plant surfaces with large surface areas whilst Gramicidin S reduces disease in regions of higher humidity, such as the base of lettuce plants and on tomato stems.

Zugl.: Marburg, Univ., Diss., 2005

Zugl.: Marburg, Univ., Diss., 2005

Computational research making use of molecular dynamics (MD) simulations has begun to expand the paradigm of structural biology to include dynamics as the mediator between structure and function. This work aims to expand the utility of MD simulations by developing Principal Component Analysis (PCA) techniques to extract the biologically relevant information in these increasingly complex data sets. Gramicidin is a simple protein with a very clear functional role and a long history of experimental, theoretical and computational study, making it an ideal candidate for detailed quantitative study and the development of new analysis techniques. First we quantify the convergence of our PCA results to underwrite the scope and validity of three 64 ns simulations of gA and two covalently linked analogs (SS and RR) solvated in a glycerol mono-oleate (GMO) membrane. Next we introduce a number of statistical measures for identifying regions of anharmonicity on the free energy landscape and highlight the utility of PCA in identifying functional modes of motion at both long and short wavelengths. We then introduce a simple ansatz for extracting physically meaningful modes of collective dynamics from the results of PCA, through a weighted superposition of eigenvectors. Applied to the gA, SS and RR backbone, this analysis results in a small number of collective modes which relate structural differences among the three analogs to dynamic properties with functional interpretations. Finally, we apply elements of our analysis to the GMO membrane, yielding two simple modes of motion from a large number of noisy and complex eigenvectors. Our results demonstrate that PCA can be used to isolate covariant motions on a number of different length and time scales, and highlight the need for an adequate structural and dynamical account of many more PCs than have been conventionally examined in the analysis of protein motion.

Computational research making use of molecular dynamics (MD) simulations has begun to expand the paradigm of structural biology to include dynamics as the mediator between structure and function. This work aims to expand the utility of MD simulations by developing Principal Component Analysis (PCA) techniques to extract the biologically relevant information in these increasingly complex data sets. Gramicidin is a simple protein with a very clear functional role and a long history of experimental, theoretical and computational study, making it an ideal candidate for detailed quantitative study and the development of new analysis techniques. First we quantify the convergence of our PCA results to underwrite the scope and validity of three 64 ns simulations of gA and two covalently linked analogs (SS and RR) solvated in a glycerol mono-oleate (GMO) membrane. Next we introduce a number of statistical measures for identifying regions of anharmonicity on the free energy landscape and highlight the utility of PCA in identifying functional modes of motion at both long and short wavelengths. We then introduce a simple ansatz for extracting physically meaningful modes of collective dynamics from the results of PCA, through a weighted superposition of eigenvectors. Applied to the gA, SS and RR backbone, this analysis results in a small number of collective modes which relate structural differences among the three analogs to dynamic properties with functional interpretations. Finally, we apply elements of our analysis to the GMO membrane, yielding two simple modes of motion from a large number of noisy and complex eigenvectors. Our results demonstrate that PCA can be used to isolate covariant motions on a number of different length and time scales, and highlight the need for an adequate structural and dynamical account of many more PCs than have been conventionally examined in the analysis of protein motion.

Atomic force microscopy (AFM) is an important technology that allows researchers to probe local surface properties at nanometer length scales. In addition to surface topography, the AFM can probe many types of tip-surface interactions (including adhesion and friction) to gain a better understanding of the chemical properties of surfaces. This thesis contains two experiments which utilize AFM to in addition to several other techniques to study (1) Self Assembled Monolayer (SAM) formation and corrosion and (2) intermolecular and surface/molecular effects on gramicidin film formation and molecular orientation. In the first experiment, N-octadecyltrichlorosilane (OTS) molecules were self-assembled onto silicon samples. We observed that OTS required a very short time (about 15 seconds) to complete the formation of the monolayer on surface. However, this SAM film was highly susceptible to corrosion by the strong oxidant (KMnO4), resulting in a chemical change to the film from hydrophobic functional groups (CH3) to hydrophilic functional groups (OH). In subsequent experiments, we observed that if the SAMs were formed using longer exposure times (about 24 hours), they were highly resistant to corrosion. Fourier Transform Infrared Spectroscopy (FTIR) and X-Ray Photoelectron Spectroscopy (XPS) also showed that the 24 hour growth SAM films were densely packed. These results indicate that SAM films based on organosilane molecules can protect the surface from corrosion, and further that more densely packed SAMs exhibit better anti-corrosion performance than less dense films. In the second experiment, the antibacterial peptide Gramicidin was used to study how intermolecular and surface energy properties can influence the aggregation and film formation of molecules on several surfaces. Gramicidin has a unique physical and chemical structure with hydrophobic side chain and hydrophilic ends. Here, we have used three different substrates (Silicon, Mica, and Graphite) to study intermolecular interactions, aggregation, and orientation of Gramicidin peptide. Langmuir-Blodgett methods were also used to study aggregation and molecular orientation at the solid-liquid interface.

Durch Ausübung physiologischer Grundfunktionen spielen Ionenkanäle eine entscheidende Rolle für die reguläre Funktion von Zellen. Zum besseren Verständnis ihrer Struktur und Funktion sind Untersuchungen natürlicher und künstlicher Ionenkanäle wichtige Werkzeuge. Großes analytisches und therapeutisches Potential ist in der Untersuchung künstlicher Kanäle in lebenden Zellen vorhanden, was bisher wenig Beachtung fand. In der vorliegenden Arbeit wurde die Wirkung der künstlichen Ionenkanäle THF-gram, THF-gram-TBDPS sowie linked-gram-TBDPS auf elektrophysiologische Eigenschaften boviner Trabekelwerkszellen des Auges anhand von Patch-Clamp-Untersuchungen im Whole-Cell-Modus analysiert. Die Untersuchung brachte folgende Erkenntnisse: 1. Die Inkorporation aller drei Verbindungen war erfolgreich, was sich durch Anstieg der Stromdichte und Verschiebung des Umkehrpotentials zeigte. 2. Einbau von THF-gram und THF-gram-TBDPS war mit dem Überleben der Zellen vereinbar, während linked-gram-TBDPS aufgrund einer sehr potenten Antwort bereits bei sehr geringen Konzentrationen zum raschen Zelltod führte. 3. Eine Asymmetrie der Stromantwort zugunsten stärkerer Auswärtsströme wurde bei THF-gram und in schwächerer Ausprägung bei THF-gram-TBDPS festgestellt. Linked-gram-TBDPS zeigte keine derartige Asymmetrie. 4. Unter Verwendung von Cs+ als Ladungsträger war der beobachtete Anstieg der Stromdichte bei allen drei Verbindungen eindeutig stärker als unter physiologischen Bedingungen (Na+/K+). 5. Die dargestellten Erkenntnisse sind ein erster Schritt zur therapeutischen Anwendung von künstlichen Ionenkanälen. Eine Weiterentwicklung in Richtung höherer Selektivität und besserer Kontrolle ist jedoch genauso erforderlich wie die Klärung der klinischen Umsetzbarkeit. / Ion channels play a pivotal role for regular cell function. To better understand their structure and function, investigation of both natural and artificial ion channels is being performed to date. Investigation of artificial channels in living cells hides a big potential, however, little attention has been paid to this field so far. In this work, the effect of the artificial ion channels THF-gram, THF-gram-TBDPS and linked-gram-TBDPS on electrophysiological properties of bovine trabecular meshwork cells was investigated with the patch-clamp-technique. Following results were obtained: 1. Incorporation of all three compounds was successful, which was proven by increase of current density and cell depolarisation. 2. The cells survived after incorporation of THF-gram and THF-gram-TBDPS but not after linked-gram-TBDPS, which resulted in cell death at very low concentrations. 3. Larger outward currents were observed with THF-gram and, at a lower extent, with THF-gram-TBDPS. Linked-gram-TBDPS did not show such an asymmetry. 4. With Cs+ as charge carrier all three compunds showed a stronger increase of current density than under physiological conditions (Na+/K+). 5. The decribed results are a first step towards therapeutic application of artificial ion channels, however, further development towards higher selectivity and better control is as necessary as clarification of clinical feasibility.

No description available.