A novel electrochemical probe has been designed, built, and used to characterize the distribution in solution potential within the metal capillary and Taylor cone of the electrospray (ES) device. Results show that the measured potential difference increases as the internal probe travels toward the ES capillary exit, with values rising sharply as the base of the Taylor cone is penetrated. Higher conductivity solutions exhibit potentials of higher magnitude at longer distances away from the counter electrode, but these same solutions show lower potentials near the ES capillary exit. Removal of easily oxidizable species from the solution causes the measured potential difference to have nonzero values at distances further within the capillary, and the values measured at all points are raised. The influence of the diameter of the spray tip employed for nano-electrospray mass spectrometry (nano-ES-MS) upon mass spectral charge state distributions was investigated. A detailed comparison of charge state distributions obtained for nanospray capillaries of varying diameters was undertaken while systematically varying experimental parameters such as sample flow rate, analyte concentration, solvent composition, and electrospray current. The general tendency to obtain higher charge states from narrow diameter capillaries was conserved throughout, but tips with smaller orifices were more sensitive to sample flow rate, while tips with larger orifices were more sensitive to analyte concentration and pH of the solution. Electrospray mass spectrometry (ES-MS) has been employed to study noncovalent associations between lipids and fusion peptides. Detailed binding specificities between selected phospholipids and model fusion peptides were investigated. Strong evidence has been compiled to demonstrate the importance of the initial hydrophobic interaction to the observation of lipid-peptide binding by ES-MS. Initial hydrophobic interactions in solution contributed heavily to the formation of these peptide-lipid complexes, particularly for [peptide+PC] complexes, whereas electrostatic interactions played a larger role for [peptide+PG] complexes. The influence of solution pH and degree of unsaturation of lipids upon the binding strength of [peptide+PC] complexes were also investigated. These experiments help to establish ES-MS as a viable new biotechnology tool capable of providing valuable information regarding the strength of hydrophobically driven, noncovalent interactions.

Disabled-2 (Dab2) is an adaptor protein that plays critical roles in various biological processes, including protein endocytosis, platelet activation and aggregation, tumor growth, and development. In platelets, Dab2 associates with membrane sulfatide at the platelet surface, modulating platelet inside-out and outside-in signaling pathways. A Dab2-derived peptide, named the sulfatide-binding peptide (SBP), is the minimal unit of Dab2 to exert its function as a negative regulator of platelet activation and aggregation. The work of this thesis refines the model of Dab2 SBP binding to sulfatide and provides structural and functional insights into the mechanism by which Dab2 SBP modulates platelet activation. Using molecular docking, lipid-protein overlay assay, nuclear magnetic resonance, and surface plasma resonance tools, this work identifies the critical residues within two major regions responsible for sulfatide interaction. First, docking a sulfatide to Dab2 SBP, a hydrophilic region, primarily mediated by Arg42, is thought to be responsible for the association with the sulfatide headgroup. We observed that Arg 42 could directly interact with sulfatide by forming hydrogen bonds with the OS atoms in the sulfatide head group. Further lipid-protein overlay assay and surface plasma resonance experiments confirmed that both the positive charge and stereochemistry of the side chain of Dab2 SBP Arg42 are required for the sulfatide binding. Moreover, Arg42 is found to be critical in the inhibition of P-selectin expression on activated platelets. The residues nearby Arg42 (i.e., Glu33, Ty38, and Lys 44) also contribute to sulfatide interaction. Second, the second polybasic motif located at the C-terminal -helix 2 is considered to interact with the acyl chain through hydrophobic interactions rather than direct binding to the charged sulfatide head group. Lysine residues in this region are suggested to exert a dual role in sulfatide association, that is, by favoring electrostatic interactions with the negatively-charged sulfatide and/or by employing their flexible hydrocarbon spacers for hydrophobic interactions with membrane lipids. Consistent with this suggestion, we found a hydrophobic patch in the wild type Dab2 SBP structure surrounded by Lys49, Lys51, and Lys53. Furthermore, the role of the second sulfatide binding motif in sulfatide binding is confirmed by mutagenesis analysis and lipid-protein overlay assays, highlighting the ability of molecular docking to accurately predict critical residues responsible for sulfatide binding. In summary, this work provides a detailed structural basis for Dab2 recognition by sulfatide through multiple biophysical methods. The corresponding biological implications in the inhibition of platelet activation are also evaluated by flow cytometry. By elucidating the underlying mechanisms of Dab2 mediating platelet activation through sulfatide binding, we provided structural and functional insights for designing a Dab2-derived peptide with altered sulfatide recognition features in platelets, which can be further employed in antiplatelet therapy. / Doctor of Philosophy / Platelets are blood cells that are fundamentally intended to help form clots to stop bleeding. They do so by being activated after getting signals from damaged blood vessels and reaching the injury site. Consequently, they form aggregates by attracting more platelets to clump on the clot. However, platelet activation induced by a tumor cell can, in turn, protect the tumor cell from immune system elimination and facilitates their growth and spread. This platelet-tumor complex formation suggests platelets as a therapeutic target for reducing tumor migration out of the bloodstream. Our study investigates the mechanism of a Disabled-2-derived peptide, named Dab2 SBP, which upon binding to a sulfatide lipid, can reduce the platelet activation extent, using molecular and cellular approaches. The results of this study may be instrumental in the generation of Dab2 SBP-derived peptides with altered sulfatide binding ability and selectivity, which may lead to a design of an antiplatelet drug that can limit the ability of tumor cells to invade other tissues.

This thesis presents applications of high resolution secondary ion mass spectrometry (NanoSIMS) analysis for stable isotope imaging in biological samples. These projects were designed to explore the potential applications of NanoSIMS analysis, and to develop protocols and novel methodologies to visualize and quantify biological processes. Working with collaborators in the UK and USA, I have applied NanoSIMS analysis to study 3 research areas, including molecule interactions, single cell metabolisms and lipid imaging in tissues. Antimicrobial peptides (AMPs) play important role in the immune system, and understanding how AMPs interact with cell membranes can provide useful information to design new therapies to control infection. The pore structures and dynamics of the interaction of AMPs with membranes has been visualized for the first time and confirmed with combined AFM and NanoSIMS analysis. A correlative backscattered electron (BSE) imaging and NanoSIMS analysis methodology has been developed to study glutamine metabolism in single cancer cells. This method enables us to measure the chemical information in specific organelles in these cells and can be widely applied to study metabolisms and to trace the uptake of labelled molecules in biological matrices. Quantitative analysis on the effects of hypoxic conditions and the PYGL gene were studied. Applying correlative BSE and NanoSIMS analysis, I also studied lipid uptake mechanisms in various mouse tissues, including brown adipose tissue, heart, intestines, liver and skeletal muscle, mainly focused on a recently discovered protein, GPIHBP1, and its function in the lipid uptake process. TRL margination was proved to depend on the GPIBP1-LPL complex, and 3 stages of lipid transport from capillary lumen to lipid droplets was also visualized by combined BSE and NanoSIMS analysis.

The complexity of multi cellular organisms demands systems that facilitate communicationbetween cells. The neurons in our brains for instance are specialized in this cell-cellcommunication. The flow of ions, through their different ion channels, across the membrane, isresponsible for almost all of the communication between neurons in the brain by changing theneurons membrane potentials. Voltage-gated ion channels open when a certain thresholdpotential is reached. This change in membrane potential is detected by voltage-sensors in the ionchannels. In this licentiate thesis the Homo sapiens voltage- and calcium-gated BK potassiumchannel (HsapBK) has been studied. The NMR solution structure of the voltage-sensor ofHsapBK was solved to shed light upon the voltage-gating in these channels. Structures of othervoltage-gated potassium channels (Kv) have been determined by other groups, enablingcomparison among different types of Kv channels. Interestingly, the peptide-lipid interactions ofthe voltage-sensor in HsapBK are crucial for its mechanism of action.Uni cellular organisms need to sense their environment too, to be able to move towardsmore favorable areas and from less favorable ones, and to adapt their gene profiles to currentcircumstances. This is accomplished by the two-component system, comprising a sensor proteinand a response regulator. The sensor protein transfers signals across the membrane to thecytoplasm. Many sensor proteins contain a HAMP domain close to the membrane that isinvolved in transmitting the signal. The mechanism of this transfer is not yet revealed. Ourstudies show that HAMP domains can be divided into two groups based on the membraneinteraction of their AS1 segments. Further, these two groups are suggested to work by differentmechanisms; one membrane-dependent and one membrane-independent mechanism.Both the voltage-gating mechanism and the signal transduction carried out by HAMPdomains in the membrane-dependent group, demand peptide-lipid interactions that can be readilymodulated. This modulation enables movement of peptides within membranes or within thelipid-water interface. These conditions make these peptides especially suitable for NMR studies.

The pathogenesis of Alzheimer’s disease (AD), the most common senile dementia, is a complex process. A crucial event in AD is the aggregation of amyloid-β protein (Aβ), a cleavage product from the Amyloid Precursor Protein (APP). Aβ40, a common component in amyloid plaques found in patients, aggregates in vitro at concentrations, much higher than the one found in vivo. But in the presence of charged lipid membranes, aggregations occurs at much lower concentration in vitro compared to the membrane-free case. This can be understood due to the ability of Aβ to get electrostatically attracted to target membranes with a pronounced surface potential. This electrostatically driven process accumulates peptide at the membrane surface at concentrations high enough for aggregation while the bulk concentration still remains below threshold. Here, we elucidated the molecular nature of this Aβ-membrane process and its consequences for Aβ misfolding by Circular Dichroism Spectroscopy, Differential Scanning Calorimetry and Nuclear Magnetic Resonance Spectroscopy. First, we revealed by NMR that Aβ40 peptide does indeed interact electrostatically with membranes of negative and positive surface potential. Surprisingly, it even binds to nominal neutral membranes if these contain lipids of opposite charge. Combined NMR and CD studies also revealed that the peptide might be shielded from aggregation when incorporated into the membrane. Moreover, CD studies of Aβ40 added to charged membranes showed that both positively and negatively membranes induce aggregation albeit at different kinetics and finally that macromolecular crowding can both speed up and slow down aggregation of Aβ.

No description available.

Peptídeos antimicrobianos são vistos como alternativas promissoras a serem empregadas pela iindústria farmacêutica no controle de infecções causadas por microrganismos, como também na indústria alimentícia, onde podem desempenhar papéis como conservantes naturais de alimentos. Plantaricina149 é um membro deste grupo, sendo composto por 22 resíduos de aminoácidos, com natureza catiônica e atividade inibitória sobre algumas bactérias patogênicas. Neste trabalho, foram sintetizados diferentes peptídeos análogos à Plantaricina149 para investigar suas ações sobre microrganismos (bactérias e fungos), a fim de correlacionar estes estudos com a ação lítica do peptídeo em modelos de membrana diversos (monocamadas e vesículas fosfolipídicas). A interação de Plantaricina149 com estes sistemas foi monitorada pelas espectroscopias de dicroísmo circular e fluorescência, ensaios de tensão superficial, calorimetria e ressonância plasmônica de superfície, e mostrou ser altamente específica para superfícies fosfolipídicas que apresentam densidade de cargas negativas, tais como a membrana celular de bactérias. A interação eletrostática inicial que se estabelece entre o peptídeo e os fosfolipídios é de extrema importância, sendo capaz de induzir uma estruturação helicoidal na região C-terminal do peptídeo, enquanto a região Nterminal contribui com as interações hidrofóbicas necessárias para a penetração do peptídeo nas camadas fosfolipídicas levando a ruptura das mesmas. De forma semelhante, a atividade antimicrobiana de Plantaricina149a (e alguns de seus análogos) também mostrou ser resultado das interações das duas regiões da molécula, e foi afetada com a retirada ou modificação da região N-terminal do peptídeo. Com a deleção desta região, o peptídeo passou a ter somente ação bacteriostática sobre Staphylococcus aureus e Pseudomonas aeruginosa, perdendo a capacidade bactericida. / Antimicrobial peptides are seen as promising alternatives to be employed in pharmaceutical industry for controlling infections caused by microorganisms, and also in food industry, where they can play roles as natural food preservatives. Plantaricina149 is a member of this group, constituted of 22 amino acid residues, cationic in nature and presenting inhibitory activity against some pathogenic bacteria. In this work, different Plantaricina149 analog peptides were synthesized to investigate their action against microorganisms (bacteria and fungi), with the aim of correlating these studies with the lytic action of the peptide on several membrane models (phospholipid monolayers and vesicles). The Plantaricina149 interaction with these systems was monitored by circular dichroism and fluorescence spectroscopies, surface tension assays, calorimetry and surface plasmon resonance, and showed to be highly specific to phospholipid surfaces that present negative charge density, such as the bacteria cell membrane. The initial peptide-phospholipids electrostatic interaction is extremely important, and it is capable of inducing a helical structure in the peptide C-terminal region, while the Nterminal region contributes with the hydrophobic interactions needed to the peptide penetration in the phospholipid layers and to the disruption of them. Similarly, the Plantaricina149 antimicrobial activity has also proved to be a result of the interactions from the two regions of the molecule, and it was strongly affected by the removal or modification of the peptide N-terminal region. Promoting the deletion of this region has left the peptide only with a bacteriostatic action against Staphylococcus aureus and Pseudomonas aeruginosa, removing its bactericide ability.

Les peptides vecteurs riches en Arginine (Arg) ont la faculté de transporter des molécules à travers les membranes cellulaires, d'une manière récepteur- et énergie indépendante, sans toxicité envers la cellule et présentent ainsi un fort potentiel pour la libération de molécules thérapeutiques ou diagnostiques. La compréhension du mécanisme d'internalisation cellulaire et de l'interaction membranaire de ces peptides vecteurs est donc primordiale pour leur développement pharmaceutique. Dans cette étude, deux peptide svecteurs riches en Arg et dérivés de la pénétratine ont été étudiés : les peptides RW16(RRWRRWWRRWWRRWRR) et RW9 (RRWWRRWRR). Dans un premier temps,l'analyse biophysique complète de l'interaction peptide/lipide (P/L) a été réalisée pour le peptide RW16 et une interaction favorisée en présence de lipides anioniques a été révélée.Dans un second temps, des peptides dérivés de RW9 ont été synthétisés dans lesquels chaque tryptophane a été systématiquement remplacé par une phenylalanine. L'internalisation cellulaire et les interactions P/L de RW9 ont été étudiées, et l'importance de la position et du nombre de tryptophane dans la séquence peptidique a été mise en évidence. / Cell-penetrating peptides (CPPs) are able to efficiently transport cargos acrosscell membranes in a receptor- and energy-independent manner, without being cytotoxic to cells and thus present a great potential in drug delivery and diagnosis. The understanding of the cellular internalization and membrane interaction mechanisms is thus fundamental for their pharmaceutical development. In this study, two Arginine-rich CPPs derived from penetratin have been investigated: the peptides RW16 (RRWRRWWRRWWRRWRR) andRW9 (RRWWRRWRR). Firstly, a complete biophysical study of the peptide/lipid (P/L)interactions of RW16 has been accomplished and a preferential interaction for anionic lipids was demonstrated. Secondly, peptides derived from RW9 have been synthesized where tryptophan residues have been systematically replaced by phenylalanine. Cellular internalization and P/L interactions have been characterized, and the importance of the number and position of tryptophan has been highlighted.

The pulsed field gradient fourier transform nuclear magnetic resonance (PFG-FTNMR) method to measure translational diffusion coefficients in multicomponent systems has been applied to amphiphilic molecules forming liquid crystalline phases. By analyzing the concentration dependence of the diffusion coefficients of water and amphiphile in a micellar system of N,N-dimethyldodecy lamine oxide (DDAO) in water it was possible to conclude that the micelles formed were polydisperse in size and shape. It was also shown that solubilization of small amounts of hydrophobic molecules into the micelles induces spherical micelles of a narrow size distribution. From the magnitude of the lateral diffusion coefficient in the cubic phase of DDAO/water it was concluded that this phase is built up of bicontinous aggregates. The lipid lateral diffusion in the cubic phase of monooleoylglycerol (MO)/water has been measured. The decrease in the lateral diffusion of MO in this phase, when the water was replaced by glycerol, was ascribed to changes in viscosity in the polar region. Measurements by electron spin resonance and time-resolved fluorescence spectroscopy showed that changes in viscosity of the solvent also affected the motions in the hydrocarbon region. The diffusion coefficients of all three components in the cubic phase located in the lowwater region of the ternary system of diacylglycerol (DAG)/soybean phosphatidylcholine (SPC)/water have been determined. Conclusive evidence was provided for that this cubic phase is built up of reversed micelles containing mainly SPC in a continous matrix of mainly DAG. The effect on the phase properties of DDAO upon incorporation of the peptide gramicidin D has been investigated. It was shown that gramicidin D induces a lamellar phase at all water contents. The change in the order parameter profile of the C-2H bonds in perdeuterated DDAO upon incorporation of gramicidin D is compatible with theoretical calculations for proteins exhibiting a positive hydrophobic mismatch. A method for using the PFG FTNMR technique in measurements of the transmembrane exchange rate of small molecules in vesicular suspensions is discussed and some preliminary data is shown. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1994, härtill 4 uppsatser</p> / digitalisering@umu

Peptídeos antimicrobianos são vistos como alternativas promissoras a serem empregadas pela iindústria farmacêutica no controle de infecções causadas por microrganismos, como também na indústria alimentícia, onde podem desempenhar papéis como conservantes naturais de alimentos. Plantaricina149 é um membro deste grupo, sendo composto por 22 resíduos de aminoácidos, com natureza catiônica e atividade inibitória sobre algumas bactérias patogênicas. Neste trabalho, foram sintetizados diferentes peptídeos análogos à Plantaricina149 para investigar suas ações sobre microrganismos (bactérias e fungos), a fim de correlacionar estes estudos com a ação lítica do peptídeo em modelos de membrana diversos (monocamadas e vesículas fosfolipídicas). A interação de Plantaricina149 com estes sistemas foi monitorada pelas espectroscopias de dicroísmo circular e fluorescência, ensaios de tensão superficial, calorimetria e ressonância plasmônica de superfície, e mostrou ser altamente específica para superfícies fosfolipídicas que apresentam densidade de cargas negativas, tais como a membrana celular de bactérias. A interação eletrostática inicial que se estabelece entre o peptídeo e os fosfolipídios é de extrema importância, sendo capaz de induzir uma estruturação helicoidal na região C-terminal do peptídeo, enquanto a região Nterminal contribui com as interações hidrofóbicas necessárias para a penetração do peptídeo nas camadas fosfolipídicas levando a ruptura das mesmas. De forma semelhante, a atividade antimicrobiana de Plantaricina149a (e alguns de seus análogos) também mostrou ser resultado das interações das duas regiões da molécula, e foi afetada com a retirada ou modificação da região N-terminal do peptídeo. Com a deleção desta região, o peptídeo passou a ter somente ação bacteriostática sobre Staphylococcus aureus e Pseudomonas aeruginosa, perdendo a capacidade bactericida. / Antimicrobial peptides are seen as promising alternatives to be employed in pharmaceutical industry for controlling infections caused by microorganisms, and also in food industry, where they can play roles as natural food preservatives. Plantaricina149 is a member of this group, constituted of 22 amino acid residues, cationic in nature and presenting inhibitory activity against some pathogenic bacteria. In this work, different Plantaricina149 analog peptides were synthesized to investigate their action against microorganisms (bacteria and fungi), with the aim of correlating these studies with the lytic action of the peptide on several membrane models (phospholipid monolayers and vesicles). The Plantaricina149 interaction with these systems was monitored by circular dichroism and fluorescence spectroscopies, surface tension assays, calorimetry and surface plasmon resonance, and showed to be highly specific to phospholipid surfaces that present negative charge density, such as the bacteria cell membrane. The initial peptide-phospholipids electrostatic interaction is extremely important, and it is capable of inducing a helical structure in the peptide C-terminal region, while the Nterminal region contributes with the hydrophobic interactions needed to the peptide penetration in the phospholipid layers and to the disruption of them. Similarly, the Plantaricina149 antimicrobial activity has also proved to be a result of the interactions from the two regions of the molecule, and it was strongly affected by the removal or modification of the peptide N-terminal region. Promoting the deletion of this region has left the peptide only with a bacteriostatic action against Staphylococcus aureus and Pseudomonas aeruginosa, removing its bactericide ability.