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The studies presented in this thesis focus on the synthesis of air-stable solid supported lipid bilayers by anhydrobiotic mechanisms. Supported lipid bilayers (SLBs) serve as platforms that mimic cellular membrane surfaces in appearance and behavior. One of the most attractive aspects of the SLB is that it exhibits two-dimensional fluidity that allows for individual components to rearrange as they would in actual cellular membranes. The one thing that would allow the SLB to become an ideal biosensor is the ability to remain stable in the absence of bulk water. As it stands now, unprotected SLBs are unstable in the presence of air causing the membrane to rearrange and delaminate from the surface. Several biological organisms utilize the process of anhydrobiosis to persevere in severe dehydrated states. Anhydrobiosis occurs when organisms employ large amounts of sugars, particularly disaccharides, to protect their cell membranes. The sugars, often released as a stress response, protect the membrane by replacing the water around the lipid headgroups while also interacting with other sugars to form a glass atop the bilayer. One of the most successful anhydrobiotic sugars has been trehalose, although other sugars have been evaluated and are capable of protecting lipid bilayers minimally. The experimental section of this thesis involves the creation of SLBs that are examined with and without the presence of sugar molecules. Essentially, the SLB was created, exposed to sugar solutions, dried, and subsequently rehydrated. Successful experiments occurred when rehydrated bilayers exhibited little damage and were mobile and functional. In addition to trehalose, several other mono- and disaccharides were used as were glycolipids, lipids with sugar headgroups. Upon the completion of all experiments it was clear that trehalose afforded the most protection of all species tested and that glycolipids do not sufficiently protect the membrane during rehydration. Therefore, the addition of a sugar such as trehalose to an SLB could allow for the creation of an air-stable biosensor that would be both practical and require little maintenance.

This dissertation focuses on the applications of solid supported phospholipid membranes as mimics of the cellular membrane using lab-on-a-chip devices in order to study biochemical events such as ligand-receptor binding and the chemical mechanism for the preservation of the biomembrane. Supported lipid bilayers (SLBs) mimic the native membrane by presenting the important property of two-dimensional lateral fluidity of the individual lipid molecules within the membrane. This is the same property that allows for the reorganization of native membrane components and facilitates multivalent ligand-receptor interactions akin to immune response, cell signaling, pathogen attack and other biochemical processes. The study is divided into two main facets. The first deals with developing a novel lipopolymer supported membrane biochip consisting of Poly(ethylene glycol) (PEG)-lipopolymer incorporated membranes. The formation and characterization of the lipopolymer membranes was investigated in terms of the polymer size, concentration and molecular conformation. The lateral diffusion of the PEG-bilayers was similar to the control bilayers. The air-stability conferred to SLBs was determined to be more effective when the PEG polymer was at, or above, the onset of the mushroom-to-brush transition. The system is able to function even after dehydration for 24 hours. Ligandreceptor binding was analyzed as a function of PEG density. The PEG-lipopolymer acts as a size exclusion barrier for protein analytes in which the binding of streptavidin was unaffected whereas the binding of the much larger IgG and IgM were either partially or completely inhibited in the presence of PEG. The second area of this study presents a molecular mechanism for in vivo biopreservation by employing solid supported membranes as a model system. The molecular mechanism of how a variety of organisms are preserved during stresses such as anhydrobiosis or cryogenic conditions was investigated. We investigated the interaction of two disaccharides, trehalose and maltose with the SLBs. Trehalose was found to be the most effective in preserving the membrane, whereas maltose exhibited limited protection. Trehalose lowers the lipid phase transition temperature and spectroscopic evidence shows the intercalation of trehalose within the membrane provides the chemical and morphological stability under a stress environment.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009. / Härtill 3 uppsatser.

The cell membrane is a highly complex structure consisting of a large diversity of phospholipids and macromolecules. There exist a variety of diseases that compromise the integrity of this key component of the cell. This thesis considers the investigation of interactions between β-amyloid peptide (Aβ₄₂) and lipid bilayers. To facilitate understanding of this complex system, it is advantageous to employ a model sample; supported lipid bilayers (SLB) and giant multilamellar vesicles (MLVs) are used as proxy cell membranes. These nanostructures are widely used as models of cellular membranes in many areas of scientific research. Phospholipid molecules self-organise into bilayer structures containing phase-separated microdomains, which are believed to be important in many biological processes. This study aims to develop model systems and experimental tools to explore hypothetical mechanisms through which the β-amyloid interacts with the lipid membranes. A lack of mechanistic understanding is the major challenge to our efforts to elucidate not only the interactions of the Aβ42 with the lipid membranes, but also the behaviour of these systems towards the changes of the environmental conditions (pH, concentration, temperature). Our results suggest that there are various different methods, such as AFM, CARS microscopy and Raman spectroscopy as well as neutron scattering that are capable of fast imaging. Overall, all these techniques contributed in a complementary study of Aβ₄₂ aggregation states under extreme and physiological conditions as well as to image Aβ₄₂ interactions with lipid bilayers consisted of specific lipids.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Virginia, 2000. / Spine title: Phospholipase A₂ binding. Includes bibliographical references (p. 245-258). Also available online through Digital Dissertations.

In recent years, advances in computer architecture and lipid force field parameters have made Molecular Dynamics (MD) a powerful tool for gaining atomistic resolution of biological membranes on timescales that other tools simply cannot explore. With many key biological processes involving membranes occurring on the nanosecond timescale, MD allows us to probe the dynamics and energetics of these interactions in molecular detail. Specifically, we can observe the interactions taking place as a peptide or protein comes into contact with a lipid bilayer, and how this may shape or alter the bilayer either locally (changes in headgroup orientation, lipid fluidity) or in bulk (lipid demixing, membrane curvature). The resolution achieved through atomistic MD can be directly compared with other tools such as NMR and EPR to gain a full perspective of how these biological systems behave over different timescales. As my background is in computational physics, this thesis not only looks into broadening our understanding of various interactions with biological membranes, but also into the development of construction and analytical software to assist in my research and benefit others in the field. One aspect of biological membranes that could vastly benefit from MD simulations is that of antimicrobial peptides (AMPs). These peptides primarily target and destroy microbes by permeabilising the cell membrane through a variety of proposed mechanisms, where each mechanism relies on the AMP to adopt specific conformations upon contact with bacterial membranes. In this thesis, I present an investigation into the interactions between a synthetic AMP and an inhibitor peptide designed to regulate antimicrobial activity through the formation of a coiled coil structure, which restricts the AMP from adopting new conformations. Simulations captured the spontaneous formation of coiled coils between these peptides, and specific residues in their sequences were identified that promote unfolding. This knowledge may lead to better design of coiled coil forming peptides. Another aspect of biological membranes that can be explored with MD is the interactions between model bacterial membranes and amphipathic helices, such as the MinD membrane targeting sequence (MinD-MTS). This 11-residue helix is responsible for anchoring the MinD protein to the inner membrane of Bacillus subtilis and plays a crucial role in bacterial cell division. MinD is known to exhibit sensitivity to transmembrane potentials (TMVs), whereby its localisation and binding affinity to bacterial membranes are disrupted upon removal of the TMV. Simulations revealed rapid insertions of MinD-MTS peptides into the headgroup region of a model bacterial membrane. Analytical software was constructed to measure the membrane properties of the lipids surrounding inserted MinDMTS peptides, which revealed splayed lipid tails and suggests the MinD-MTS may be capable of inducing membrane curvature. Additional simulations were conducted to investigate the influence of a TMV on model bacterial membranes, where software was constructed to measure changes in membrane properties. An analysis of these simulations suggests that a TMV is capable of lowering the transition temperature of a model bacterial membrane by a few degrees, yielding increased fluidity in the lipids and increased perturbations on the membrane surface. Finally, another aspect of biological membranes that can be explored through MD is that of electroporation. This induction of transient water pores in cell membrane provides an exciting aspect for drug delivery applications into cells, whereby electric fields are applied to cells to increase the uptake of therapeutic drugs. Simulations of membranes with high voltage TMVs were conducted that sought to investigate the implications of electroporation across a variety of bilayer compositions at different temperatures. Software was constructed to measure changes in membrane and system properties, which revealed that pore formation occurred at the same threshold voltage for different bilayer compositions in the fluid phase (~1.9 V) and a higher voltage for DPPC bilayers in the gel phase (~2.4 V). The TMV was found to be highly dependent on the area per lipid (APL), implying that bilayers with bulkier lipids or those transitioning from gel to fluid will experience smaller TMVs and fewer pore formations. These simulations also revealed lipid flip-flopping through pores, where charged lipids tended to translocate in the direction of the electric field to produce an asymmetrically charged bilayer. Finally, simulations utilising charged peptides with membranes yielded electroporation effects, whereby the charged peptides generate an identical TMV to those produced by an ion imbalance of equal magnitude. This suggests that charged peptides, such as AMPs, may be capable of permeabilising cell membranes through electroporation mechanisms.

Lipid molecules are amphiphilic in nature consisting of a hydrophilic head group and hydrophobic hydrocarbon tails. The lipid bilayer consists of two layers of lipid molecules arranged with their hydrophobic tails facing each other and their hydrophilic head groups solvated by water. Lipid bilayers with hydrophilic polymer chains grafted onto the head groups have applications in various fields, such as stabilization of liposomes designed for targeted drug delivery, synthesis of supported bilayers for biomaterial applications, surface modification of implanted medical devices to prevent biological fouling and design of in vitro biosensors. The focus of this thesis lies in understanding the effects of polymer grafting on the thermodynamics and mechanical properties of lipid bilayers. Dissipative particle dynamics (DPD) has evolved as a promising method to study complex soft matter systems. The basic DPD algorithm, and its implementation are discussed in Chapter 2 of this thesis. It is important to achieve a tensionless state while studying phase transitions and deducing the mechanical properties of the bilayer. We proposed a modification of the Andersen barostat which can be incorporated in a DPD simulation to achieve the tensionless state as well as carry out simulations at a prescribed tension. In Chapter 3 of this thesis the effect of polymer grafting on single tailed lipid bilayers is studied. Simulations are carried out by varying the grafting fraction, Gf, defined as the ratio of the number of polymer molecules to the number of lipid molecules. At lowGf, the bilayer shows a sharp transition from the gel (Lβ) to the liquid crystalline (Lα) phase. This main melting transition temperature is lowered as Gf is increased. Corresponding to this, an increase in the area per head group is also observed. Above a critical value of Gf the interdigitated, LβI phase is observed prior to the main transition for the longer lipid tails. The analysis for two tailed lipids as a function of polymer chain length is extensively studied in Chapter 5. For the case of two tailed lipids, an intermediate interdigitated phase was not observed and the decrease in the melting temperature is more pronounced as the length of the polymer chain is increased. The scaling for fractional change in the area per head group, as well as the decrease in transition temperature as a function of polymer grafting are in good agreement with mean field theory predictions. The bending modulus (k) and area stretch modulus (kA) are essential for determining the shape and the mechanical stability of biological cells or lipid based vesicles. In simulations, the bending modulus k is evaluated from the Fourier transform of the out-of-plane fluctuations of the bilayer mid-plane. In Chapter 4 of this thesis, we illustrate that a surface representation based on Delanuay triangulation provides a robust parameter free representation of the bilayer surface. By evaluating the bending modulus for single tail lipids of different tail lengths, the continuum scaling relation d2 is verified. To our knowledge this is the first systematic investigation and verification of this scaling relationship using computer simulations. Using the continuum relation, =kAd2/ we find that α depends weakly on the tail lengths of the bilayer. Nevertheless we illustrate that a value of α=130 can be used to reliably estimate the bending modulus from the area stretch modulus for polymer free bilayers. Using our method, we are also able to capture the low q scalings and obtain the bending modulus of the gel (Lβ) phase. Grafted polymer was found to increase the value of the bending modulus for single tail lipids. Although the presence of polymer directly increases the area per head group, the suppressed height fluctuations dominate and the bending modulus increases for the single tail lipids. For two tail lipids a small decrease in the bending modulus was observed at low grafting fractions and short polymer chains. For large polymer lengths the bending modulus was found to increase monotonically.

A associação lipídio/citocromo c é importante e deve ser estudada, pois repercute na atividade peroxidática da proteína abordada e pode contribuir para o processo apoptótico, ou morte programada da célula, e também desempenha um papel significativo na cadeia respiratória. A natureza e a especificidade da interação do citocromo c com bicamadas lipídicas têm sido bastante investigadas ultimamente, mas informações detalhadas e precisas sobre tais assuntos ainda não existem. É aceito que ocorre primeiramente uma interação eletrostática entre a proteína citocromo c e as membranas fosfolipídicas. Em seguida, há uma interação hidrofóbica. Entretanto, ainda não é bem compreendido o papel da cadeia fosfolipídica. A associação do citocromo c com membranas lipídicas induz mudanças no estado de spin do átomo de ferro. A interação entre as vesículas carregadas e o citocromo c induz mudanças estruturais na proteína, as quais são refletidas no seu centro ativo, ou grupo heme. As mudanças do campo cristalino no sítio do ferro hemínico de forte para fraco são acompanhadas por mudanças do estado de spin de baixo para alto, respectivamente. Neste trabalho, estuda-se sistematicamente a natureza da interação entre o citocromo c e a cadeia fosfolipídica. As mudanças estruturais no grupo heme foram correlacionadas com a natureza do lipídio, ou seja, com a carga da cabeça e com o tamanho e o tipo da cadeia fosfolipídica. Foram utilizados treze lipídios diferentes, naturais e sintetizados, com cabeças polares negativas e neutras e com cadeias carbônicas saturadas e insaturadas de diferentes comprimentos. Para tal investigação, utilizamos as técnicas: Ressonância Paramagnética Eletrônica (RPE) Onda Contínua (CW) e Pulsada (PW) e Dicroísmo Circular Magnético (MCD). As técnicas enunciadas avaliam as mudanças de estado de spin e a simetria do citocromo c nos seus estados férrico e ferroso. A interação lipoprotéica lipídio/citocromo c foi avaliada com lipídios diferentes, inclusive com o lipossomo PCPECL, que mimetiza a membrana interna da mitocôndria nos eucariontes. A partir dos resultados experimentais, sugerimos um modelo para esse tipo de associação. / This association lipid/cytochrome c is interesting to study in order to understand the peroxidase activity of this protein, that plays an important role in the respiratory chain and in the apoptosis process or the programmed cell death. The nature and specificity of the interaction of cytochrome c with lipid bilayers have been major goals in recent studies, but detailed information on that issue is not yet widely available. In this regard, it is generally accepted that the electrostatic interaction is an important factor in the association of cytochrome c with phospholipid membranes, followed by a hydrophobic interaction. However, the role played by the phospholipid chain is not well understood. The association of cytochrome c with negative membranes induces a change in the heme iron spin state. The interaction between the charged vesicles and cytochrome c leads to structural changes in the active central or heme group. The changing of the crystalline field of the heme iron from strong to weak is accompanied by spin states changes from low to high spin, respectively. These facts concerned us to investigate more systematically the nature of the interaction between cytochrome c and the phospholipid chains. The lipid-induced effects in the heme iron crystalline field are correlated to the nature of the charged head group and to the size and type of the phospholipid chain. Thirteen different lipids, nature and synthetic, were used, with negative and neutra1 polar head group and saturated and unsaturated acyl chains with different length. This work investigates the change of heme iron spin state and symmetry of ferric cytochrome c using Continuous Wave (CW) and pulsed (PW) Electron Paramagnetic Resonance (EPR) and Magnetic Circular Dichroism (MCD) techniques. These techniques analyze the spin state change and the symmetry of the iron cytochrome c in its ferric and ferrous states. The effect of the different lipids were analyzed, including PCPECL membrane that mimetics the inner mitocondrial membrane in eukaryotes.