Bacterial infections are and likely always will be a serious and costly complication to treatment in a healthcare environment. However consistent rises in the number of both healthcare associated and antibiotic resistant infections over the last of decades has the potential to turn a serious problem into a catastrophe. Control of infections in hospital wards has improved over the last five years but data from the European Centre for Disease Prevention and Control suggests a stale mate. While the numerical rise in drug resistant organisms has slowed, the severity of drug resistance appears to be on the increase with the prolific emergence of multiple drug resistant isolates. On the front lines of the threat that these organisms represent are some of the most susceptible. In hospitals those who are already sick are more vulnerable, those with co-morbidities, those with surgical or other wounds, the very old and the very young. Children especially show high susceptibility as they are often incapable of communicating clinical complications in the way an adult might. This coupled with higher commonality of specific aetiologies in children such as scalds, open wounds that are prone to infection without proper treatment, creates population in need. Antibiotics are often thought to be part of the problem in drug resistance, indeed to an extent they are. However their real downfall may be improper use. In order to improve treatment outcomes and simultaneously decrease antimicrobial resistance a combination of rapid diagnosis and prophylaxis can be utilised to decrease selection of resistance. As such, this study focuses on the development of a novel vesicle based sensor system for the detection of bacterial infections in burn wounds. Additionally an organometallic antimicrobial system has been developed with the potential for surface attachment. Work with the vesicle based biosensor demonstrates high sensitivity to both Staphylococcus aureus and Pseudomonas aeruginosa. The toxins involved in activation of the sensor have been determined in both cases and an in-depth study into the activity of the staphylococcal agents of lysis (Phenol Soluble Modulins and delta haemolysin), shows a high degree of plasticity and tunability in the sensors function. Work with the zinc based antimicrobial reveals a highly complex system which demonstrates possible functions as a not only an antimicrobial but as a sensor system in its own right.

Due to the escalating challenge of antibiotic resistance in bacteria over the past several decades, interest in the identification and development of antibiotic alternatives has intensified. Antimicrobial peptides (AMPs), which serve as part of the innate immune systems of most eukaryotic organisms, are being researched extensively as potential alternatives. However, the mechanism behind their bactericidal capabilities is not well understood. Previous studies have suggested that AMPs may first attach to the cell membranes, leading to pore formation caused by peptide insertion, lipid removal in the form of peptide-lipid aggregates, or a combination of both mechanisms. In addition to the lack of mechanistic knowledge, a significant hurdle in AMP-based drug development is their potential cytotoxicity to mammalian cells. Understanding AMP interactions with eukaryotic model membranes would allow therapeutics to be tailored for preferential action toward specific classes of bacterial membranes. In this study, we developed novel methods of quartz crystal microbalance with dissipation monitoring (QCM-D) data analysis to determine the fundamental mechanism of action between eukaryotic and bacterial membrane mimics and select membrane-active AMPs. A new technique for creating supported membranes composed entirely of anionic lipids was developed to model Gram-positive bacterial membranes. Atomic force microscopy (AFM) imaging was also used to capture the progression of AMP-induced changes in supported lipid membranes over time and to validate our method of QCM-D analysis. QCM-D and AFM were used to investigate the molecular-scale interactions of four peptides, alamethicin, chrysophsin-3, sheep myeloid antimicrobial peptide (SMAP-29) and indolicidin, with a supported zwitterionic membrane, which served as a model for eukaryotic cell membranes. Since established methods of QCM-D analysis were not sufficient to provide information about these interaction mechanisms, we developed a novel method of using QCM-D overtones to probe molecular events occurring within supported lipid membranes. Also, most previous studies that have used AFM imaging to investigate AMP-membrane interactions have been inconclusive due to AFM limitations and poor image quality. We were able to capture high-resolution AFM images that clearly show the progression of AMP-induced defects in the membrane. Each AMP produced a unique QCM-D signature that clearly distinguished their mechanism of action and provided information on peptide addition to and lipid removal from the membrane. Alamethicin, an alpha-helical peptide, predominantly demonstrated a pore formation mechanism. Chrysophsin-3 and SMAP-29, which are also alpha-helical peptides of varied lengths, inserted into the membrane and adsorbed to the membrane surface. Indolicidin, a shorter peptide that forms a folded, boat-shaped structure, was shown to adsorb and partially insert into the membrane. An investigation of rates at which the peptide actions were initiated revealed that the highest initial interaction rate was demonstrated by SMAP-29, the most cationic peptide in this study. The mechanistic variations in peptide action were related to their fundamental structural properties including length, net charge, hydrophobicity, hydrophobic moment, accessible surface area and the probability of alpha-helical secondary structures. Due to the charges associated with anionic lipids, previous studies have not been successful in forming consistent anionic supported lipid membranes, which were required to mimic Gram-positive bacterial membranes. We developed a new protocol for forming anionic supported lipid membranes and supported vesicle films using a vesicle fusion process. Chrysophsin-3 was shown to favor insertion into the anionic lipid bilayer and did not adsorb to the surface as it did with zwitterionic membranes. When introduced to supported anionic vesicle films, chrysophsin-3 caused some vesicles to rupture, likely through lipid membrane disruption. This study demonstrated that molecular-level interactions between antimicrobial peptides and model cell membranes are largely determined by peptide structure, peptide concentration, and membrane lipid composition. Novel techniques for analyzing QCM-D overtone data were also developed, which could enable the extraction of more molecular orientation and interaction dynamics information from other QCM-D studies. A new method of forming supported anionic membranes was also designed, which may be used to further investigate the behavior of bacterial membranes in future studies. Insight into AMP-membrane interactions and development of AMP structure-activity relationships will facilitate the selection and design of more efficient AMPs for use in therapeutics that could impact the lives of millions of people per year who are threatened by antibiotic-resistant organisms.

Supported lipid bilayers (SLBs) are one of the most common model membranes used in the field of cell membrane biology as they provide a well-defined model membrane platform for determination of molecular-level interactions between different biomolecules (e.g. proteins, peptides) and lipid membrane. Compared to model organisms, the use of SLB is preferable since it mimics cell plasma membrane in a very simple and well-controlled way. Therefore, molecular structure of membrane and experimental conditions (e.g. solution chemistry, temperature, and pH) can be easily adjusted to the required conditions of any systematic research. In addition, SLBs are typically easy to form, cheap and very reproducible and they are compatible with different surface characterization techniques, such as quartz crystal microbalance with dissipation (QCM-D), ellipsometry and atomic force microscopy (AFM). This study demonstrates that QCM-D analysis of SLBs serve as powerful tool to investigate and characterize the mechanisms of interactions between lipid membrane and gold nanoparticles (NPs), environmentally relevant polymers, and disease-inducing peptides. Due to many critical applications of gold NPs in drug delivery and diagnostics, understanding of membrane-NP interactions is crucial especially for determination of NPs cytotoxicity. In this study we focus on membrane disruption as one of the different mechanisms by which metal NPs induce cytotoxicity. The use of SLB is beneficial for this goal as it elucidates the unique mechanism of membrane disruption without interference of other mechanisms taking place simultaneously in biological cells. For NP-membrane interaction studies, a SLB composed of L-α-phosphatidylcholine (egg PC) was formed on a SiO2-coated crystal and QCM-D analysis was performed to obtain information about mass and viscoelastic changes of SLB resulting from interactions with gold NPs. For better understanding of the mechanisms of NP-membrane interactions, we systematically changed the NPs properties and the experimental conditions. In order to understand the effect of NP size, gold NPs with diameters of 2,5,10, and 40 nm were tested and compared to each other. NPs were tested in their citric acid-stabilized state as well as in the presence of poly (methacrylic acid) (PMAA), representing an organic coating that could become associated with NPs in the environment. The results indicated that when dissolved in water, gold NPs with the dimeters of 2, 5, 10, and 40 nm did not perturb the membrane, but in the presence of environmentally relevant polymer, the larger nanoparticles were found to disrupt the membrane. In order to elucidate the effect of surface chemistry, 10 nm - gold NPs with various functionalizations (i.e. anionic, cationic and non-ionic ligands) were tested. Control experiments were designed to test the effect of NPs in the absence of humic substances which means the NPs were dissolved in water. In these cases, regardless of the type of NP functionalization, no substantial bilayer mass changes were observed. This suggests that the charge and chemistry of the ligands had a minor effect on NP-membrane interactions. Furthermore, in both the control and humic acid experiments, there were small dissipation changes (less than 1 unit) indicating that the overall membrane structure was not perturbed. In order to mimic environmentally-relevant conditions, mass and viscoelasticity of SLB was characterized in the presence of four different natural polymers, also known as natural organic materials (NOMs): Fulvic and humic acids extracted from Suwannee River (SRFA and SRHA), which had relatively lower molecular weights and a commercial humic acid (HA) and the humic acid extracted from Elliott soil (ESHA) with higher molecular weight. The results showed that NOMs with lower molecular weights, adsorbed to the bilayer, while higher molecular weight components, did not induce any changes to the bilayers. In addition, the NPs in SRFA and SRHA increased the mass of the bilayer by 20-30 ng, while the NPs in HA and ESHA changed the mass of the bilayer by < 10 ng. It was concluded that the presence of humic substances as well as their physical and chemical properties exert a direct impact on the interactions between cell membrane and the nanoparticles. In addition to the field of NP toxicity, SLBs play a pivotal role in the field of neurodegenerative diseases, such as Alzheimer’s disease (AD), in which the pathological cascade of events starts from interactions of a misfolded peptide with cell membrane. In this thesis, we confirm the validity of QCM-D analysis of SLB as an important platform for investigation of amyloid β (the peptide associated with AD) interactions with lipid membrane. Adsorption of Aβ peptide to cell membrane is known to take place on the so-called “lipid raftâ€� which are membrane microdomains enriched with cholesterol, sphingomyelin and ganglioside. The formation of SLBs containing lipid rafts is not only important for the field of AD research, but also it is important for other in vitro studies of cell biology as the lipid rafts are responsible for a variety of biological functions such as association of some membrane proteins and cellular signaling. However, the presence of lipid raft components such as sphingomyelin and cholesterol makes the formation of the bilayer more challenging which leads to adsorption of intact vesicles on the substrate without formation of the bilayer. In this study, the formation of lipid bilayer composed of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl- sn-glycero-3-phospho-L-serine (DOPS), cholesterol (Chol), sphingomyelin (SM), and ganglioside (GM) was investigated using QCM-D. A challenge was that the raft-containing vesicles remained intact on the SiO2 crystal. Therefore, different experimental conditions were tested to induce vesicle fusion, such as pH, temperature, osmotic pressure, and vesicle size. The key parameter in forming the bilayer was found to be applying osmotic pressure to the vesicles by having the vesicles exterior concentration of NaCl higher than interior concentration. When this concentration gradient was applied to the vesicles before flowing them on the substrate, vesicle rupture was favored and formation of a complete bilayer could occur. Here, we report the effects of each tested variable on the adsorption and fusion of the raft-containing vesicles, and the results are discussed based on the mechanisms of vesicle-vesicle and vesicle-substrate interactions.After developing the robust method for formation of SLB with lipid rafts, we used that as a template to characterize the mechanism of interactions between Aβ peptide and cell membrane which leads to onset of AD. The mechanism of Aβ toxicity leading to AD has not fully discovered yet, due to the complexity of the process including several steps of Aβ peptide adsorption on membrane, conformational change from disordered in solution to a membrane-bound α-helix structure and then formation of β-sheet aggregates that serve as fibrillation seeds. In this study, we showed that QCM-D technique as a promising tool to conduct systematic studies on the mechanism of interactions between Aβ peptide with lipid membrane. To our knowledge, this was the first time QCM-D was utilized for characterization of Aβ fibrillation starting from monomer states until formation of mature fibrils. The data indicated that peptide-membrane interactions follow a two-step kinetic pathway starting with the adsorption of small (low-n) oligomers until covering all the adsorption sites on the surface. In the second step, the membrane structure is destabilized as the result of interaction with oligomers which leads to lipid loss from the surface. Consistency of the results with the data obtained via other techniques substantiates QCM-D technique as a robust approach to answer the remaining unanswered questions in the field of Alzheimer’s disease.

The thesis is divided into two major parts. The first part focuses on the localisation of probes in lipid/polymeric bilayers and in GM1 micelles. Included in this thesis is a new approach based on electronic energy transfer/migration (FRET/DDEM), which efficiently determines transversal positions of fluorescent molecules in lipid bilayers. This approach has been used to locate newly synthesized lipid probes in DOPC bilayers. The label was introduced at the end of sn-2 acyl chains of variable length. Analytical models accounting for FRET exist for a limited number of basic geometries. Here, a combination of FRET and Monte Carlo simulations enables the localisation of probes in bicelles and in bilayers containing pores, i.e. in lipid systems with variable curvature, or in non-homogenous lipid systems. This approach has been used to test whether conical-like fluorescence probes have an increased affinity to highly curved regions, which would enable preferential labelling of membrane pores. A simplified FRET model has been applied to localize 2-pyridones, a class of potential drugs, in GM1 micelles. Since the localisation of drugs within nanoparticles might influence the release kinetics and loading efficiency, knowledge about the drug location is highly relevant. It turned out that all derivatives were localised at the core-shell interface of GM1 micelles. The second part of the thesis focuses mainly on the estimation of lipid nanodomain size by means of FRET, which still remains the most powerful method in this field. Limitations of FRET in the determination of domain size have been explored. We showed that the limitations of FRET are mainly caused by a low probes affinity to either the liquid-ordered or liquid-disordered phase. In the continuing work we provided a detailed dynamic and structural study of crosslinker-triggered formation of nanodomains. Here, two different domains have been revealed, i.e. i) domains whose size grows with increasing amount of added cholera toxin (CTxB), and to which CTxB binds tightly; ii) domains formed in membranes containing a slightly increased amount of sphingomyelin (as compared to i) whose size does not change during titration by additional CTxB and to which CTxB binds less tightly. / Disertace je rozdělena do dvou hlavníchčástí. Prvníčást se zabývá lokalizací značek v lipidových/polymerních dvojvrstvách a v GM1micelách. V práci prezentujeme nový přístup založený na přenosu/migraci elektronické energie (FRET/DDEM), jež umožňuje efektivně určovat vertikální pozici fluorescenčních molekul uvnitř lipidové dvojvrstvy. Tato metoda byla použita k lokalizaci nově syntetizovaných lipidových značek značených na konci sn-2 acylového řetězce s různou délkou v DOPC dvojvrstvách. Analytické modely popisující FRET existují pouze pro limitovaný počet základních geometrií. Kombinace FRETu s Monte Carlo simulacemi nicméně umožňuje lokalizaci značek v bicelách a v dvojvrstvách obsahujících póry, tj. v lipidových systémech s proměnlivým zakřivením a v nehomogenních lipidových útvarech. Tento přístup umožnil např. zjistit, zda kuželovitětvarované značky mají zvýšenou afinitu k vysoce zakřiveným oblastem dvojvrstvy, což by umožnilo preferenční značení pórů. Lokalizovány byly rovněž tři deriváty 2-pyridonů(potencionálních léčiv) v GM1micelách za použití jednoduchého modelu zohledňujícího FRET mezi donory a akceptory nacházejícími se v micelách. Lokalizace léčiv v nanočásticích ovlivňuje kinetiku uvolňování (release kinetics) a množství látky solubilizované v micelách (loading efficiency). Druhá část se především zabývá určováním velikostí lipidových nanodomén pomocí FRETu, který stále zůstává nejvíce výkonnou metodou v této oblasti. Zkoumány byly limitace FRETu v určování lipidových nanodomén. Ukázalo se, že tato omezení jsou především způsobena nízkou afinitou značek buď k Lonebo k Ldfázi. V navazující studii jsme poskytnuli detailní dynamickou a strukturní studii formace nanodomén indukované crosslinkerem. Objevili jsme dva typy domén: a) domény, jejichž velikost se zvětšuje s rostoucím množstvím přidaného cholera toxinu (CTxB) a k nimž se CTxB váže pevně a b) domény vzniklé v membránách se zvýšeným množstvím sfingomyelinu (ve srovnání s a)), jejichž velikost se nemění během titrace dodatečným CTxB a k nimž se CTxB váže méně pevně. / This thesis has been elaborated within the framework of the Agreement on JointSupervision (co-tutelle) of an International Doctoral Degree Programmebetween Charles University in Prague, Czech Republic and the Department of Chemistry at Umeå University, Sweden.

In this thesis we study the interactions of ions and cationic peptides with a negatively charged lipid bilayer in an ionic solution where the electrostatic interactions are screened. <br /><br /> We first examine the problem of charge renormalization and inversion of a highly charged bilayer with low dielectric constant. To be specific, we consider an asymmetrically charged lipid bilayer, in which only one layer is negatively charged. In particular, we study how dielectric discontinuities and charge correlations among lipid charges and condensed counterions influence the effective charge of the surface. When counterions are monovalent, e. g. , Na⁺, our mean-field approach implies that dielectric discontinuities can enhance counterion condensation. A simple scaling picture shows how the effects of dielectric discontinuities and surface-charge distributions are intertwined: Dielectric discontinuities diminish condensation if the backbone charge is uniformly smeared out while counterions are localized in space; they can, however, enhance condensation when the backbone charge is discrete. In the presence of asymmetric salts such as CaCl₂, we find that the correlation effect, treated at the Gaussian level, is more pronounced when the surface has a lower dielectric constant, inverting the sign of the charge at a smaller value of Ca²⁺ concentration. <br /><br /> In the last chapter we study binding of cationic peptides onto a lipid-bilayer membrane. The peptide not only interacts electrostatically with anionic lipids, rearranging their spatial distributions, but it can also insert hydrophobically into the membrane, expanding the area of its binding layer (i. e. , the outer layer). We examine how peptide charges and peptide insertion (thus area expansion) are intertwined. Our results show that, depending on the bilayer's surface charge density and peptide hydrophobicity, there is an optimal peptide charge yielding the maximum peptide penetration. Our results shed light on the physics behind the activity and selective toxicity of antimicrobial peptides, i. e. , they selectively rupture bacterial membranes while leaving host cells intact.

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.

The behavior of a protein molecule at the solid-liquid interface is a worthy scientific problem for at least three reasons. The main driving force for studying this problem is a practical one, as many areas of bio-related technologies, such as medical implants, biosensing, and drug delivery, require the understanding of protein-surface interactions. In this dissertation, the nature of the precursive weakly adsorbed state of proteins during binding is reviewed. From this perspective, the adsorption and binding of proteins to a solid block copolymer thin film was achieved with regular spacing. Further efforts produced a monolayer of green fluorescent protein (GFP) covalently bound with regular spacing and orientation to a diblock copolymer thin film. This protein could be folded and refolded by changing solvent characteristics. We also explored the binding of DC-SIGN to mannose and mannotriose bearing lipid membranes. While no binding was observed, the usefulness of the lipid-based glycan microarray was proven using the well-studied CTB-GM1 binding motif. / text

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.

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.