The temperature dependence of lipid membrane permeability, its quantized nature, and the influence of anesthetics

We investigate the permeability of lipid membranes for fluorescence dyes and ions. We find that permeability reaches a maximum close to the chain melting transition of the membranes. Close to transitions, fluctuations in area and compressibility are high, leading to an increased likelihood of spontaneous lipid pore formation. Fluorescence Correlation Spectroscopy (FCS) reveals the permeability for rhodamine dyes across 100 nm vesicles. Using FCS, we find that the permeability of vesicle membranes for fluorescence dyes is within error proportional to the excess heat capacity. To estimate defect size we measure the conductance of solvent-free planar lipid bilayer. Microscopically, we show that permeation events appear as quantized current events. Further, we demonstrate that anesthetics lead to a change in membrane permeability that can be predicted from their effect on heat capacity profiles. Depending on temperature, the permeability can be enhanced or reduced. We demonstrate that anesthetics decrease channel conductance and ultimately lead to 'blocking' of the lipid pores in experiments performed at or above the chain melting transition. Our data suggest that the macroscopic increase in permeability close to transitions and microscopic lipid channel formation are the same physical process.Comment: 12 pages, 6 figure

Untersuchung der Wirkung von Anästhetika auf Membranen und der Adsorption von Proteinen an Festkörperoberflächen mittels Moleküldynamik-Simulationen

In this thesis, the mechanisms underlying anesthesia and the adsorption of proteins on solid surfaces have been studied using the method of molecular dynamics simulations. It is generally assumed that biological membranes are the site of anesthetic action. However, there is no consensus whether anesthetics act directly by binding to membrane proteins, thereby inhibiting their function, or indirectly by modulating the physical properties of the lipid part of the membrane. In the simulations presented here, distinct changes of lipid bilayer properties in response to the presence of alkanols, a group of anesthetics, have been observed. An anesthetic-induced shift of the equilibrium between different membrane protein conformations, modeled by simple geometric shapes, has been found. In simulations with the ion channel gramicidin A embedded in a lipid bilayer, alkanols distributed inhomogeneously in the bilayer, with almost no alkanol molecules residing in close vicinity to the gramicidin. These results provide evidence for an indirect mode of anesthetic action. Spontaneous protein adsorption on solid-liquid interfaces is the first step in the formation of biofilms. Here, a coarse-grained molecular dynamics scheme has been applied to study this complex process at high resolution, but still reaching the necessary time and length scales. Changes in protein structure and dynamics after adsorption and preferred orientations of proteins on the surface were observed.In dieser Arbeit wurde die Wirkungsweise von Anästhetika und die Adsorption von Proteinen an Festkörperoberflächen mittels Moleküldynamik-Simulationen untersucht. Es wird allgemein angenommen, dass Anästhetika auf biologische Membranen wirken. Umstritten ist jedoch, ob Anästhetika direkt an Membranproteine binden und damit deren Funktion hemmen, oder ob sie indirekt wirken, indem sie die physikalischen Eigenschaften der Lipiddoppelschicht der Membran verändern. Solche indirekten Effekte wurden in den hier vorgestellten Simulationen bei Anwesenheit von Alkanolen, einer Gruppe von Anästhetika, beobachtet. Gleichzeitig wurde eine durch Anästhetika verursachte Verschiebung des Gleichgewichts zwischen unterschiedlichen, vereinfacht dargestellten Proteinkonformationen gefunden. Simulationen eines in einer Lipiddoppelschicht eingebetteten Ionenkanals zeigten eine sehr geringe Konzentration von Alkanolen in unmittelbarer Nähe des Kanals. Diese Ergebnisse deuten auf eine indirekte Wirkungsweise von Anästhetika hin. Spontane Adsorption von Proteinen an fest-flüssig Grenzflächen ist der erste Schritt bei der Bildung von Biofilmen. Um diesen Prozess der Proteinadsorption mit hoher Auflösung auf ausreichend langen Zeit- und Längenskalen zu untersuchen, wurde ein "coarse-grained" Moleküldynamik-Schema verwendet. Es wurden Veränderungen in der Proteinstruktur und -dynamik und bevorzugte Ausrichtungen der Proteine auf der Oberfläche beobachtet

PRESSURIZED SOLVENTS IN WHOLE-CELL BIOPROCESSING: METABOLIC AND STRUCTURAL PERTURBATIONS

Compressed and supercritical fluids, such as pressurized CO2, ethane, orpropane, provide a versatile and environmentally acceptable alternative to conventionalliquid organic solvents in bioprocessing applications – specifically in the areas ofproduct extraction, protein purification, microbial sterilization, and enzymatic and wholecellbiocatalysis. While their advantages have been well demonstrated, the effects ofcompressed and supercritical fluids on whole cells are largely unknown.Metabolic and structural perturbations of whole cells by compressed andsupercritical fluid solvents were examined. These perturbations exist as cell metabolismand membrane structure are influenced by pressure and the presence of a solventphase. Continuous cultures of Clostridium thermocellum (a model ethanol-producingthermophilic bacterium) were conducted under elevated hydrostatic and hyperbaricpressure to elucidate pressure- and solvent-effects on metabolism and growth.Fluorescence anisotropy was employed to study liposome fluidization due to thepresence of compressed and supercritical fluids and their partitioning/accumulation inthe phospholipid bilayer.Under elevated hydrostatic pressure (7.0 and 13.9 MPa; 333 K), significantchanges in product selectivity (towards ethanol) and growth were observed in C.thermocellum in conjunction with reduced maximum theoretical growth yields andincreased maintenance requirements. Similarly, metabolism and growth were greatlyinfluenced under hyperbaric pressure (1.8 and 7.0 MPa N2, ethane, and propane; 333K); however, severe inhibition was observed in the presence of supercritical ethane andliquid propane. These changes were attributed to mass-action effects on metabolicpathways, alterations in membrane fluidity, and the dominant role of phase toxicityassociated with compressed and supercritical fluids.Fluorescence anisotropy revealed fluidization and melting point depression ofdipalmitoylphosphatidylcholine liposomes in the presence of CO2, ethane, and propane(1.8 to 20.7 MPa; 295 to 333 K). The accumulation of these fluids within the bilayerupon pressurization and the ordering effects of pressure influenced liposome fluidity, themelting temperature, and the gel-fluid phase transition region. These resultsdemonstrate the disordering effects of compressed and supercritical fluids on biologicalmembranes and the ability to manipulate liposomes

Action of anesthetics on membranes and protein adsorption on solid surfaces studied by molecular dynamics simulations

In this thesis, the mechanisms underlying anesthesia and the adsorption of proteins on solid surfaces have been studied using the method of molecular dynamics simulations. It is generally assumed that biological membranes are the site of anesthetic action. However, there is no consensus whether anesthetics act directly by binding to membrane proteins, thereby inhibiting their function, or indirectly by modulating the physical properties of the lipid part of the membrane. In the simulations presented here, distinct changes of lipid bilayer properties in response to the presence of alkanols, a group of anesthetics, have been observed. An anesthetic-induced shift of the equilibrium between different membrane protein conformations, modeled by simple geometric shapes, has been found. In simulations with the ion channel gramicidin A embedded in a lipid bilayer, alkanols distributed inhomogeneously in the bilayer, with almost no alkanol molecules residing in close vicinity to the gramicidin. These results provide evidence for an indirect mode of anesthetic action. Spontaneous protein adsorption on solid-liquid interfaces is the first step in the formation of biofilms. Here, a coarse-grained molecular dynamics scheme has been applied to study this complex process at high resolution, but still reaching the necessary time and length scales. Changes in protein structure and dynamics after adsorption and preferred orientations of proteins on the surface were observed.In dieser Arbeit wurde die Wirkungsweise von Anästhetika und die Adsorption von Proteinen an Festkörperoberflächen mittels Moleküldynamik-Simulationen untersucht. Es wird allgemein angenommen, dass Anästhetika auf biologische Membranen wirken. Umstritten ist jedoch, ob Anästhetika direkt an Membranproteine binden und damit deren Funktion hemmen, oder ob sie indirekt wirken, indem sie die physikalischen Eigenschaften der Lipiddoppelschicht der Membran verändern. Solche indirekten Effekte wurden in den hier vorgestellten Simulationen bei Anwesenheit von Alkanolen, einer Gruppe von Anästhetika, beobachtet. Gleichzeitig wurde eine durch Anästhetika verursachte Verschiebung des Gleichgewichts zwischen unterschiedlichen, vereinfacht dargestellten Proteinkonformationen gefunden. Simulationen eines in einer Lipiddoppelschicht eingebetteten Ionenkanals zeigten eine sehr geringe Konzentration von Alkanolen in unmittelbarer Nähe des Kanals. Diese Ergebnisse deuten auf eine indirekte Wirkungsweise von Anästhetika hin. Spontane Adsorption von Proteinen an fest-flüssig Grenzflächen ist der erste Schritt bei der Bildung von Biofilmen. Um diesen Prozess der Proteinadsorption mit hoher Auflösung auf ausreichend langen Zeit- und Längenskalen zu untersuchen, wurde ein "coarse-grained" Moleküldynamik-Schema verwendet. Es wurden Veränderungen in der Proteinstruktur und -dynamik und bevorzugte Ausrichtungen der Proteine auf der Oberfläche beobachtet

Connexin channels and phospholipids: association and modulation

<p>Abstract</p> <p>Background</p> <p>For membrane proteins, lipids provide a structural framework and means to modulate function. Paired connexin hemichannels form the intercellular channels that compose gap junction plaques while unpaired hemichannels have regulated functions in non-junctional plasma membrane. The importance of interactions between connexin channels and phospholipids is poorly understood.</p> <p>Results</p> <p>Endogenous phospholipids most tightly associated with purified connexin26 or connexin32 hemichannels or with junctional plaques in cell membranes, those likely to have structural and/or modulatory effects, were identified by tandem electrospray ionization-mass spectrometry using class-specific interpretative methods. Phospholipids were characterized by headgroup class, charge, glycerol-alkyl chain linkage and by acyl chain length and saturation. The results indicate that specific endogenous phospholipids are uniquely associated with either connexin26 or connexin32 channels, and some phospholipids are associated with both. Functional effects of the major phospholipid classes on connexin channel activity were assessed by molecular permeability of hemichannels reconstituted into liposomes. Changes to phospholipid composition(s) of the liposome membrane altered the activity of connexin channels in a manner reflecting changes to the surface charge/potential of the membrane and, secondarily, to cholesterol content. Together, the data show that connexin26 and connexin32 channels have a preference for tight association with unique anionic phospholipids, and that these, independent of headgroup, have a positive effect on the activity of both connexin26 and connexin32 channels. Additionally, the data suggest that the likely in vivo phospholipid modulators of connexin channel structure-function that are connexin isoform-specific are found in the cytoplasmic leaflet. A modulatory role for phospholipids that promote negative curvature is also inferred.</p> <p>Conclusion</p> <p>This study is the first to identify (endogenous) phospholipids that tightly associate with connexin channels. The finding that specific phospholipids are associated with different connexin isoforms suggests connexin-specific regulatory and/or structural interactions with lipid membranes. The results are interpreted in light of connexin channel function and cell biology, as informed by current knowledge of lipid-protein interactions and membrane biophysics. The intimate involvement of distinct phospholipids with different connexins contributes to channel structure and/or function, as well as plaque integrity, and to modulation of connexin channels by lipophilic agents.</p

Microfluidic Transport Studies on Lipid Vesicles

The plasma membrane is the outermost layer of a cell and separates it from the extracellular environment. The membrane, as well as the proteins that are anchored in it, plays a crucial role in the uptake and efflux of molecules. Lipid bilayers are therefore an important object of research in many fields of science, such as biophysics, pharmacology and synthetic biology. In this thesis, we develop new microfluidic methods that allow us to study the laws that govern the transport processes through lipid bilayers. We use the novel microfluidic Octanol-Assisted Liposome Assembly (OLA) technique to obtain lipid vesicles which serve as model membrane for our studies. We perform a literature review, where we discuss OLA and other techniques to obtain liposomes in detail, before we provide a biophysical analysis of liposomes generated with OLA and compare them to vesicles obtained via traditional techniques. The biophysical analysis represents the first systematic evaluation of the properties of GUVs produced with the OLA technique to date. Using a fluorescence intensity assay, we show that OLA allows for the production of GUVs with binary lipid mixtures of DOPC-DOPG and DOPC-DOPE in the 1:3, 2:2 and 3:1 lipid ratio. GUVs with binary lipid mixtures of DOPG-DOPE are only stable in the 1:3 and 2:2 mixing ratio, but not in the 3:1 ratio. We attribute this behaviour to the high charge density of this mixture and the lipid polymorphism which makes it energetically unfavourable for certain lipid compositions to form lamellar structures. We furthermore investigate the lateral lipid diffusivity of DOPC and POPC vesicles produced with OLA using fluorescence recovery after photobleaching (FRAP) and compare it to that of vesicles obtained via the established electroformation technique. We find the lateral diffusion coefficients to be quantitatively similar and in the range of 1 μm²/s for the different lipid systems and techniques tested. Finally, using a dithionite bleaching assay, we quantitatively show the unilamellarity of OLA vesicles, confirming previous results. After examining OLA vesicles for their suitability for transport measurements, we expand the OLA technique and develop a platform that allows for the on-chip fabrication and controlled exposure of liposomes to a solute of interest. This novel microfluidic platform combines OLA with a flow through system that enables us to measure transport and other membrane-active processes that occur in time scales of tens of seconds on chip. In this platform, we either use fluorescent labels or exploit the solute molecule’s autofluorescence to visualise the transport across the vesicle membrane. Using this new method, we investigate the permeability of small antibiotic molecules of the fluoroquinolone family and quantify the transport of the drugs ciprofloxacin and norfloxacin through liposome membranes. For PGPC membranes, we measure median permeability coefficients of 3.57 × 10⁻⁶ cm/s for norfloxacin and 4.83 × 10⁻⁶ cm/s for ciprofloxacin in a PBS buffer at pH 7.4. These values correlate with the partition coefficients of the drugs, as well as previous studies on their permeation into lipid vesicles. In a second series of experiments, we investigate whether or not DNA nanostructures can act as ion channels and increase the membrane’s permeability towards protons. For these studies, we use a microfluidic perfusion assay that was previously developed by the Keyser group. In this assay, OLA-generated vesicles are immobilised on chip via vesicle traps and are either incubated with DNA nanopores or a buffer control, before being perfused with a low pH solution. By means of the fluorescent pH indicator HPTS, encapsulated inside the GUVs, the intravesicular proton concentration is monitored throughout this exposure. Our results do not suggest substantial enhancement of proton flux as a result of the incubation with the DNA nanopore. We attribute this behaviour to the low insertion efficiency of the DNA nanopore and the temporal resolution of the assay, which might be too low when put in context with the timescale of passive proton permeation to show a potentially flux enhancing effect of the DNA nanostructure. Finally, we widen the scope of our technique by taking the first steps towards a new visualisation method based on the deep UV absorbance of molecules rather than fluorescence. Our results indicate that this new visualisation method can in principle be incorporated into a microfluidic assay and used to measure membrane permeability, however this imaging mode features a substantially lower signal to noise ratio compared to the design based on fluorescence, limiting its capabilities. The issue of low signal to noise ratio must be overcome before the absorbance assay can provide a true alternative to fluorescence-based microfluidic assays as a means to study membrane permeation. All in all, our studies show that GUVs obtained with the novel OLA technique are viable alternative to vesicles generated with established methods such as electroformation. We successfully use OLA-generated vesicles to study a series of biophysical membrane parameters and conduct measurements on membrane permeability. We also demonstrate the versatility of the OLA technique by successfully incorporating it into complex microfluidic assays, exemplifying its potential for the investigation of membrane properties and its use in transport studies.Friedrich-Naumann-Stiftung für die Freiheit, Cambridge Philosophical Society, Jane Bourque-Driscoll Fund (Jesus College Cambridge

Microbial transformation of tetralin

Biocatalytic oxidation of cyclic hydrocarbons has many potential applications in the production of fine chemicals. Especially regioselective hydroxylation of aromatics and the stereospecific formation of secondary alcohols is of interest for the pharmaceutical and flavoring industries. Hydroxylating enzymes are active under mild reaction conditions allowing the controlled transformation of less stable substrates and formation of easily oxidizable products (e.g., catechols). Furthermore, application of microorganisms for the removal of cyclic hydrocarbons from waste streams provides a highly versatile method for the removal of toxic cyclic hydrocarbons.Major drawbacks in the application of biocatalysts, for these processes are the need for cofactor regeneration and the low stability as a result of inhibitory effects of the hydrocarbon substrates. The investigations described in this thesis have dealt with microbiological aspects of the design of a biocatalytic hydroxylation process. As a model for microbial oxy-functionalization, the dioxygenation of tetralin to 1,2,5,6,7,8-hexahydro- cis - naphthalene diol and the subsequent chemical rearrangement to 5,6,7,8-tetrahydro-1-naphthol has been studied. Tetralin provides a perfect model compound for specific hydroxylation since different sites of initial oxidative attack can be envisaged (Chapter 4). Moreover, different oxygenated derivatives of tetralin are of interest to pharmaceutical industries as precursors for the production of hormone-analogs, sedatives, and tranquilizers. Also for the production of fragrance compounds, oxygenated tetralins may be useful (Chapter 1 and 4). Special attention has been given to the mechanism of the toxicity of cyclic hydrocarbon substrates. In Chapter 2 literature data concerning inhibitory effects of cyclic hydrocarbons and other lipophilic compounds on microorganisms has been reviewed.Selection of suitable biocatalysts . Chapter 3 describes the procedure that has been followed to obtain microorganisms that are able to use tetralin as sole source of carbon and energy. Enrichment cultures on tetralin were set up with soil samples from polluted areas, and also cyclic hydrocarbon-utilizing strains from culture collections were tested. Initial attempts were unsuccessful, which was attributed to substrate inhibition. By lowering the concentration of tetralin in the incubation media, growth occurred in several enrichment cultures, but no pure strains were isolated. Eventually, a pure culture was isolated by supplying tetralin in subsaturating concentrations (lower than 125 μmol/liter). Initial studies on the inhibitory action of tetralin on this strain, Arthrobacter sp. strain T2, indicated that an aqueous concentration of approximately 100 μmol/liter). already impaired growth, whereas quantities above the saturation concentration (approximately 125 μmol/liter) fully inhibited growth of the starved cells. These findings were taken into consideration in new attempts to isolate tetralin-utilizing strains. Addition of tetralin via the vapor phase, thus limiting the aqueous concentration, resulted in the selection of another bacterium from an enrichment culture set up with soil from a land farming facility. Furthermore, four strains that were previously isolated on o -xylene, styrene, or mesitylene respectively were also shown to degrade tetralin. Alternatively, enrichment cultures were set up with tetralin added in a non-miscible, non-biodegradable, and non-toxic organic solvent (Fluorocompound 40) which limits the aqueous concentration by serving as a reservoir for the toxic substrate. From these enrichment cultures, two different strains were obtained that were able to use tetralin as sole source of carbon and energy.In Chapter 4 a survey of initial oxidation steps that may be involved in the biotransformation of tetralin is presented together with experimental data on the accumulation of intermediates oxygenated intermediates from tetralin. The knowledge on the initial oxidative steps has been used to evaluate the potentialities of the selected strains. It appeared that five strains started with an initial oxidation of the benzylic carbon atom, resulting in the formation of a-tetralol and a-tetralone. Two strains exhibited aspecific oxidations yielding products characteristic of both oxidation of the aromatic and the alicyclic moiety. Only one strain, Corynebacterium sp. strain C125, degraded tetralin by initially oxidizing the benzene nucleus. The metabolic pathway of tetralin in Corynebacterium sp. strain C125 has been studied in detail. The results, which are presented in Chapter 5, show that the aromatic moiety is attacked by a dioxygenase at the carbon atoms proximal to the cycloalkane substituent. The cis-dihydro diol that is formed in this reaction is further metabolized via a dehydrogenase to 5,6,7,8-tetrahydro-1,2-dihydroxynaphthalene, which is a substrate for an extra-diol cleaving catechol dioxygenase. Acidrearrangement of 1,2,5,6,7,8-hexahydro- cis -1,2-naphthalene diol yielded the corresponding phenols, 5,6,7,8-tetrahydro-1-naphthol and 5,6,7,8-tetrahydro-2-naphthol, in a ratio of 1:6. The apparent preference for the 2-naphthol limits the feasability of this system for the formation of the desired fragrance compound, 5,6,7,8-tetrahydro-1-naphthol However, the specificities of the different tetralin- transforming strains enable the formation of some high-value precursors for the synthesis of pharamaceuticals (Chapter 1 and 4).Mechanism of the inhibitory action of tetralin and other cyclic hydrocarbons .From the results presented in Chapter 3 it is clear that tetralin has a deleterious effect not only on tetralin-utilizing strains but also on other organisms. From incubations with possible intermediates of tetralin metabolism it appeared that tetralin, and not an intermediairy reaction product, was responsible for the observed toxicity. Similar observations for other cyclic hydrocarbons suggested that the inhibitory action of these compounds resulted from interaction with the membrane(s) of microbial cells (Chapter 2).In Chapter 6 the mechanism of the toxic action of tetralin on microorganisms has been investigated in intact cells of tetralin-utilizing and non-utilizing bacteria, as well as in liposomes. The results of these investigations indicated that tetralin was accumulated in the membrane, which lead to a significant increase in membrane surface area. Similar studies with other cyclic hydrocarbons, as described in Chapter 7, showed that in addition to the increase in surface area also an increased membrane fluidity was observed. The effective concentrations of the cyclic hydrocarbons necessary for disturbing membrane integrity decreased with increasing hydrophobicity (measured as partition coefficient in an octanol/water system). The hydrophobicity of the hydrocarbon compounds provides a good measure for the partition coefficients of these compounds between the aqueous environment and the membrane (Chapter 7). From the estimated membrane/buffer partition coefficients the actual concentrations of the different in the membrane could be calculated and related to their effects on the membrane properties.As a result of the membrane expansion and increase in bilayer fluidity, the integrity of the membrane was impaired. Consequently, the passive permeability of the membrane to protons (ions) was increased and the activity of the membrane embedded proton-pump cytochrome c oxidase was reduced. As a result of an increased permeability for protons and impairment of proton-pumping activity, the proton motive force was dissipated and internal pH homeostasis was disturbed. The dissipation of the proton motive force may result in the depletion of metabolic energy, but lowering of the internal pH may lead to complete inactivation of enzymes.In Chapter 8 some implications of the postulated mechanism of the toxic action of cyclic hydrocarbons have been discussed in relation to the application of microorganisms for the biotransformation of such compounds. Also some aspects of the adapation of the cells have been treated in connection with a general response of cells to stress. Finally, some methods to prevent deleterious effects of cyclic hydrocarbons have been discussed in view of the proposed toxicity mechanism