Lipid membranes for the fabrication of functional micro- and nano-structures

The central goal of this thesis work is to fabricate novel, functional fluorescent nanostructures in confined systems offered by phospholipid membranes, which are known to have highly ordered, thermotropic and lyotropic structures. In separate approaches, we have used three different lipid systems: multilamellar planar lipid membranes, unilamellar vesicular membranes as well as lipid monolayers for the development of functional fluorescent nano-, micro- and meso-scopic structures. Techniques like fluorescence microscopy, single particle imaging, electron microscopy, electron diffraction were used to achieve fundamental understanding of the resulting structures. Multilayer stacks of phospholipid membranes have been used as an effective template for the growth of high aspect ratio fluorescent nanowires. The room temperature synthesis was achieved in the confined nanometer-sized interlamellar water space of lipid multilayers where supersaturating CdCl2 concentrations were induced by acidification leading to controlled unidirectional growth of nanowires. The possibility to render the nanowires fluorescent by doping with CdS quantum dots (QDs) and the light waveguiding along hundreds of micrometers together with the possibility of lateral manipulation make these nanowires attractive candidates for future optoelectronic applications. Novel organic-inorganic functional nanocontainers have been designed and tested by making use of vesicle forming lipid bilayers in combination with semiconductor QDs. Hydrophobic QDs can be integrated into bilayers of lipid vesicles and such lipid/QD hybrid vesicles are capable to fuse with live cells, thereby stain the cell's plasma membrane selectively with fluorescent QDs and transfer the vesicle's cargo into the cell. Modification of the membrane of such hybrid vesicles on the other hand, made them capable to enter the cytoplasm of live cells. Additionally, these hybrid vesicles were found extremely useful for long-term model membrane imaging studies. The results described in this thesis imply that cell and lipid membranes can integrate any kind of hydrophobic nanoparticle whose size matches the membrane thickness, opening novel possibilities to manipulate them as individuals or in ensemble with wide-ranging applications for nanobiotechnology. In a further step, the ability of phospholipid molecules to exhibit lamellar to non-lamellar transition using external stimuli enabled a directed self-assembly of QDs into mesoscale fluorescent structures. An easy and versatile method for the surface modification of TOPO coated CdSe QDs using 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA) have been achieved. DPPA predominantly form non-lamellar phases when dispersed in water, for example, upon addition of Ca2+ or at a pH below 6 are known to form hexagonal II phases. This particular property of DPPA has been exploited to form mesoscale self-assemblies of QD based structures both in solution and in confined systems. Potential applications include the detection or removal of Ca2+ ions in attoliter volumes, the construction of functional devices where QDs are reversibly organized in different forms as well as use of fluorescently labeled DPPA molecules for cellular studies using fluorescence microscopy.

Activation and Spatial Organization of the Adenosine A2A Receptor in Supported Plasma Membrane Sheets

Sophie Roizard

G protein-coupled receptors (GPCRs) are ubiquitous mediators of signal transduction across cell membranes and constitute a very important class of therapeutic targets. The main mechanisms involving GPCR activation and signal transduction to the cell interior are understood but many of the processes which fine-tune GPCRs signalling need to be unraveled. The work performed in this thesis intended to develop new methods to study GPCRs in their native environment but in a simplified system compared to live cells. In this context, the tethering GPCR-containing membrane fragments on solid surfaces appeared to be interesting: it enabled studying the receptors in their real native environment with several high resolution imaging techniques and with the possibility to engineer and control the downstream intracellular signaling components. Two procedures have been established to produce plasma membrane supported on micrometer-sized porous beads or on microscope grids for fluorescence or electron microscopy studies. They have been applied in this thesis to study the Adenosine A2A receeptor (A2A) as a prototypical GPCR. The first procedure enabled the transfer of native membrane patches from live cells with an inside-out orientation onto lectin-coated beads. Using heterologously expressed A2ARs carrying a yellow fluorescent protein (A2AR-Citrine) we showed that the tethered membranes were simultaneously accessible from both sides and comprised fully functional receptors in terms of ligand and G protein binding. These beads were investigated by confocal (Chapter 2) and single molecule (Chapter 3) fluorescence microscopies to study agonist and antagonist binding to the A2AR and the assembly and disassembly of the ternary complexes agonist-A2AR-Gαβγ proteins. The interactions between the different signalling partners could be observed in real time using multicolor fluorescence microscopy. These beads were also used in collaboration with colleagues for screening ligand binding to other transmembrane receptors. The second procedure produced GPCRs-containing plasma membrane fragments ripped-off on electron microscope grids for investigation by transmission electron microscopy (TEM) (Chapter 4). This procedure was improved from an existing protocol in order to produce reproducibly and with a high yield large membrane sheets and to preserve the membrane-associated cytoskeleton during the ripping-off. A2AR-Citrine were specifically gold immuno-labelled on their extracellular terminus using the FLAG-tag or their intracellular side by targeting the protein Citrine. The high resolution of TEM showed that the gold immuno-labelled A2AR-Citrine clustered and compartmentalized on actin-based cytoskeletton. This approach provided the ability to sample multiple single cells on a single grid and presented excellent potential to probe and unravel the organization of protein signalling networks in direct association with the plasma membrane-associated cytoskeleton.

EPFL2013

Influence of the protein environment on spectroscopy and ultrafast dynamics of retinal in bacteriorhodopsin

Selma Schenkl

The environment is important for the exact course of a chemical reaction. As an example of the strong influence of the environment on the reaction, this work studies the isomerization reaction of the chromophore retinal in the binding pocket of the protein bacteriorhodopsin. Three selected distance scales with reference to the chromophore are addressed: The distant protein surface at a length scale of 3-5 nm, the fuzzy environment of the binding pocket (1-2 nm) and the immediate neighborhood represented by the amino acid tryptophan Trp86 (5 Å). First, static absorption and fluorescence spectroscopies are applied to three dimensionally crystallized bacteriorhodopsin. An especially adapted set-up was developed for each task. The analysis of data is carried out in comparison with the measured spectra of the native protein membrane in solution. While the absorption spectrum shows a broadening on the high-energy side, the fluorescence spectrum deviates in the general shape of that measured in solution. The synoptic analysis of all spectra reveals the existence of three spectroscopically distinct species in the crystal: BR490 (absorption maximum at 490 nm) with a relative contribution of 28%, BR570 (native spectrum of solution, 68%), and BR610 ("blue membrane", 4%). BR490 appears particularly in the additional fluorescence band at 550 nm and is assigned to the dehydrated species of bacteriorhodopsin. The presence of that particular species indicates the suppression of rehydration in a partially dehydrated crystal and shows that the three dimensional arrangement hinders water diffusion. A simple model, close domains are assumed to form, eventually suppressing free water diffusion in a crystal. The lack of water on the protein surface modifies the protonation states of the amino acids in the binding pocket and therefore the optical properties of the retinal. The chromophore actually reacts on the alteration of its distant environment upon incorporation in the protein crystal. The second distance scale - the fuzzy environment - is addressed by a time-integrated fluorescence spectroscopy on native bacteriorhodopsin as function of the excitation wavelength (430 nm to 650 nm). The optimized experimental set-up guaranteed that the fluorescence of only the excited state I460 contributed. The measured spectra show maxima at 740 nm, and exhibit a Stokes shift of 5000 cm-1, independent of the excitation wavelength. The similarity of the fluorescence excitation spectrum with the spectrum of absorbed photons excludes energy-dependent loss channels. However, the spectra show a growing asymmetric broadening on the high-energy side with decreasing excitation wavelength. The additional fluorescence originates from vibrational hot states, also supported by the decomposition by Gaussian functions. A comparison with the fluorescence spectrum of blue membranes shows that the low-energy part also arises from vibrational hot states. Consequently, the first fast intramolecular vibrational energy relaxation does not entirely distribute the access-energy to other modes. The remaining energy is found in a quasi-static occupation of vibrational modes. The subsequent second vibrational relaxation — probably not only involving the retinal but also the protein — takes place on a time scale similar to that of the isomerization. The fast isomerization reaction prevents dissipation of energy relaxation of the excited state of the retinal into the protein environment. On a third distance scale, femtosecond spectroscopy in the UV offers a direct view into the electrostatic interaction of the chromophore with its immediate neighborhood. For the first time, the retinal isomerization was studied within the spectral window of the tryptophan absorption from 265 nm to 310 nm. For this, a set-up adapted to UV wavelengths was realized, basing on non-linear optics. The developed single-shot detection system yields a sensitivity of 10-5 in optical density. In the transient absorption measurements, a time resolution of 90 fs was achieved. At short probe wavelengths (265 nm - 294 nm), the measured transients show first a fast reduction of the absorption which subsequently recovers on three time scales (380 fs, 3 ps, > 16 ps). With increasing probe wavelength an induced transient absorption becomes dominant (282 nm - 294 nm). It has a rise time of 280 fs, and decays with a time constant of 4.3 ps. With further increasing probe wavelength the absorption signal decays faster. In response to the complex signature of the transients, an iterative and model-independent transient-decomposition is introduced. A set of three basis elements were identified: one bleach, and two absorption transients. The two induced absorption transients are assigned to retinal. The dynamics of the excited state (I460) in the first retinal-transient is described by two time constants (280 fs and 1 ps). The fast time constant leads to the photoproduct, while the slower one leads back to the ground state, presumably by internal conversion. The 250 fs delayed onset of the second transient (photo-intermediate J) is connected to a wave packet motion in the excited state. This delayed onset of the transient was observed for the first time. It demands a rise time of the J photo-intermediate of only 280 fs. This contrasts common estimates of 500 fs given in literature. For the first time, the temporal behavior of the permanent dipole moment of retinal is observed experimentally. Its evolution is imprinted in the dominant bleach feature (265 nm - 294 nm), originating from tryptophan Trp86. In the excited state, the permanent dipole moment increases strongly within the first 250 fs, and decays exponentially afterwards, following the formation of the photoproduct. However, the related time constant of 380 fs is slightly greater than that of 280 fs of the photoproduct J. This discrepancy may indicate a remaining long-lived polarization of the binding pocket. In brief, by using the intrinsic tryptophan as a probe, the local evolution of the electric field during the isomerization reaction was investigated. Indeed the isomerization reaction is reflected in the immediate environment. The variety of optical spectroscopy provides methods especially adapted for studying the influences of the environment on a chemical reaction. The results achieved here highlight the importance of these influences.

EPFL2004

A new class of thiolipids for the attachment of lipid bilayers on gold surfaces

Claus Duschl, Horst Vogel

A new class of lipid mols. is synthesized, based on two dipalmitoylphosphatidic mols., each extended at the lipid phosphate by a hydrophilic spacer chain of ethoxy groups of variable length, which are then coupled as a bilipid via a terminal disulfide group at the hydrophilic spacer. These anchor-bearing "thiolipids" can attach to gold substrates by forming stable gold-sulfur bonds. In this way, the authors can couple lipid bilayers to gold surfaces, with the possibility of preserving a water layer between the support and the first monolayer. The thiolipid mols. are characterized on a Langmuir film balance using fluorescence microscopy. The mol. areas of the thiolipids on the water surface are 80-90 .ANG.2 at a fully compressed state. The thiolipid monolayers show a typical first-order phase transition on the water surface with regular, starlike domains. The formation of thiolipid-attached mono- and bilayers on gold surfaces was studied by surface plasmon resonance (SPR), impedance measurements, and cyclic voltammetry. Four different supported membrane systems are studied in detail: (1) pure thiolipid layers; (2) mixed lipid bilayers contg. a first pure thiolipid monolayer and a second one of conventional phospholipids; (3) bilayers, where the first gold-attached monolayer is composed of a mixt. of thio- and conventional phospholipids with another second phospholipid layer on top; (4) monolayers of pure 1-hexadecanethiol and layers with a second phospholipid film on top of the 1-hexadecanethiol. The electrochem. expts. reveal elec. blocking layers for all lipid systems investigated with specific resistances of 104-105 W cm2. The capacitance values for pure thiolipid bilayers are in the range of 0.5-0.7 mF/cm2 for the pure thiolipid bilayers and 0.7-0.8 mF/cm2 for the mixed thiolipid/phospholipid bilayers, which is comparable to the values found for unsupported, so-called black lipid membranes. SPR measurements confirm qual. the results of the electrochem. expts. [on SciFinder (R)]

1994