Alexandra Graff

Virus-assisted loading of polymer nanocontainer

We present a DNA-containing polymeric nanocontainer using the self-assembled superstructure of amphiphilic block copolymers in aqueous solutions. To demonstrate that DNA translocation is possible across a completely synthetic block copolymer membrane, we have used a phage transfection strategy as a DNA-transfer model system. For this purpose the bacterial channel forming protein LamB was reconstituted in ABA-triblock copolymer vesicles. The outer membrane protein LamB is a specific transporter for maltodextrins but also serves as a receptor for λ phage to trigger the ejection of λ phage DNA. We demonstrate that the functionality of the LamB protein is fully preserved despite the artificial surrounding. This leads to a type of polymeric vehicle for DNA that could be useful for gene therapy.

Disease Modeling and Phenotypic Drug Screening for Diabetic Cardiomyopathy using Human Induced Pluripotent Stem Cells

Diabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC) model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance.

Ion-carrier controlled precipitation of calcium phosphate in giant ABA triblock copolymer vesicles

An ionophore assisted metal-ion transport across block copolymer membranes has been used to control the local Ca2+ concentration during precipitation of calcium phosphate in giant block copolymer vesicles.

Stabilization of planar lipid membranes: A stratified layer approach

Here we briefly summarize our recent efforts in stabilization of giant planar lipid membranes and provide the first promising results achieved with a new technique. A water-soluble polymer can be coupled to lipid membranes either electrostatically or ia a hydrophobic linker. Such coated membranes are significantly destabilized and the rupture process is slowed down. In contrast, partitioning of hydrophobic styrene monomer into the lipid membrane and its polymerization lead to an increase in stability. A short electric field pulse under controlled conditions was applied to quantify the stability. Voltages above 1.2 V are required to induce an electrical discharge. Within less than 100 μs these defects reseal. However, after resealing, the stability of this particular lipid membrane corresponds to that of a lipid membrane without a 2-D polymer network. We suggest the use of this technique to stabilize self-assembled lipid membrane structures.

Large scale expression and purification of the mouse 5-HT3 receptor

Receptors of the Cys-loop family are central to neurotransmission and primary therapeutic targets. In order to decipher their gating and modulation mechanisms, structural data is essential. However, structural studies require large amounts of pure, functional receptors. Here, we present the expression and purification of the mouse serotonin 5-HT3 receptor to high purity and homogeneity levels. Inducible expression in human embryonic kidney 293 cells in suspension cultures with orbital shaking resulted in yields of 6-8mg receptor per liter of culture. Affinity purification using a strep tag provided pure protein in active form. Further deglycosylation and removal of the purification tag led to a pentameric receptor after size-exclusion chromatography, at the milligram scale. This material is suitable for crystallography, as demonstrated by X-ray diffraction of receptor crystals at low resolution.

Thermal Unfolding of a Mammalian Pentameric Ligand-gated Ion Channel Proceeds at Consecutive, Distinct Steps

Background: The 5-hydroxytryptamine receptor (5-HT3R) is a prototypical pentameric ligand-gated ion channel. Results: The receptors thermal stability was investigated in native plasma membranes, in detergent solution, and in reconstituted lipid bilayers. Conclusion: Unfolding of the 5-HT3R occurs via hierarchical, consecutive structural transitions, which can be distinguished experimentally. Significance: Our findings serve as an important base to create receptors of increased stability for structural/functional studies. Pentameric ligand-gated ion channels (LGICs) play an important role in fast synaptic signal transduction. Binding of agonists to the β-sheet-structured extracellular domain opens an ion channel in the transmembrane α-helical region of the LGIC. How the structurally distinct and distant domains are functionally coupled for such central transmembrane signaling processes remains an open question. To obtain detailed information about the stability of and the coupling between these different functional domains, we analyzed the thermal unfolding of a homopentameric LGIC, the 5-hydroxytryptamine receptor (ligand binding, secondary structure, accessibility of Trp and Cys residues, and aggregation), in plasma membranes as well as during detergent extraction, purification, and reconstitution into artificial lipid bilayers. We found a large loss in thermostability correlating with the loss of the lipid bilayer during membrane solubilization and purification. Thermal unfolding of the 5-hydroxytryptamine receptor occurred in consecutive steps at distinct protein locations. A loss of ligand binding was detected first, followed by formation of different transient low oligomeric states of receptor pentamers, followed by partial unfolding of helical parts of the protein, which finally lead to the formation receptor aggregates. Structural destabilization of the receptor in detergents could be partially reversed by reconstituting the receptor into lipid bilayers. Our results are important because they quantify the stability of LGICs during detergent extraction and purification and can be used to create stabilized receptor proteins for structural and functional studies.

Expression, Purification and Stabilization of the Mouse 5HT3 Receptor

Reference EPFL-CONF-201047View record in Web of Science Record created on 2014-08-29, modified on 2016-08-09