Oxidized polyethylene films for orienting polar molecules for linear dichroism spectroscopy

Stretched polyethylene (PE) films have been used to orient small molecules for decades by depositing solutions on their surface and allowing the solvent to evaporate leaving the analyte absorbed on the polymer film. However, the non-polar hydrophobic nature of PE is an obstacle to aligning polar molecules and biological samples. In this work PE film was treated with oxygen plasma in order to increase surface hydrophilicity. Different treatment conditions were evaluated using contact angle measurement and X-ray photoelectron spectroscopy. Treated PE (PEOX) films are shown to be able to align molecules of different polarities including progesterone, 1-pyrenecarboxaldehyde, 4′,6-diamidino-2-phenylindole (DAPI) and anthracene. The degree of alignment of each molecule was studied by running series of linear dichroism (LD) experiments and the polarizations of electronic transition moments were determined. For the first time optimal conditions (such as stretching factor and concentration of the sample) for stretched film LD were determined. PEOX aligning ability was compared to that of normal PE films. Progesterone showed a slightly better alignment on PEOX than PE. 1-Pyrenecarboxaldehyde oriented differently on the two different films which enabled transition moment assignment for this low symmetry molecule. DAPI (which does not align on PE) aligned well on PEOX and enabled us to obtain better LD data than had previously been collected with polyvinyl alcohol. Anthracene alignment and formation of dimers and higher order structures were studied in much more detail than previously possible, showing a variety of assemblies on PE and PEOX films

Synaptic vesicle mimics affect the aggregation of wild-type and A53T α-synuclein variants differently albeit similar membrane affinity

Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: [email protected]. α-Synuclein misfolding results in the accumulation of amyloid fibrils in Parkinson\u27s disease. Missense protein mutations (e.g. A53T) have been linked to early onset disease. Although α-synuclein interacts with synaptic vesicles in the brain, it is not clear what role they play in the protein aggregation process. Here, we compare the effect of small unilamellar vesicles (lipid composition similar to synaptic vesicles) on wild-type (WT) and A53T α-synuclein aggregation. Using biophysical techniques, we reveal that binding affinity to the vesicles is similar for the two proteins, and both interact with the helix long axis parallel to the membrane surface. Still, the vesicles affect the aggregation of the variants differently: effects on secondary processes such as fragmentation dominate for WT, whereas for A53T, fibril elongation is mostly affected. We speculate that vesicle interactions with aggregate intermediate species, in addition to monomer binding, vary between WT and A53T, resulting in different consequences for amyloid formation.\ua0\ua9 The Author(s) 2019

Targeting the endoplasmic reticulum with a membrane-interactive luminescent ruthenium(II) polypyridyl complex

The characterization and bioactivity of the dinuclear ruthenium(II) complex [(Ru(DIP)2)2(tpphz)]4+ (DIP = 4,7-diphenyl-1,10-phenanthroline and tpphz = tetrapyrido[3,2-a:2′,3′-c:3′′,2′′-h:2′′′,3′′′-j]phenazine) is reported. This new complex is found to be luminescent in acetonitrile, where excitation into MLCT (metal-to-ligand charge-transfer) bands in the visible area of the spectrum (λex = 450 nm, ε = 45 000 M−1 cm−1) result in red emission (λem,max = 620 nm, ΦMLCT = 0.017). Aqueous in vitro binding studies indicate that this complex binds to duplex DNA with an affinity of 1.8 × 106 M−1 through a non-classical groove-binding interaction, however, unlike the parent complex [(Ru(phen)2)2(tpphz)]4+ (phen = 1,10-phenanthroline), it also displays an increase in MLCT luminescence on addition of liposomes. Confocal microscopy and TEM studies show that this lipophilic complex targets the endoplasmic reticulum of eukaryotic cells, where it functions as an imaging agent for this organelle, and cytotoxicity studies in human cancer cell lines indicate a comparable potency to the anti-cancer drug cisplatin

Flow-Alignment of Extracellular Vesicles: Structure and Orientation of Membrane Associated Biomacromolecules Studied with Polarized Light

Extracellular vesicles (EVs) are currently in the scientific focus having a great potential to revolutionize the diagnosis and therapy of various diseases. However, numerous aspects of these species are still poorly understood, thus additional insight to molecular level properties, membrane-protein interactions, or membrane rigidity is still needed. We here demonstrate using red blood cell derived EVs (REVs) that polarized light spectroscopy techniques, linear and circular dichroism, can provide molecular level structural information on these systems. Flow-linear dichroism (flow-LD) measurements show that EVs can be oriented by shear force, and indicate that hemoglobin molecules are associated to the lipid bilayer in freshly released REVs. During storage this interaction ceases coupled to major protein conformational changes relative to the initial state. Further on, the degree of orientation gives insight to vesicle rigidity, which decreases in time parallel to changes in protein conformation. In overall, we propose that both LD and circular dichroism (CD) spectra provide simple, rapid, yet efficient ways to track changes in membrane-protein interactions of EV components at the molecular level which may also give insight to processes occurring during vesiculation

Membrane Orientation Studies with Polarized Light Spectroscopy

Lipid vesicles are versatile tools in modern biophysical science. They can be used as models of the cell membrane, to examine permeability, stability and behavior of molecules in the bilayer. They may act as transporters in a pharmacological context, designed to deliver their cargo to a certain place in the patient. The thesis has a methodological approach to the problem of assessing the orientation of guest molecules in a bilayer environment, a problem pertinent to drug development as well as to the understanding of biochemical and biological processes connected to cell membranes. Lipid vesicles can be deformed in a shear flow into ellipsoidal shapes and these macroscopically oriented samples studied with polarized light. The differential absorption between light polarized parallel and perpendicular to the flow orientation direction tells whether a transition moment in the molecule of interest is oriented preferentially parallel or perpendicular to the lipid chains of the vesicle bilayer. Molecules studied range from small aromatic molecules (pyrene, anthracene) over membrane-spanning substituted aromatic groups to large polypyridyl ruthenium complexes, for which small changes of peripheral substituents are found to cause dramatic changes in the orientational behavior. Refractive index matching of the liposomes, by adding sucrose in the surrounding solution, reduces light scattering dramatically, and for the first time enables study of transitions in the ultraviolet region of the spectrum. An alternative way to achieve a membrane host with anisotropic features is to orient a lamellar lyotropic liquid crystal, consisting of planar bilayers with interstitial water layers, between glass plates. Upon introducing a guest molecule into this system, its orientation can be assessed by tilting the sample and measuring linear dichroism. At normal incidence, the circular dichroism of the oriented molecule can be used to reveal the absolute configuration and as an aid for the assignment of electronic transitions

Membrane Orientation Studies with Polarized Light Spectroscopy

Lipid vesicles are versatile tools in modern biophysical science. They can be used as models of the cell membrane, to examine permeability, stability and behavior of molecules in the bilayer. They may act as transporters in a pharmacological context, designed to deliver their cargo to a certain place in the patient. <p />The thesis has a methodological approach to the problem of assessing the orientation of guest molecules in a bilayer environment, a problem pertinent to drug development as well as to the understanding of biochemical and biological processes connected to cell membranes. Lipid vesicles can be deformed in a shear flow into ellipsoidal shapes and these macroscopically oriented samples studied with polarized light. The differential absorption between light polarized parallel and perpendicular to the flow orientation direction tells whether a transition moment in the molecule of interest is oriented preferentially parallel or perpendicular to the lipid chains of the vesicle bilayer. Molecules studied range from small aromatic molecules (pyrene, anthracene) over membrane-spanning substituted aromatic groups to large polypyridyl ruthenium complexes, for which small changes of peripheral substituents are found to cause dramatic changes in the orientational behavior. Refractive index matching of the liposomes, by adding sucrose in the surrounding solution, reduces light scattering dramatically, and for the first time enables study of transitions in the ultraviolet region of the spectrum. <p />An alternative way to achieve a membrane host with anisotropic features is to orient a lamellar lyotropic liquid crystal, consisting of planar bilayers with interstitial water layers, between glass plates. Upon introducing a guest molecule into this system, its orientation can be assessed by tilting the sample and measuring linear dichroism. At normal incidence, the circular dichroism of the oriented molecule can be used to reveal the absolute configuration and as an aid for the assignment of electronic transitions