scholarly journals Terminal lipophilization of a unique DNA dodecamer by various nucleolipid headgroups: Their incorporation into artificial lipid bilayers and hydrodynamic properties

2015 ◽  
Vol 11 ◽  
pp. 913-929 ◽  
Author(s):  
Emma Werz ◽  
Helmut Rosemeyer
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Head Group ◽  
Artificial Lipid Bilayers ◽  
Decisive Importance ◽  
Head Groups ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Dna Dodecamer ◽  
And Diffusion

A series of six cyanine-5-labeled oligonucleotides (LONs 10–15), each terminally lipophilized with different nucleolipid head groups, were synthesized using the recently prepared phosphoramidites 4b–9b. The insertion of the LONs within an artificial lipid bilayer, composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), was studied by single molecule fluorescence spectroscopy and microscopy with the help of an optically transparent microfluidic sample carrier with perfusion capabilities. The incorporation of the lipo-oligonucleotides into the bilayer was studied with respect to efficiency (maximal bilayer brightness) as well as stability against perfusion (final stable bilayer brightness). Attempts to correlate these parameters with the log P values of the corresponding nucleolipid head groups failed, a result which clearly demonstrates that not only the lipophilicity but mainly the chemical structure and topology of the head group is of decisive importance for the optimal interaction of a lipo-oligonucleotide with an artificial lipid bilayer. Moreover, fluorescence half-live and diffusion time values were measured to determine the diffusion coefficients of the lipo-oligonucleotides.

scholarly journals Specific DNA duplex formation at an artificial lipid bilayer: fluorescence microscopy after Sybr Green I staining

2014 ◽  
Vol 10 ◽  
pp. 2307-2321 ◽  
Author(s):  
Emma Werz ◽  
Helmut Rosemeyer
Keyword(s):  
Lipid Bilayer ◽  
Single Molecule ◽  
Dna Sequences ◽  
Sybr Green ◽  
Sybr Green I ◽  
Racemic Mixture ◽  
Dna Duplexes ◽  
Target Dna ◽  
Single Molecule Fluorescence Spectroscopy ◽  
Duplex Formation

The article describes the immobilization of different probe oligonucleotides (4, 7, 10) carrying each a racemic mixture of 2,3-bis(hexadecyloxy)propan-1-ol (1a) at the 5’-terminus on a stable artificial lipid bilayer composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The bilayer separates two compartments (cis/trans channel) of an optical transparent microfluidic sample carrier with perfusion capabilities. Injection of unlabeled target DNA sequences (6, 8, or 9), differing in sequence and length, leads in the case of complementarity to the formation of stable DNA duplexes at the bilayer surface. This could be verified by Sybr Green I double strand staining, followed by incubation periods and thorough perfusions, and was visualized by single molecule fluorescence spectroscopy and microscopy. The different bilayer-immobilized complexes consisting of various DNA duplexes and the fluorescent dye were studied with respect to the kinetics of their formation as well as to their stability against perfusion.

scholarly journals Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity

2016 ◽  
Vol 113 (46) ◽  
pp. E7185-E7193 ◽  
Author(s):  
Rahul Grover ◽  
Janine Fischer ◽  
Friedrich W. Schwarz ◽  
Wilhelm J. Walter ◽  
Petra Schwille ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Gliding Motility ◽  
Supported Lipid Bilayers ◽  
Transport Efficiency ◽  
Collective Transport ◽  
Wide Range ◽  
Motor Density

In eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors, such as kinesin-1, have been well characterized in vitro as single molecules or as ensembles rigidly attached to nonbiological substrates. However, the collective transport by membrane-anchored motors, that is, motors attached to a fluid lipid bilayer, is poorly understood. Here, we investigate the influence of motors’ anchorage to a lipid bilayer on the collective transport characteristics. We reconstituted “membrane-anchored” gliding motility assays using truncated kinesin-1 motors with a streptavidin-binding peptide tag that can attach to streptavidin-loaded, supported lipid bilayers. We found that the diffusing kinesin-1 motors propelled the microtubules in the presence of ATP. Notably, we found the gliding velocity of the microtubules to be strongly dependent on the number of motors and their diffusivity in the lipid bilayer. The microtubule gliding velocity increased with increasing motor density and membrane viscosity, reaching up to the stepping velocity of single motors. This finding is in contrast to conventional gliding motility assays where the density of surface-immobilized kinesin-1 motors does not influence the microtubule velocity over a wide range. We reason that the transport efficiency of membrane-anchored motors is reduced because of their slippage in the lipid bilayer, an effect that we directly observed using single-molecule fluorescence microscopy. Our results illustrate the importance of motor–cargo coupling, which potentially provides cells with an additional means of regulating the efficiency of cargo transport.

scholarly journals Non-vesicular transfer of membrane proteins from nanoparticles to lipid bilayers

2011 ◽  
Vol 137 (2) ◽  
pp. 217-223 ◽  
Author(s):  
Sourabh Banerjee ◽  
Crina M. Nimigean
Keyword(s):  
Membrane Proteins ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Lipid Membranes ◽  
Single Channel ◽  
Black Lipid Membranes ◽  
Potassium Channel Kcsa ◽  
Lipid Environment ◽  
Biochemical Preparation

Discoidal lipoproteins are a novel class of nanoparticles for studying membrane proteins (MPs) in a soluble, native lipid environment, using assays that have not been traditionally applied to transmembrane proteins. Here, we report the successful delivery of an ion channel from these particles, called nanoscale apolipoprotein-bound bilayers (NABBs), to a distinct, continuous lipid bilayer that will allow both ensemble assays, made possible by the soluble NABB platform, and single-molecule assays, to be performed from the same biochemical preparation. We optimized the incorporation and verified the homogeneity of NABBs containing a prototypical potassium channel, KcsA. We also evaluated the transfer of KcsA from the NABBs to lipid bilayers using single-channel electrophysiology and found that the functional properties of the channel remained intact. NABBs containing KcsA were stable, homogeneous, and able to spontaneously deliver the channel to black lipid membranes without measurably affecting the electrical properties of the bilayer. Our results are the first to demonstrate the transfer of a MP from NABBs to a different lipid bilayer without involving vesicle fusion.

scholarly journals Interaction of metalloporphyrins with lipid bilayers, a calorimetric study

2011 ◽  
Vol 34 (1) ◽  
pp. 11-14
Author(s):  
Katarzyna Pamin ◽  
Jan Połtowicz ◽  
Joanna Kiełkowicz ◽  
Andrzej Hendrich
Keyword(s):  
Experimental Data ◽  
Phase Transitions ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Electrostatic Interactions ◽  
Acyl Chain ◽  
Head Group ◽  
Calorimetric Study ◽  
Polar Head Group ◽  
Polar Head

Interaction of metalloporphyrins with lipid bilayers, a calorimetric studyThe interaction of three metalloporphyrins, containing manganese, iron and cobalt atoms, with lipid bilayers composed of neutral (DPPC) or charged (DMPG) phospholipids were studied by means of scanning differential calorimetry. We found only minute effects exerted by studied compounds on DPPC, while phase transitions of charged DMPG were seriously affected by porphyrins. Analysis of experimental data revealed that due to the electrostatic interactions DMPG bilayers are perturbed not only in the polar head group region. Putative rearrangements of the polar heads packing affects also the acyl chain region of this lipid bilayer. Perturbation of DMPG polar heads induced by porphyrin in complex with manganese atoms is bigger than that induced by other porphyrins.

Study on Molecular Thermal Energy Transfer in a Lipid Bilayer

2007 ◽  
Author(s):  
Takeo Nakano ◽  
Taku Ohara ◽  
Gota Kikugawa
Keyword(s):  
Energy Transfer ◽  
Thermal Resistance ◽  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Thermal Energy ◽  
Hydrocarbon Chain ◽  
Bilayer Membrane ◽  
Head Group ◽  
Total Thermal Resistance ◽  
Dynamics Simulations

In recent studies, lipid bilayers attract a great deal of interest as a material for nanoscale structure. Some devices utilizing lipid bilayers, which include various kinds of sensors and molecular sorting devices, have been proposed. Understanding of thermal energy transfer in the lipid bilayers plays an important role in developing such devices with nano structures. In this study, we have investigated the energy transfer along and across the bilayer membrane by molecular dynamics simulations of the lipid bilayer in liquid water. We found that along the bilayer, the thermal energy is transferred by the interaction principally between water molecules and barely between lipid molecules. On the other hand, in the latter case, total thermal resistance of the lipid bilayer structure is composed of the thermal resistance of the structure’s various parts, including water, head group of lipid, and tail hydrocarbon chain of lipid, which show different values. It is found that the tail hydrocarbon chains have the highest thermal resistance.

scholarly journals Fast Formation of Lipid Bilayer Membranes for Simultaneous Analysis of Molecular Transport Using Parylene Coated Chips

Proceedings ◽  
2018 ◽  
Vol 2 (13) ◽  
pp. 920 ◽  
Author(s):  
Tanzir Ahmed ◽  
Sander van den Driesche ◽  
Martin Oellers ◽  
Roland Hemmler ◽  
Karsten Gall ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Molecular Transport ◽  
Bilayer Membrane ◽  
Average Lifetime ◽  
Lipid Bilayer Membranes ◽  
Parylene C ◽  
Silicon Nitride Membrane ◽  
Artificial Lipid Bilayers ◽  
Parallel Assembly

Artificial lipid bilayers are an essential tool to investigate channel forming proteins. A particular challenge is to study antibiotic uptake through bacterial porins requiring low volume and parallelization. Here, we present a lipid bilayer silicon chip having a Parylene-C coated silicon nitride membrane with different aperture sizes. The Parylene-C allows very fast lipid bilayer membrane fabrication, 30 to 130 s. The realization-success is very high and an average lifetime of at least 9 h was established. Furthermore, a 3D-printed holder is realized where parallel assembly of the chips, including fluid inlets for the pipetting robot, is demonstrated.

Creating conjugated hydrogel encapsulated membranes

2006 ◽  
Vol 950 ◽  
Author(s):  
Tae-Joon Jeon ◽  
Noah Malmstadt ◽  
Jacob Schmidt
Keyword(s):  
Ion Channel ◽  
Lipid Bilayer ◽  
Single Molecule ◽  
Lipid Bilayer Membranes ◽  
Single Molecule Level ◽  
Channel Proteins ◽  
Head Groups ◽  
Order Of Magnitude ◽  
Previous State ◽  
Ion Channel Proteins

ABSTRACTDevice engineering for ion channel proteins requires developing systems that incorporate mechanically stable, long-lived lipid bilayer membranes. Building on our previous work, we have further increased lipid bilayer longevity through covalent conjugation of lipid molecules in the bilayer to an encapsulating hydrogel. This is accomplished by polymerizing the hydrogel in situ around a gigaohm-seal membrane containing vinyl-modified lipid head groups, forming a conjugated hydrogel encapsulated membrane (cgHEM). Membranes formed in this manner show remarkable stability, maintaining gigaohm-level resistance for over 270 hours, better than an order-of-magnitude improvement over the previous state of the art. They also demonstrate the capacity to support the incorporation and measurement of ion channel proteins at the single-molecule level.

Influence of Brain Gangliosides on the Formation and Properties of Supported Lipid Bilayers for Binding Kinetics Studies

2018 ◽  
Author(s):  
Luke Jordan ◽  
Nathan Wittenberg
Keyword(s):  
Lipid Bilayers ◽  
Binding Kinetics ◽  
Supported Lipid Bilayers ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Supported Lipid Bilayer ◽  
Head Groups ◽  
Lipid Diffusion ◽  
Kinetics Of ◽  
Brain Gangliosides

This is a comprehensive study of the effects of the four major brain gangliosides (GM1, GD1b, GD1a, and GT1b) on the adsorption and rupture of phospholipid vesicles on SiO2 surfaces for the formation of supported lipid bilayer (SLB) membranes. Using quartz crystal microbalance with dissipation monitoring (QCM-D) we show that gangliosides GD1a and GT1b significantly slow the SLB formation process, whereas GM1 and GD1b have smaller effects. This is likely due to the net ganglioside charge as well as the positions of acidic sugar groups on ganglioside glycan head groups. Data is included that shows calcium can accelerate the formation of ganglioside-rich SLBs. Using fluorescence recovery after photobleaching (FRAP) we also show that the presence of gangliosides significantly reduces lipid diffusion coefficients in SLBs in a concentration-dependent manner. Finally, using QCM-D and GD1a-rich SLB membranes we measure the binding kinetics of an anti-GD1a antibody that has similarities to a monoclonal antibody that is a hallmark of a variant of Guillain-Barre syndrome.

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