The Mitochondrial Oxidase Assembly Protein1 (Oxa1) Insertase Forms a Membrane Pore in Lipid Bilayers

ABSTRACT We used site-directed mutagenesis to probe the function of four alternating arginines located at amino acid positions 525, 527, 529, and 531 in a highly conserved region of domain III in the Cry1Ac toxin of Bacillus thuringiensis. We created 10 mutants: eight single mutants, with each arginine replaced by either glycine (G) or aspartic acid (D), and two double mutants (R525G/R527G and R529G/R531G). In lawn assays of the 10 mutants with a cultured Choristoneura fumiferana insect cell line (Cf1), replacement of a single arginine by either glycine or aspartic acid at position 525 or 529 decreased toxicity 4- to 12-fold relative to native Cry1Ac toxin, whereas replacement at position 527 or 531 decreased toxicity only 3-fold. The reduction in toxicity seen with double mutants was 8-fold for R525G/R527G and 25-fold for R529G/R531G. Five of the mutants (R525G, R525D, R527G, R529D, and R525G/R527G) were tested in bioassays with Plutella xylostella larvae and ion channel formation in planar lipid bilayers. In the bioassays, R525D, R529D, and R525G/R527G showed reduced toxicity. In planar lipid bilayers, the conductance and the selectivity of the mutants were similar to those of native Cry1Ac. Toxins with alteration at position 527 or 529 tended to remain in their subconducting states rather than the maximally conducting state. Our results suggest that the primary role of this conserved region is to maintain both the structural integrity of the native toxin and the full functionality of the formed membrane pore.

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Energy landscape steering in SecYEG mediates dynamic coupling in ATP driven protein translocation

10.1101/793943 ◽

2019 ◽

Author(s):

Joel A. Crossley ◽

Tomas Fessl ◽

Matthew A. Watson ◽

Daniel W. Watkins ◽

Robin A. Corey ◽

...

Keyword(s):

Lipid Bilayers ◽

Conformational Changes ◽

Protein Translocation ◽

Energy Landscape ◽

Membrane Pore ◽

Reaction Coordinates ◽

Allosteric Communication ◽

Pore Opening ◽

Lateral Gate ◽

Allosteric Model

AbstractThe Sec translocon is a transmembrane assembly highly conserved among all forms of life as the principal route for transport of polypeptides across or into lipid bilayers. In bacteria translocation involves allosteric communication between the membrane pore SecYEG and the associated SecA ATPase. Using singlemolecule fluorescence we reveal that slow conformational changes associated with the ATPase SecA modulate fast opening and closure of the SecY lateral gate. Such a mismatch of timescales is not compatible with direct coupling between SecA and SecYEG. A dynamic allosteric model is proposed in which the SecA ATPase cycle ‘steers’ the energy landscape for SecY pore opening. We map the experimental traces onto reduced reaction coordinates derived from molecular dynamics trajectories, providing a model for the energy landscape and a structural interpretation of the associated dynamics. Dynamic allostery may be common among motor ATPases that drive conformational changes in molecular machines.Graphical TOC Entry

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Membrane pore formation by pentraxin proteins from Limulus, the American horseshoe crab

Biochemical Journal ◽

10.1042/bj20071357 ◽

2008 ◽

Vol 413 (2) ◽

pp. 305-313 ◽

Cited By ~ 12

Author(s):

John M. Harrington ◽

Hui-Ting Chou ◽

Thomas Gutsmann ◽

Christoph Gelhaus ◽

Henning Stahlberg ◽

...

Keyword(s):

Lipid Bilayers ◽

Pore Formation ◽

Horseshoe Crab ◽

Planar Lipid Bilayers ◽

C Reactive Protein ◽

Membrane Pore ◽

Reactive Protein ◽

Independent Form ◽

Transmembrane Pores ◽

Amyloid P

The pentraxins are a family of highly conserved plasma proteins of metazoans known to function in immune defence. The canonical members, C-reactive protein and serum amyloid P component, have been identified in arthropods and humans. Mammalian pentraxins are known to bind lipid bilayers, and a pentraxin representative from the American horseshoe crab, Limulus polyphemus, binds and permeabilizes mammalian erythrocytes. Both activities are Ca2+-dependent. Utilizing model liposomes and planar lipid bilayers, in the present study we have investigated the membrane-active properties of the three pentraxin representatives from Limulus and show that all of the Limulus pentraxins permeabilize lipid bilayers. Mechanistically, Limulus C-reactive protein forms transmembrane pores in asymmetric planar lipid bilayers that mimic the outer membrane of Gram-negative bacteria and exhibits a Ca2+-independent form of membrane binding that may be sufficient for pore formation.

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Observation of Concanavalin-A receptors on hydrated planar erythrocyte membrane film by in situ electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100076640 ◽

1983 ◽

Vol 41 ◽

pp. 608-609

Author(s):

Neng-Bo He ◽

S.W. Hui

Keyword(s):

Lipid Bilayers ◽

Erythrocyte Membrane ◽

Lipid Membranes ◽

Surface Film ◽

Biological Membranes ◽

Electron Microscopic Observation ◽

Electron Microscopic ◽

Black Lipid Membranes ◽

Fine Mesh ◽

Planar Films

Monolayers and planar "black" lipid membranes have been widely used as models for studying the structure and properties of biological membranes. Because of the lack of a suitable method to prepare these membranes for electron microscopic observation, their ultrastructure is so far not well understood. A method of forming molecular bilayers over the holes of fine mesh grids was developed by Hui et al. to study hydrated and unsupported lipid bilayers by electron diffraction, and to image phase separated domains by diffraction contrast. We now adapted the method of Pattus et al. of spreading biological membranes vesicles on the air-water interfaces to reconstitute biological membranes into unsupported planar films for electron microscopic study. hemoglobin-free human erythrocyte membrane stroma was prepared by hemolysis. The membranes were spreaded at 20°C on balanced salt solution in a Langmuir trough until a surface pressure of 20 dyne/cm was reached. The surface film was repeatedly washed by passing to adjacent troughs over shallow partitions (fig. 1).

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Effect of Integral Proteins on Frozen Membrane Fracture

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600003287 ◽

1980 ◽

Vol 38 ◽

pp. 756-759 ◽

Cited By ~ 1

Author(s):

S. Kirchanski ◽

D. Branton

Keyword(s):

Lipid Bilayers ◽

Freeze Fracture ◽

Covalent Bond ◽

Erythrocyte Membranes ◽

Glycophorin A ◽

Experimental Protocol ◽

Transmembrane Glycoprotein ◽

Bond Breakage ◽

Human Erythrocyte Membranes ◽

Integral Proteins

We have investigated the effect of integral membrane proteins upon the fracturing of frozen lipid bilayers. This investigation has been part of an effort to develop freeze fracture labeling techniques and to assess the possible breakage of covalent protein bonds during the freeze fracture process. We have developed an experimental protocol utilizing lectin affinity columns which should detect small amounts of covalent bond breakage during the fracture of liposomes containing purified (1) glycophorin (a transmembrane glycoprotein of human erythrocyte membranes). To fracture liposomes in bulk, frozen liposomes are ground repeatedly under liquid nitrogen. Failure to detect any significant covalent bond breakage (contrary to (2)) led us to question the effectiveness of our grinding procedure in fracturing and splitting lipid bilayers.

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Late Stages of the “Pearling" Instability in Lipid Bilayers

Journal de Physique II ◽

10.1051/jp2:1997180 ◽

1997 ◽

Vol 7 (9) ◽

pp. 1185-1204 ◽

Cited By ~ 6

Author(s):

J. L. Coveas ◽

S. T. Milner ◽

W. B. Russel

Keyword(s):

Lipid Bilayers ◽

Late Stages

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Improved micro-impedance spectroscopy to determine cell barrier properties

The EuroBiotech Journal ◽

10.2478/ebtj-2020-0017 ◽

2020 ◽

Vol 4 (3) ◽

pp. 150-155 ◽

Cited By ~ 1

Author(s):

Md. Mehadi Hasan Sohag ◽

Olivier Nicoud ◽

Racha Amine ◽

Abir Khalil-Mgharbel ◽

Jean-Pierre Alcaraz ◽

...

Keyword(s):

Impedance Spectroscopy ◽

Epithelial Cells ◽

Lipid Bilayers ◽

Cultured Cells ◽

Cell Model ◽

Measurement Techniques ◽

Cell Monolayer ◽

Microfluidic Channel ◽

Small Surface ◽

Resistance Pathway

AbstractThe goal of this study was to determine whether the Tethapod system, which was designed to determine the impedance properties of lipid bilayers, could be used for cell culture in order to utilise micro-impedance spectroscopy to examine further biological applications. To that purpose we have used normal epithelial cells from kidney (RPTEC) and a kidney cancer cell model (786-O). We demonstrate that the Tethapod system is compatible with the culture of 10,000 cells seeded to grow on a small area gold measurement electrode for several days without affecting the cell viability. Furthermore, the range of frequencies for EIS measurements were tuned to examine easily the characteristics of the cell monolayer. We demonstrate significant differences in the paracellular resistance pathway between normal and cancer kidney epithelial cells. Thus, we conclude that this device has advantages for the study of cultured cells that include (i) the configuration of measurement and reference electrodes across a microfluidic channel, and (ii) the small surface area of 6 parallel measurement electrodes (2.1 mm2) integrated in a microfluidic system. These characteristics might improve micro-impedance spectroscopy measurement techniques to provide a simple tool for further studies in the field of the patho-physiology of biological barriers.

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Direct Measurement of Charge Reversal on Lipid Bilayers using Heterodyne-Detected Second Harmonic Generation Spectroscopy

10.26434/chemrxiv.8678450.v1 ◽

2019 ◽

Author(s):

HanByul Chang ◽

Paul Ohno ◽

Yangdongling Liu ◽

Franz Geiger

Keyword(s):

Lipid Bilayers ◽

Surface Potential ◽

Second Harmonic ◽

Fluid Phase ◽

Cationic Polyelectrolyte ◽

Charge Reversal ◽

Buried Interfaces ◽

Phase Transition Temperatures ◽

Complementary Techniques ◽

Order Susceptibility

We report the detection of charge reversal induced by the adsorption of a cationic polyelectrolyte, poly(allylamine) hydrochloride (PAH), to buried supported lipid bilayers (SLBs), used as idealized model biological membranes. We observe changes in the surface potential in isolation from other contributors to the total SHG response by extracting the phase-shifted potential-dependent third-order susceptibility from the overall SHG signal. We demonstrate the utility of this technique in detecting both the sign of the surface potential and the point of charge reversal at buried interfaces without any prior information or complementary techniques<i>.</i>Furthermore, isolation of the second-order susceptibility contribution from the overall SHG response allows us to directly monitor changes in the Stern Layer. Finally, we characterize the Stern and Diffuse Layers over single-component SLBs formed from three different zwitterionic lipids of different gel-to-fluid phase transition temperatures (T<sub>m</sub>s). We determine whether the surface potential changes with the physical phase state (gel, transitioning, or fluid) of the SLB and incorporate 20 percent of negatively charged lipids to the zwitterionic SLB to investigate how the surface potential changes with surface charge.

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

10.26434/chemrxiv.7505330 ◽

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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