Reconstituted Membrane Systems for Assaying Membrane Proteins in Controlled Lipid Environments

2020 ◽  
pp. 93-121
Author(s):  
Gaya P. Yadav ◽  
Qiu-Xing Jiang
Keyword(s):  
Membrane Proteins ◽  
Membrane Systems

scholarly journals bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles

2015 ◽  
Vol 147 (1) ◽  
pp. 77-93 ◽  
Author(s):  
Hui Zheng ◽  
Sungsoo Lee ◽  
Marc C. Llaguno ◽  
Qiu-Xing Jiang
Keyword(s):  
Ion Channels ◽  
Membrane Proteins ◽  
Lipid Bilayers ◽  
Lipid Composition ◽  
Targeted Delivery ◽  
Membrane System ◽  
Membrane Systems ◽  
Giant Vesicles ◽  
Planar Electrode ◽  
New System

Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid composition of membranes. Here, using a newly developed chemical engineering procedure, we report on a bead-supported unilamellar membrane (bSUM) system that allows good control over membrane dimension, protein orientation, and lipid composition. Our new system uses specific ligands to facilitate the unidirectional incorporation of membrane proteins into lipid bilayers. Cryo–electron microscopic imaging demonstrates the unilamellar nature of the bSUMs. Electrical recordings from voltage-gated ion channels in bSUMs of varying diameters demonstrate the versatility of the new system. Using KvAP as a model system, we show that compared with other in vitro membrane systems, the bSUMs have the following advantages: (a) a major fraction of channels are orientated in a controlled way; (b) the channels mediate the formation of the lipid bilayer; (c) there is one and only one bilayer membrane on each bead; (d) the lipid composition can be controlled and the bSUM size is also under experimental control over a range of 0.2–20 µm; (e) the channel activity can be recorded by patch clamp using a planar electrode; and (f) the voltage-clamp speed (0.2–0.5 ms) of the bSUM on a planar electrode is fast, making it suitable to study ion channels with fast gating kinetics. Our observations suggest that the chemically engineered bSUMs afford a novel platform for studying lipid–protein interactions in membranes of varying lipid composition and may be useful for other applications, such as targeted delivery and single-molecule imaging.

Self-assembling layers created by membrane proteins on gold

2007 ◽  
Vol 35 (3) ◽  
pp. 522-526 ◽  
Author(s):  
D.S. Shah ◽  
M.B. Thomas ◽  
S. Phillips ◽  
D.A. Cisneros ◽  
A.P. Le Brun ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Self Assembly ◽  
Three Dimensions ◽  
Membrane Systems ◽  
Electrical Measurements ◽  
Supported Bilayers ◽  
Force Microscopy ◽  
Planar Surfaces ◽  
Self Assembling ◽  
Atomic Force

Membrane systems are based on several types of organization. First, amphiphilic lipids are able to create monolayer and bilayer structures which may be flat, vesicular or micellar. Into these structures membrane proteins can be inserted which use the membrane to provide signals for lateral and orientational organization. Furthermore, the proteins are the product of highly specific self-assembly otherwise known as folding, which mostly places individual atoms at precise places in three dimensions. These structures all have dimensions in the nanoscale, except for the size of membrane planes which may extend for millimetres in large liposomes or centimetres on planar surfaces such as monolayers at the air/water interface. Membrane systems can be assembled on to surfaces to create supported bilayers and these have uses in biosensors and in electrical measurements using modified ion channels. The supported systems also allow for measurements using spectroscopy, surface plasmon resonance and atomic force microscopy. By combining the roles of lipids and proteins, highly ordered and specific structures can be self-assembled in aqueous solution at the nanoscale.

Fabrication of Hybrid Bionanodevices Based on Coupled Protein Functionality

2004 ◽  
Author(s):  
D. Ho ◽  
B. Chu ◽  
H. Lee ◽  
K. Kuo ◽  
C. D. Montemagno
Keyword(s):  
Membrane Proteins ◽  
Proton Pumping ◽  
Protein Functionality ◽  
Single Chain ◽  
Membrane Systems ◽  
Large Area ◽  
Property A ◽  
Langmuir Blodgett ◽  
Membrane Fabrication ◽  
Natural Cell

Block copolymer-based membrane technology represents a versatile class of nanoscale materials in which biomolecules, such as membrane proteins, can be reconstituted. Among its many advantages over conventional lipid-based membrane systems, block copolymers can mimic natural cell biomembrane environments in a single chain, enabling large-area membrane fabrication using methods like Langmuir-Blodgett deposition, or spontaneous protein-functionalized nanovesicle formation. Based on this unique membrane property, a wide variety of membrane proteins possessing unique functionalities including pH/voltage gatable porosity, photon-activated proton pumping, and gradient-dependent production of electricity have been successfully inserted into these biomimetic systems.

Bimolecular and Monomolecular Lipid Films: Selected Area Electron Diffraction

1969 ◽  
Vol 27 ◽  
pp. 338-339
Author(s):  
Robert M. Glaeser ◽  
David W. Deamer
Keyword(s):  
Electron Diffraction ◽  
Membrane Structure ◽  
Molecular Organization ◽  
Membrane Material ◽  
X Ray Diffraction ◽  
Membrane Systems ◽  
X Ray ◽  
Selected Area Electron Diffraction ◽  
Lipid Films ◽  
Ultimate Objective

In the investigation of the molecular organization of cell membranes it is often supposed that lipid molecules are arranged in a bimolecular film. X-ray diffraction data obtained in a direction perpendicular to the plane of suitably layered membrane systems have generally been interpreted in accord with such a model of the membrane structure. The present studies were begun in order to determine whether selected area electron diffraction would provide a tool of sufficient sensitivity to permit investigation of the degree of intermolecular order within lipid films. The ultimate objective would then be to apply the method to single fragments of cell membrane material in order to obtain data complementary to the transverse data obtainable by x-ray diffraction.

Changes Occur in Plasma Membrane Turnover when K562 Leukemia Cells are Induced to Differentiate

1982 ◽  
Vol 40 ◽  
pp. 286-287
Author(s):  
L. M. Marshall
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Intracellular Transport ◽  
Parent Cell ◽  
Membrane Turnover ◽  
Coated Pits ◽  
Surface Distribution ◽  
Specific Proteins ◽  
Erythroleukemic Cell ◽  
Specific Membrane

A human erythroleukemic cell line, metabolically blocked in a late stage of erythropoiesis, becomes capable of differentiation along the normal pathway when grown in the presence of hemin. This process is characterized by hemoglobin synthesis followed by rearrangement of the plasma membrane proteins and culminates in asymmetrical cytokinesis in the absence of nuclear division. A reticulocyte-like cell buds from the nucleus-containing parent cell after erythrocyte specific membrane proteins have been sequestered into its membrane. In this process the parent cell faces two obstacles. First, to organize its erythrocyte specific proteins at one pole of the cell for inclusion in the reticulocyte; second, to reduce or abolish membrane protein turnover since hemoglobin is virtually the only protein being synthesized at this stage. A means of achieving redistribution and cessation of turnover could involve movement of membrane proteins by a directional lipid flow. Generation of a lipid flow towards one pole and accumulation of erythrocyte-specific membrane proteins could be achieved by clathrin coated pits which are implicated in membrane endocytosis, intracellular transport and turnover. In non-differentiating cells, membrane proteins are turned over and are random in surface distribution. If, however, the erythrocyte specific proteins in differentiating cells were excluded from endocytosing coated pits, not only would their turnover cease, but they would also tend to drift towards and collect at the site of endocytosis. This hypothesis requires that different protein species are endocytosed by the coated vesicles in non-differentiating than by differentiating cells.

Image Analysis of Intramembrane Particles in Freeze-Fractured Biomembranes Using Computer Simulation Techniques

1975 ◽  
Vol 33 ◽  
pp. 214-215
Author(s):  
D.J. Benefiel ◽  
R.S. Weinstein
Keyword(s):  
Membrane Proteins ◽  
Freeze Fracture ◽  
Integral Membrane Proteins ◽  
Systematic Evaluation ◽  
Intramembrane Particles ◽  
Modeling Methods ◽  
Simulation Techniques ◽  
Freeze Fracture Electron Microscopy ◽  
Fracture Electron ◽  
Computer Simulation Techniques

Intramembrane particles (IMP or MAP) are components of most biomembranes. They are visualized by freeze-fracture electron microscopy, and they probably represent replicas of integral membrane proteins. The presence of MAP in biomembranes has been extensively investigated but their detailed ultrastructure has been largely ignored. In this study, we have attempted to lay groundwork for a systematic evaluation of MAP ultrastructure. Using mathematical modeling methods, we have simulated the electron optical appearances of idealized globular proteins as they might be expected to appear in replicas under defined conditions. By comparing these images with the apearances of MAPs in replicas, we have attempted to evaluate dimensional and shape distortions that may be introduced by the freeze-fracture technique and further to deduce the actual shapes of integral membrane proteins from their freezefracture images.

A Novel Reconstitution Method for Inducing the Formation of Regular 2-Dimensional Arrays of Membrane Proteins and Lipids

1988 ◽  
Vol 46 ◽  
pp. 152-153 ◽  
Author(s):  
A. Engel ◽  
A. Holzenburg ◽  
K. Stauffer ◽  
J. Rosenbusch ◽  
U. Aebi
Keyword(s):  
Membrane Proteins ◽  
Flow Rate ◽  
Lipid Membranes ◽  
Functional Characterization ◽  
Dimensional Structure ◽  
Electron Crystallography ◽  
Peltier Element ◽  
Protein Ratio ◽  
Precise Control ◽  
3 Dimensional

Reconstitution of solubilized and purified membrane proteins in the presence of phospholipids into vesicles allows their functions to be studied by simple bulk measurements (e.g. diffusion of differently sized solutes) or by conductance measurements after transformation into planar membranes. On the other hand, reconstitution into regular protein-lipid arrays, usually forming at a specific lipid-to-protein ratio, provides the basis for determining the 3-dimensional structure of membrane proteins employing the tools of electron crystallography.To refine reconstitution conditions for reproducibly inducing formation of large and highly ordered protein-lipid membranes that are suitable for both electron crystallography and patch clamping experiments aimed at their functional characterization, we built a flow-dialysis device that allows precise control of temperature and flow-rate (Fig. 1). The flow rate is generated by a peristaltic pump and can be adjusted from 1 to 500 ml/h. The dialysis buffer is brought to a preselected temperature during its travel through a meandering path before it enters the dialysis reservoir. A Z-80 based computer controls a Peltier element allowing the temperature profile to be programmed as function of time.

The structure of membranes and membrane proteins embedded in amorphous ice

1992 ◽  
Vol 50 (1) ◽  
pp. 432-433
Author(s):  
Uwe Lücken ◽  
Joachim Jäger
Keyword(s):  
Phase Transition ◽  
Transition Temperature ◽  
Membrane Proteins ◽  
Phase Transition Temperature ◽  
Lipid Vesicles ◽  
Freezing Stress ◽  
Amorphous Ice ◽  
Different Temperatures ◽  
Band Pass ◽  
Lipid Polymorphism

TEM imaging of frozen-hydrated lipid vesicles has been done by several groups Thermotrophic and lyotrophic polymorphism has been reported. By using image processing, computer simulation and tilt experiments, we tried to learn about the influence of freezing-stress and defocus artifacts on the lipid polymorphism and fine structure of the bilayer profile. We show integrated membrane proteins do modulate the bilayer structure and the morphology of the vesicles.Phase transitions of DMPC vesicles were visualized after freezing under equilibrium conditions at different temperatures in a controlled-environment vitrification system. Below the main phase transition temperature of 24°C (Fig. 1), vesicles show a facetted appearance due to the quasicrystalline areas. A gradual increase in temperature leads to melting processes with different morphology in the bilayer profile. Far above the phase transition temperature the bilayer profile is still present. In the band-pass-filtered images (Fig. 2) no significant change in the width of the bilayer profile is visible.

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