scholarly journals Fluorescence Approaches for Characterizing Ion Channels in Synthetic Bilayers

Membranes ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 857
Author(s):  
Md. Sirajul Islam ◽  
James P. Gaston ◽  
Matthew A. B. Baker
Keyword(s):  
Ion Channels ◽  
Membrane Proteins ◽  
Lipid Bilayers ◽  
Lipid Membranes ◽  
Protein Composition ◽  
Cellular Processes ◽  
Model Lipid Membranes ◽  
Wide Range ◽  
Lipid Environment

Ion channels are membrane proteins that play important roles in a wide range of fundamental cellular processes. Studying membrane proteins at a molecular level becomes challenging in complex cellular environments. Instead, many studies focus on the isolation and reconstitution of the membrane proteins into model lipid membranes. Such simpler, in vitro, systems offer the advantage of control over the membrane and protein composition and the lipid environment. Rhodopsin and rhodopsin-like ion channels are widely studied due to their light-interacting properties and are a natural candidate for investigation with fluorescence methods. Here we review techniques for synthesizing liposomes and for reconstituting membrane proteins into lipid bilayers. We then summarize fluorescence assays which can be used to verify the functionality of reconstituted membrane proteins in synthetic liposomes.

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 Investigating the structural properties of hydrophobic solvent-rich lipid bilayers.

Soft Matter ◽  
2021 ◽  
Author(s):  
Valeria Zoni ◽  
Pablo Campomanes ◽  
Stefano Vanni
Keyword(s):  
Structural Properties ◽  
Lipid Bilayers ◽  
Lipid Membranes ◽  
Cellular Processes ◽  
Indispensable Tool ◽  
Hydrophobic Solvent

In vitro reconstitutions of lipid membranes have proven to be an indispensable tool to rationalize the complexity of lipid membranes and to understand their role in countless cellular processes. However,...

scholarly journals Fake It ‘Till You Make It—The Pursuit of Suitable Membrane Mimetics for Membrane Protein Biophysics

2020 ◽  
Vol 22 (1) ◽  
pp. 50
Author(s):  
Johannes Thoma ◽  
Björn M. Burmann
Keyword(s):  
Membrane Proteins ◽  
Lipid Bilayers ◽  
Lipid Membranes ◽  
Cubic Phase ◽  
Advantages And Disadvantages ◽  
Membrane Mimetic ◽  
Lipid Environment ◽  
Protein Biophysics ◽  
And Function ◽  
Biological Methods

Membrane proteins evolved to reside in the hydrophobic lipid bilayers of cellular membranes. Therefore, membrane proteins bridge the different aqueous compartments separated by the membrane, and furthermore, dynamically interact with their surrounding lipid environment. The latter not only stabilizes membrane proteins, but directly impacts their folding, structure and function. In order to be characterized with biophysical and structural biological methods, membrane proteins are typically extracted and subsequently purified from their native lipid environment. This approach requires that lipid membranes are replaced by suitable surrogates, which ideally closely mimic the native bilayer, in order to maintain the membrane proteins structural and functional integrity. In this review, we survey the currently available membrane mimetic environments ranging from detergent micelles to bicelles, nanodiscs, lipidic-cubic phase (LCP), liposomes, and polymersomes. We discuss their respective advantages and disadvantages as well as their suitability for downstream biophysical and structural characterization. Finally, we take a look at ongoing methodological developments, which aim for direct in-situ characterization of membrane proteins within native membranes instead of relying on membrane mimetics.

scholarly journals Membrane proteins: always an insoluble problem?

2016 ◽  
Vol 44 (3) ◽  
pp. 790-795 ◽  
Author(s):  
Andrea E. Rawlings
Keyword(s):  
Membrane Proteins ◽  
Lipid Membranes ◽  
Drug Targets ◽  
Functional Characterization ◽  
Water Soluble ◽  
High Yield ◽  
Cellular Processes ◽  
Native Sequence ◽  
Protein Research ◽  
New Methodologies

Membrane proteins play crucial roles in cellular processes and are often important pharmacological drug targets. The hydrophobic properties of these proteins make full structural and functional characterization challenging because of the need to use detergents or other solubilizing agents when extracting them from their native lipid membranes. To aid membrane protein research, new methodologies are required to allow these proteins to be expressed and purified cheaply, easily, in high yield and to provide water soluble proteins for subsequent study. This mini review focuses on the relatively new area of water soluble membrane proteins and in particular two innovative approaches: the redesign of membrane proteins to yield water soluble variants and how adding solubilizing fusion proteins can help to overcome these challenges. This review also looks at naturally occurring membrane proteins, which are able to exist as stable, functional, water soluble assemblies with no alteration to their native sequence.

scholarly journals The Fusion of Lipid and DNA Nanotechnology

Genes ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 1001 ◽  
Author(s):  
Es Darley ◽  
Jasleen Kaur Daljit Singh ◽  
Natalie A. Surace ◽  
Shelley F. J. Wickham ◽  
Matthew A. B. Baker
Keyword(s):  
Membrane Proteins ◽  
Lipid Membranes ◽  
Dna Nanotechnology ◽  
Bilayer Membrane ◽  
Diverse Range ◽  
Impermeable Barrier ◽  
Self Assembling ◽  
Associated Proteins ◽  
Fundamental Biological Process

Lipid membranes form the boundary of many biological compartments, including organelles and cells. Consisting of two leaflets of amphipathic molecules, the bilayer membrane forms an impermeable barrier to ions and small molecules. Controlled transport of molecules across lipid membranes is a fundamental biological process that is facilitated by a diverse range of membrane proteins, including ion-channels and pores. However, biological membranes and their associated proteins are challenging to experimentally characterize. These challenges have motivated recent advances in nanotechnology towards building and manipulating synthetic lipid systems. Liposomes—aqueous droplets enclosed by a bilayer membrane—can be synthesised in vitro and used as a synthetic model for the cell membrane. In DNA nanotechnology, DNA is used as programmable building material for self-assembling biocompatible nanostructures. DNA nanostructures can be functionalised with hydrophobic chemical modifications, which bind to or bridge lipid membranes. Here, we review approaches that combine techniques from lipid and DNA nanotechnology to engineer the topography, permeability, and surface interactions of membranes, and to direct the fusion and formation of liposomes. These approaches have been used to study the properties of membrane proteins, to build biosensors, and as a pathway towards assembling synthetic multicellular systems.

scholarly journals RNA G-Quadruplex Structures Mediate Gene Regulation in Bacteria

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiaolong Shao ◽  
Weitong Zhang ◽  
Mubarak Ishaq Umar ◽  
Hei Yuen Wong ◽  
Zijing Seng ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Cell Envelope ◽  
Bacterial Species ◽  
Virulence Gene ◽  
Content Type ◽  
Cellular Processes ◽  
Wide Range ◽  
G Quadruplex ◽  
Eukaryotic Organisms

ABSTRACT Guanine (G)-rich sequences in RNA can fold into diverse RNA G-quadruplex (rG4) structures to mediate various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4s in prokaryotes are still elusive. We used QUMA-1, an rG4-specific fluorescent probe, to detect rG4 structures in a wide range of bacterial species both in vitro and in live cells and found rG4 to be an abundant RNA secondary structure across those species. Subsequently, to identify bacterial rG4 sites in the transcriptome, the model Escherichia coli strain and a major human pathogen, Pseudomonas aeruginosa, were subjected to recently developed high-throughput rG4 structure sequencing (rG4-seq). In total, 168 and 161 in vitro rG4 sites were found in E. coli and P. aeruginosa, respectively. Genes carrying these rG4 sites were found to be involved in virulence, gene regulation, cell envelope synthesis, and metabolism. More importantly, biophysical assays revealed the formation of a group of rG4 sites in mRNAs (such as hemL and bswR), and they were functionally validated in cells by genetic (point mutation and lux reporter assays) and phenotypic experiments, providing substantial evidence for the formation and function of rG4s in bacteria. Overall, our study uncovers important regulatory functions of rG4s in bacterial pathogenicity and metabolic pathways and strongly suggests that rG4s exist and can be detected in a wide range of bacterial species. IMPORTANCE G-quadruplex in RNA (rG4) mediates various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4 are still elusive in prokaryotes. Here, we found that rG4 is an abundant RNA secondary structure across a wide range of bacterial species. Subsequently, the transcriptome-wide rG4 structure sequencing (rG4-seq) revealed that the model E. coli strain and a major human pathogen, P. aeruginosa, have 168 and 161 in vitro rG4 sites, respectively, involved in virulence, gene regulation, cell envelope, and metabolism. We further verified the regulatory functions of two rG4 sites in bacteria (hemL and bswR). Overall, this finding strongly suggests that rG4s play key regulatory roles in a wide range of bacterial species.

scholarly journals Sensing through Non-Sensing Ocular Ion Channels

2020 ◽  
Vol 21 (18) ◽  
pp. 6925
Author(s):  
Meha Kabra ◽  
Bikash Ranjan Pattnaik
Keyword(s):  
Ion Channels ◽  
Visual Processing ◽  
Signal Transmission ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cone Dystrophy ◽  
Wide Range ◽  
The Impact

Ion channels are membrane-spanning integral proteins expressed in multiple organs, including the eye. In the eye, ion channels are involved in various physiological processes, like signal transmission and visual processing. A wide range of mutations have been reported in the corresponding genes and their interacting subunit coding genes, which contribute significantly to an array of blindness, termed ocular channelopathies. These mutations result in either a loss- or gain-of channel functions affecting the structure, assembly, trafficking, and localization of channel proteins. A dominant-negative effect is caused in a few channels formed by the assembly of several subunits that exist as homo- or heteromeric proteins. Here, we review the role of different mutations in switching a “sensing” ion channel to “non-sensing,” leading to ocular channelopathies like Leber’s congenital amaurosis 16 (LCA16), cone dystrophy, congenital stationary night blindness (CSNB), achromatopsia, bestrophinopathies, retinitis pigmentosa, etc. We also discuss the various in vitro and in vivo disease models available to investigate the impact of mutations on channel properties, to dissect the disease mechanism, and understand the pathophysiology. Innovating the potential pharmacological and therapeutic approaches and their efficient delivery to the eye for reversing a “non-sensing” channel to “sensing” would be life-changing.

scholarly journals Lanthionine synthetase C–like protein 2 (LanCL2) is a novel regulator of Akt

2014 ◽  
Vol 25 (24) ◽  
pp. 3954-3961 ◽  
Author(s):  
Min Zeng ◽  
Wilfred A. van der Donk ◽  
Jie Chen
Keyword(s):  
Insulin Receptor ◽  
Insulin Receptor Substrate ◽  
Akt Phosphorylation ◽  
Akt Activation ◽  
Cellular Processes ◽  
Human Liver Cells ◽  
Wide Range ◽  
Physical Interactions ◽  
Protein Kinase Akt

The serine/threonine protein kinase Akt controls a wide range of biochemical and cellular processes under the modulation of a variety of regulators. In this study, we identify the lanthionine synthetase C–like 2 (LanCL2) protein as a positive regulator of Akt activation in human liver cells. LanCL2 knockdown dampens serum- and insulin-stimulated Akt phosphorylation, whereas LanCL2 overexpression enhances these processes. Neither insulin receptor phosphorylation nor the interaction between insulin receptor substrate and phosphatidylinositide 3-kinase (PI3K) is affected by LanCL2 knockdown. LanCL2 also does not function through PP2A, a phosphatase of Akt. Instead, LanCL2 directly interacts with Akt, with a preference for inactive Akt. Moreover, we show that LanCL2 also binds to the Akt kinase mTORC2, but not phosphoinositide-dependent kinase 1. Whereas LanCL2 is not required for the Akt-mTORC2 interaction, recombinant LanCL2 enhances Akt phosphorylation by target of rapamycin complex 2 (mTORC2) in vitro. Finally, consistent with a function of Akt in regulating cell survival, LanCL2 knockdown increases the rate of apoptosis, which is reversed by the expression of a constitutively active Akt. Taken together, our findings reveal LanCL2 as a novel regulator of Akt and suggest that LanCL2 facilitates optimal phosphorylation of Akt by mTORC2 via direct physical interactions with both the kinase and the substrate.

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.

scholarly journals Imaging analysis to quantitate the Interplay of membrane and cytoplasm protein dynamics

2021 ◽  
Author(s):  
N Kislev ◽  
M Egozi ◽  
D Benayahu
Keyword(s):  
Membrane Proteins ◽  
Protein Expression ◽  
Glucose Transporter ◽  
Imaging Method ◽  
Protein Distribution ◽  
Cellular Functions ◽  
Membrane Expression ◽  
Cellular Processes ◽  
Imaging Tool

AbstractPlasma membrane proteins are extremely important in cell signaling and cellular functions. Protein expression and localization alter in response to various signals in a way that is dependent on cell type and niche. Compartmental quantification of the expression of particular proteins is a very useful means of understanding their role in cellular processes. Immunofluorescence staining is frequently used to investigate the distribution of proteins of interest. Here, we present an imaging method for quantifying the membrane to cytoplasm ratio (MCR) of proteins analyzed at single-cell resolution. This technique provides a robust quantification of membrane proteins and contributes new insights into membrane expression dynamics. We have developed a protocol that uses immunostaining to assess protein expression according to the fluorescent cellular distribution and to compute the MCR. The method was applied to measure the MCR of glucose transporter 4 (GLUT4) in response to insulin in 3T3-L1 cells, an in-vitro model for adipocyte function and adipogenesis. The results revealed informative changes in the subcellular localization of GLUT4 following insulin induction. MCR analysis is a powerful imaging tool that can be generally applied to membrane proteins to provide a rapid and efficient quantitative analysis of protein distribution and sub-cellular processes in cells.

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