scholarly journals Functional solubilisation of the β2-adrenoceptor (β2AR) using Diisobutylene maleic acid (DIBMA)

2020 ◽  
Author(s):  
C. R. Harwood ◽  
D. A. Sykes ◽  
B. Hoare ◽  
F. M. Heydenreich ◽  
R. Uddin ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Membrane Proteins ◽  
Maleic Acid ◽  
Amphiphilic Copolymers ◽  
Promising Strategy ◽  
Biophysical Studies ◽  
Lipid Particle ◽  
Unique Method ◽  
Lipid Environment

AbstractThe β2-adrenoceptor (β2AR) is a well-established target in asthma and a prototypical GPCR for biophysical studies. Solubilisation of membrane proteins has classically involved the use of detergents. However, the detergent environment differs from the native membrane environment and often destabilises membrane proteins. Use of amphiphilic copolymers is a promising strategy to solubilise membrane proteins within their native lipid environment in the complete absence of detergents. Here we show the isolation of the β2AR in the polymer Diisobutylene maleic acid (DIBMA). We demonstrate that β2AR remains functional in the DIBMA lipid particle (DIBMALP) and shows improved thermal stability compared to the n-Dodecyl-β-D-Maltopyranoside (DDM) detergent solubilised β2AR. This unique method of extracting β2AR offers significant advantages over previous methods routinely employed such as the introduction of thermostabilising mutations and the use of detergents, particularly for functional biophysical studies.

scholarly journals Diisobutylene Maleic Acid Copolymer (DIBMA) Lipid Particle: A “Stealth” Membrane Mimetic for Neutron Scattering

2020 ◽  
Author(s):  
Rong Guo ◽  
Jacob Sumner ◽  
Shuo Qian
Keyword(s):  
Membrane Proteins ◽  
Neutron Scattering ◽  
Maleic Acid ◽  
Contrast Variation ◽  
Acid Copolymer ◽  
Membrane Mimetic ◽  
Lipid Particles ◽  
Lipid Particle ◽  
Contrast Matching ◽  
Maleic Acid Copolymer

Diisobutylene maleic acid (DIBMA) has been shown to solubilize and purify membrane proteins from a native lipid bilayer into nanodiscs without the need for a detergent. To explore DIBMA lipid particles as a suitable membrane mimetic system for neutron scattering studies of membrane proteins, we measured and determined the contrast matching point of DIBMA to be ~12% (v/v) D2O—similar to that of most protiated lipid molecules, but distinct from that of regular protiated proteins, providing a natural contrast for separating neutron scattering signals. Using SANS contrast variation, we demonstrated that the scattering from the whole lipid particle can be annihilated. Further, the lipid part of the particle shows a well-defined discoidal shape with DIBMA contrast matched. These results demonstrate that the DIBMA lipid particle is an outstanding “stealth” membrane mimetic for membrane proteins.<br>

scholarly journals Diisobutylene Maleic Acid Copolymer (DIBMA) Lipid Particle: A “Stealth” Membrane Mimetic for Neutron Scattering

2020 ◽  
Author(s):  
Rong Guo ◽  
Jacob Sumner ◽  
Shuo Qian
Keyword(s):  
Membrane Proteins ◽  
Neutron Scattering ◽  
Maleic Acid ◽  
Contrast Variation ◽  
Acid Copolymer ◽  
Membrane Mimetic ◽  
Lipid Particles ◽  
Lipid Particle ◽  
Contrast Matching ◽  
Maleic Acid Copolymer

Diisobutylene maleic acid (DIBMA) has been shown to solubilize and purify membrane proteins from a native lipid bilayer into nanodiscs without the need for a detergent. To explore DIBMA lipid particles as a suitable membrane mimetic system for neutron scattering studies of membrane proteins, we measured and determined the contrast matching point of DIBMA to be ~12% (v/v) D2O—similar to that of most protiated lipid molecules, but distinct from that of regular protiated proteins, providing a natural contrast for separating neutron scattering signals. Using SANS contrast variation, we demonstrated that the scattering from the whole lipid particle can be annihilated. Further, the lipid part of the particle shows a well-defined discoidal shape with DIBMA contrast matched. These results demonstrate that the DIBMA lipid particle is an outstanding “stealth” membrane mimetic for membrane proteins.<br>

Thermal gravimetric analysis of in-situ crosslinked nanocellulose whiskers • poly(methyl vinyl ether-co-maleic acid) • polyethylene glycol

TAPPI Journal ◽  
2011 ◽  
Vol 10 (4) ◽  
pp. 29-33
Author(s):  
LEE A. GOETZ ◽  
AJI P. MATHEW ◽  
KRISTIINA OKSMAN ◽  
ARTHUR J. RAGAUSKAS
Keyword(s):  
Thermal Stability ◽  
Polyethylene Glycol ◽  
Vinyl Ether ◽  
Thermal Gravimetric Analysis ◽  
Maleic Acid ◽  
Gravimetric Analysis ◽  
Thermal Gravimetric ◽  
Methyl Vinyl ◽  
Methyl Vinyl Ether

The thermal stability and decomposition of in-situ crosslinked nanocellulose whiskers – poly(methyl vinyl ether-co-maleic acid) – polyethylene glycol formulations (PMVEMA-PEG), (25%, 50%, and 75% whiskers) – were investigated using thermal gravimetric analysis (TGA) methods. The thermal degradation behavior of the films varied according to the percent cellulose whiskers in each formulation. The presence of cellulose whiskers increased the thermal stability of the PMVEMA-PEG matrix.

scholarly journals Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an E. coli Based Cell-Free Protein Synthesis System

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1325 ◽  
Author(s):  
Ke Yue ◽  
Tran Nam Trung ◽  
Yiyong Zhu ◽  
Ralf Kaldenhoff ◽  
Lei Kai
Keyword(s):  
Functional Analysis ◽  
Membrane Proteins ◽  
Fluorescent Protein ◽  
Water Channel ◽  
New Approach ◽  
E Coli ◽  
Straightforward Method ◽  
Lipid Environment ◽  
Green Fluorescent ◽  
Eukaryotic Organisms

Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from Escherichia coli and Nicotiana tabacum using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and NtPIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.

Evaluation of commercially available styrene-co-maleic acid polymers for the extraction of membrane proteins from spinach chloroplast thylakoids

2019 ◽  
Vol 114 ◽  
pp. 485-500 ◽  
Author(s):  
Olena Korotych ◽  
Jyotirmoy Mondal ◽  
Kerim M. Gattás-Asfura ◽  
Jessica Hendricks ◽  
Barry D. Bruce
Keyword(s):  
Membrane Proteins ◽  
Maleic Acid ◽  
Spinach Chloroplast

scholarly journals Lipid-protein interactions are unique fingerprints for membrane proteins

2017 ◽  
Author(s):  
Valentina Corradi ◽  
Eduardo Mendez-Villuendas ◽  
Helgi I. Ingólfsson ◽  
Ruo-Xu Gu ◽  
Iwona Siuda ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Protein Interactions ◽  
Cell Membranes ◽  
Spatiotemporal Resolution ◽  
Lipid Dynamics ◽  
Lipid Species ◽  
Lipid Environment ◽  
Lipid Protein Interactions ◽  
Dynamics Simulations ◽  
Asymmetrically Distributed

ABSTRACTCell membranes contain hundreds of different proteins and lipids in an asymmetric arrangement. Understanding the lateral organization principles of these complex mixtures is essential for life and health. However, our current understanding of the detailed organization of cell membranes remains rather elusive, owing to the lack of experimental methods suitable for studying these fluctuating nanoscale assemblies of lipids and proteins with the required spatiotemporal resolution. Here, we use molecular dynamics simulations to characterize the lipid environment of ten membrane proteins. To provide a realistic lipid environment, the proteins are embedded in a model plasma membrane, where more than 60 lipid species are represented, asymmetrically distributed between leaflets. The simulations detail how each protein modulates its local lipid environment through local lipid composition, thickness, curvature and lipid dynamics. Our results provide a molecular glimpse of the complexity of lipid-protein interactions, with potentially far reaching implications for the overall organization of the cell membrane.

scholarly journals Stability and flexibility of marginally hydrophobic–segment stalling at the endoplasmic reticulum translocon

2016 ◽  
Vol 27 (6) ◽  
pp. 930-940 ◽  
Author(s):  
Yuichiro Kida ◽  
Yudai Ishihara ◽  
Hidenobu Fujita ◽  
Yukiko Onishi ◽  
Masao Sakaguchi
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Lipid Phase ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Transmembrane Segment ◽  
Conducting Channel ◽  
Transmembrane Segments ◽  
Lipid Environment ◽  
Sec61 Channel

Many membrane proteins are integrated into the endoplasmic reticulum membrane through the protein-conducting channel, the translocon. Transmembrane segments with insufficient hydrophobicity for membrane integration are frequently found in multispanning membrane proteins, and such marginally hydrophobic (mH) segments should be accommodated, at least transiently, at the membrane. Here we investigated how mH-segments stall at the membrane and their stability. Our findings show that mH-segments can be retained at the membrane without moving into the lipid phase and that such segments flank Sec61α, the core channel of the translocon, in the translational intermediate state. The mH-segments are gradually transferred from the Sec61 channel to the lipid environment in a hydrophobicity-dependent manner, and this lateral movement may be affected by the ribosome. In addition, stalling mH-segments allow for insertion of the following transmembrane segment, forming an Ncytosol/Clumen orientation, suggesting that mH-segments can move laterally to accommodate the next transmembrane segment. These findings suggest that mH-segments may be accommodated at the ER membrane with lateral fluctuation between the Sec61 channel and the lipid phase.

Cryo-Electron Microscopy: Moving Beyond X-Ray Crystal Structures for Drug Receptors and Drug Development

2020 ◽  
Vol 60 (1) ◽  
pp. 51-71 ◽  
Author(s):  
Javier García-Nafría ◽  
Christopher G. Tate
Keyword(s):  
Membrane Proteins ◽  
G Protein ◽  
Rational Design ◽  
Protein Complexes ◽  
Host Cells ◽  
X Ray ◽  
X Ray Crystallography ◽  
Receptor Modulation ◽  
Lipid Environment ◽  
Drug Receptors

Electron cryo-microscopy (cryo-EM) has revolutionized structure determination of membrane proteins and holds great potential for structure-based drug discovery. Here we discuss the potential of cryo-EM in the rational design of therapeutics for membrane proteins compared to X-ray crystallography. We also detail recent progress in the field of drug receptors, focusing on cryo-EM of two protein families with established therapeutic value, the γ-aminobutyric acid A receptors (GABAARs) and G protein–coupled receptors (GPCRs). GABAARs are pentameric ion channels, and cryo-EM structures of physiological heteromeric receptors in a lipid environment have uncovered the molecular basis of receptor modulation by drugs such as diazepam. The structures of ten GPCR–G protein complexes from three different classes of GPCRs have now been determined by cryo-EM. These structures give detailed insights into molecular interactions with drugs, GPCR–G protein selectivity, how accessory membrane proteins alter receptor–ligand pharmacology, and the mechanism by which HIV uses GPCRs to enter host cells.

Membrane proteins: is the future disc shaped?

2016 ◽  
Vol 44 (4) ◽  
pp. 1011-1018 ◽  
Author(s):  
Sarah C. Lee ◽  
Naomi L. Pollock
Keyword(s):  
Membrane Proteins ◽  
Maleic Acid ◽  
Membrane Lipids ◽  
Biological Membranes ◽  
Structure And Function ◽  
Amphiphilic Copolymer ◽  
New Approach ◽  
Analysis Techniques ◽  
Lipid Particles ◽  
And Function

The use of styrene maleic acid lipid particles (SMALPs) for the purification of membrane proteins (MPs) is a rapidly developing technology. The amphiphilic copolymer of styrene and maleic acid (SMA) disrupts biological membranes and can extract membrane proteins in nanodiscs of approximately 10 nm diameter. These discs contain SMA, protein and membrane lipids. There is evidence that MPs in SMALPs retain their native structures and functions, in some cases with enhanced thermal stability. In addition, the method is compatible with biological buffers and a wide variety of biophysical and structural analysis techniques. The use of SMALPs to solubilize and stabilize MPs offers a new approach in our attempts to understand, and influence, the structure and function of MPs and biological membranes. In this review, we critically assess progress with this method, address some of the associated technical challenges, and discuss opportunities for exploiting SMA and SMALPs to expand our understanding of MP biology.

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