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Special Issue "Membrane Proteins: Structure, Function and Motion"

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: closed (31 May 2022) | Viewed by 35270

Special Issue Editor

Prof. Dr. Masoud Jelokhani-Niaraki
E-Mail Website
Guest Editor
Department of Chemistry & Biochemistry, Wilfrid Laurier University, Waterloo, ON, Canada
Interests: biophysical chemistry of membrane proteins and membrane interacting peptides; peptide and protein ion transport; antimicrobial peptides
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Membrane proteins are present in all cells and are essential for their homeostasis and response to environmental changes. Membrane proteins are involved in a vast variety of dynamic cellular processes such as ionic and molecular transport, electron transport, signal transduction, enzymatic reactions, and intercellular communication. Despite their relative abundance and important roles in life processes, there is considerably less structural and functional information about membrane proteins in comparison to other types of proteins. The first high-resolution structure for a membrane protein was published in 1985, and, currently, more than1000 membrane protein structures (vs. more than 150,000 structures of other protein classes) have been determined. The main two challenges in determining membrane protein structures is in optimizing experimental conditions for their extraction/purification from cell membranes and their reconstitution in cell-like membranes. In their native environments, membrane proteins interact with both membrane lipids and other (membrane) proteins. This close interaction influences their biological function, which is not easily reproducible under in vitro conditions. In addition to technical problems in isolation and purification of membrane proteins in their native conformations, there are many questions about the molecular behavior of membrane proteins that are not yet answered or are only partially answered. Some of these problems include the specific and non-specific interactions of membrane proteins with lipids and other proteins, dynamic conformational changes and oligomerization of membrane proteins, membrane protein folding, modes of action of infectious structures (such as viruses) with membrane surface and membrane proteins, and the action of membrane proteins in their complex in vivo environment. As a result of these challenging problems, membrane protein studies have become a flourishing research field in molecular biophysics. Visualizing the dynamic nature of membrane proteins and their interconnections in the cell is a key element in understanding the complex, yet efficient, molecular machinery of life.

Dr. Masoud Jelokhani-Niaraki
Guest Editor

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Keywords

  • membrane protein isolation and purification
  • membrane protein structure
  • dynamic conformation
  • membrane protein folding
  • protein–lipid and protein–protein interactions
  • membrane protein oligomerization
  • membrane transport
  • signal transduction and cellular communication
  • interaction with infectious agents
  • membrane proteins in vivo

Published Papers (25 papers)

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Communication
The Small Heat Shock Protein, HSPB1, Interacts with and Modulates the Physical Structure of Membranes
Int. J. Mol. Sci. 2022, 23(13), 7317; https://doi.org/10.3390/ijms23137317 - 30 Jun 2022
Viewed by 551
Abstract
Small heat shock proteins (sHSPs) have been demonstrated to interact with lipids and modulate the physical state of membranes across species. Through these interactions, sHSPs contribute to the maintenance of membrane integrity. HSPB1 is a major sHSP in mammals, but its lipid interaction [...] Read more.
Small heat shock proteins (sHSPs) have been demonstrated to interact with lipids and modulate the physical state of membranes across species. Through these interactions, sHSPs contribute to the maintenance of membrane integrity. HSPB1 is a major sHSP in mammals, but its lipid interaction profile has so far been unexplored. In this study, we characterized the interaction between HSPB1 and phospholipids. HSPB1 not only associated with membranes via membrane-forming lipids, but also showed a strong affinity towards highly fluid membranes. It participated in the modulation of the physical properties of the interacting membranes by altering rotational and lateral lipid mobility. In addition, the in vivo expression of HSPB1 greatly affected the phase behavior of the plasma membrane under membrane fluidizing stress conditions. In light of our current findings, we propose a new function for HSPB1 as a membrane chaperone. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Article
Hot Spot Mutagenesis Improves the Functional Expression of Unique Mammalian Odorant Receptors
Int. J. Mol. Sci. 2022, 23(1), 277; https://doi.org/10.3390/ijms23010277 - 28 Dec 2021
Viewed by 989
Abstract
Vertebrate animals detect odors through olfactory receptors (ORs), members of the G protein-coupled receptor (GPCR) family. Due to the difficulty in the heterologous expression of ORs, studies of their odor molecule recognition mechanisms have progressed poorly. Functional expression of most ORs in heterologous [...] Read more.
Vertebrate animals detect odors through olfactory receptors (ORs), members of the G protein-coupled receptor (GPCR) family. Due to the difficulty in the heterologous expression of ORs, studies of their odor molecule recognition mechanisms have progressed poorly. Functional expression of most ORs in heterologous cells requires the co-expression of their chaperone proteins, receptor transporting proteins (RTPs). Yet, some ORs were found to be functionally expressed without the support of RTP (RTP-independent ORs). In this study, we investigated whether amino acid residues highly conserved among RTP-independent ORs improve the functional expression of ORs in heterologous cells. We found that a single amino acid substitution at one of two sites (NBW3.39 and 3.43) in their conserved residues (E and L, respectively) significantly improved the functional expression of ORs in heterologous cells. E3.39 and L3.43 also enhanced the membrane expression of RTP-dependent ORs in the absence of RTP. These changes did not alter the odorant responsiveness of the tested ORs. Our results showed that specific sites within transmembrane domains regulate the membrane expression of some ORs. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Article
Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane
Int. J. Mol. Sci. 2021, 22(21), 11981; https://doi.org/10.3390/ijms222111981 - 05 Nov 2021
Viewed by 757
Abstract
Transmembrane receptor proteins are located in the plasma membranes of biological cells where they exert important functions. Archaerhodopsin (Arch) proteins belong to a class of transmembrane receptor proteins called photoreceptors that react to light. Although the light sensitivity of proteins has been intensely [...] Read more.
Transmembrane receptor proteins are located in the plasma membranes of biological cells where they exert important functions. Archaerhodopsin (Arch) proteins belong to a class of transmembrane receptor proteins called photoreceptors that react to light. Although the light sensitivity of proteins has been intensely investigated in recent decades, the electrophysiological properties of pore-forming Archaerhodopsin (Arch), as studied in vitro, have remained largely unknown. Here, we formed unsupported bilayers between two channels of a microfluidic chip which enabled the simultaneous optical and electrical assessment of the bilayer in real time. Using a cell-free expression system, we recombinantly produced a GFP (green fluorescent protein) labelled as a variant of Arch-3. The label enabled us to follow the synthesis of Arch-3 and its incorporation into the bilayer by fluorescence microscopy when excited by blue light. Applying a green laser for excitation, we studied the electrophysiological properties of Arch-3 in the bilayer. The current signal obtained during excitation revealed distinct steps upwards and downwards, which we interpreted as the opening or closing of Arch-3 pores. From these steps, we estimated the pore radius to be 0.3 nm. In the cell-free extract, proteins can be modified simply by changing the DNA. In the future, this will enable us to study the photoelectrical properties of modified transmembrane protein constructs with ease. Our work, thus, represents a first step in studying signaling cascades in conjunction with coupled receptor proteins. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Article
Loose Morphology and High Dynamism of OSER Structures Induced by the Membrane Domain of HMG-CoA Reductase
Int. J. Mol. Sci. 2021, 22(17), 9132; https://doi.org/10.3390/ijms22179132 - 24 Aug 2021
Cited by 1 | Viewed by 826
Abstract
The membrane domain of eukaryotic HMG-CoA reductase (HMGR) has the conserved capacity to induce endoplasmic reticulum (ER) proliferation and membrane association into Organized Smooth Endoplasmic Reticulum (OSER) structures. These formations develop in response to overexpression of particular proteins, but also occur naturally in [...] Read more.
The membrane domain of eukaryotic HMG-CoA reductase (HMGR) has the conserved capacity to induce endoplasmic reticulum (ER) proliferation and membrane association into Organized Smooth Endoplasmic Reticulum (OSER) structures. These formations develop in response to overexpression of particular proteins, but also occur naturally in cells of the three eukaryotic kingdoms. Here, we characterize OSER structures induced by the membrane domain of Arabidopsis HMGR (1S domain). Immunochemical confocal and electron microscopy studies demonstrate that the 1S:GFP chimera co-localizes with high levels of endogenous HMGR in several ER compartments, such as the ER network, the nuclear envelope, the outer and internal membranes of HMGR vesicles and the OSER structures, which we name ER-HMGR domains. After high-pressure freezing, ER-HMGR domains show typical crystalloid, whorled and lamellar ultrastructural patterns, but with wide heterogeneous luminal spaces, indicating that the native OSER is looser and more flexible than previously reported. The formation of ER-HMGR domains is reversible. OSER structures grow by incorporation of ER membranes on their periphery and progressive compaction to the inside. The ER-HMGR domains are highly dynamic in their formation versus their disassembly, their variable spherical-ovoid shape, their fluctuating borders and their rapid intracellular movement, indicating that they are not mere ER membrane aggregates, but active components of the eukaryotic cell. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Article
CD82 and Gangliosides Tune CD81 Membrane Behavior
Int. J. Mol. Sci. 2021, 22(16), 8459; https://doi.org/10.3390/ijms22168459 - 06 Aug 2021
Cited by 1 | Viewed by 644
Abstract
Tetraspanins are a family of transmembrane proteins that form a network of protein–protein interactions within the plasma membrane. Within this network, tetraspanin are thought to control the lateral segregation of their partners at the plasma membrane through mechanisms involving specific lipids. Here, we [...] Read more.
Tetraspanins are a family of transmembrane proteins that form a network of protein–protein interactions within the plasma membrane. Within this network, tetraspanin are thought to control the lateral segregation of their partners at the plasma membrane through mechanisms involving specific lipids. Here, we used a single molecule tracking approach to study the membrane behavior of tetraspanins in mammary epithelial cells and demonstrate that despite a common overall behavior, each tetraspanin (CD9, CD81 and CD82) has a specific signature in terms of dynamics. Furthermore, we demonstrated that tetraspanin dynamics on the cell surface are dependent on gangliosides. More specifically, we found that CD82 expression increases the dynamics of CD81 and alters its localization at the plasma membrane, this has no effect on the behavior of CD9. Our results provide new information on the ability of CD82 and gangliosides to differentially modulate the dynamics and organization of tetraspanins at the plasma membrane and highlight that its lipid and protein composition is involved in the dynamical architecture of the tetraspanin web. We predict that CD82 may act as a regulator of the lateral segregation of specific tetraspanins at the plasma membrane while gangliosides could play a crucial role in establishing tetraspanin-enriched areas. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Article
Direct Measurement of the Affinity between tBid and Bax in a Mitochondria-Like Membrane
Int. J. Mol. Sci. 2021, 22(15), 8240; https://doi.org/10.3390/ijms22158240 - 31 Jul 2021
Cited by 1 | Viewed by 753
Abstract
The execution step in apoptosis is the permeabilization of the outer mitochondrial membrane, controlled by Bcl-2 family proteins. The physical interactions between the different proteins in this family and their relative abundance literally determine the fate of the cells. These interactions, however, are [...] Read more.
The execution step in apoptosis is the permeabilization of the outer mitochondrial membrane, controlled by Bcl-2 family proteins. The physical interactions between the different proteins in this family and their relative abundance literally determine the fate of the cells. These interactions, however, are difficult to quantify, as they occur in a lipid membrane and involve proteins with multiple conformations and stoichiometries which can exist both in soluble and membrane. Here we focus on the interaction between two core Bcl-2 family members, the executor pore-forming protein Bax and the truncated form of the activator protein Bid (tBid), which we imaged at the single particle level in a mitochondria-like planar supported lipid bilayer. We inferred the conformation of the proteins from their mobility, and detected their transient interactions using a novel single particle cross-correlation analysis. We show that both tBid and Bax have at least two different conformations at the membrane, and that their affinity for one another increases by one order of magnitude (with a 2D-KD decreasing from ≃1.6μm2 to ≃0.1μm2) when they pass from their loosely membrane-associated to their transmembrane form. We conclude by proposing an updated molecular model for the activation of Bax by tBid. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Article
Angulin-1 (LSR) Affects Paracellular Water Transport, However Only in Tight Epithelial Cells
Int. J. Mol. Sci. 2021, 22(15), 7827; https://doi.org/10.3390/ijms22157827 - 22 Jul 2021
Cited by 1 | Viewed by 1139
Abstract
Water transport in epithelia occurs transcellularly (aquaporins) and paracellularly (claudin-2, claudin-15). Recently, we showed that downregulated tricellulin, a protein of the tricellular tight junction (tTJ, the site where three epithelial cells meet), increased transepithelial water flux. We now check the hypothesis that another [...] Read more.
Water transport in epithelia occurs transcellularly (aquaporins) and paracellularly (claudin-2, claudin-15). Recently, we showed that downregulated tricellulin, a protein of the tricellular tight junction (tTJ, the site where three epithelial cells meet), increased transepithelial water flux. We now check the hypothesis that another tTJ-associated protein, angulin-1 (alias lipolysis-stimulated lipoprotein receptor, LSR) is a direct negative actuator of tTJ water permeability depending on the tightness of the epithelium. For this, a tight and an intermediate-tight epithelial cell line, MDCK C7 and HT-29/B6, were stably transfected with CRISPR/Cas9 and single-guide RNA targeting angulin-1 and morphologically and functionally characterized. Water flux induced by an osmotic gradient using 4-kDa dextran caused water flux to increase in angulin-1 KO clones in MDCK C7 cells, but not in HT-29/B6 cells. In addition, we found that water permeability in HT-29/B6 cells was not modified after either angulin-1 knockout or tricellulin knockdown, which may be related to the presence of other pathways, which reduce the impact of the tTJ pathway. In conclusion, modulation of the tTJ by knockout or knockdown of tTJ proteins affects ion and macromolecule permeability in tight and intermediate-tight epithelial cell lines, while the transepithelial water permeability was affected only in tight cell lines. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Article
MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa
Int. J. Mol. Sci. 2021, 22(10), 5328; https://doi.org/10.3390/ijms22105328 - 18 May 2021
Cited by 2 | Viewed by 1786
Abstract
One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, [...] Read more.
One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Article
Transient Excursions to Membrane Core as Determinants of Influenza Virus Fusion Peptide Activity
Int. J. Mol. Sci. 2021, 22(10), 5301; https://doi.org/10.3390/ijms22105301 - 18 May 2021
Cited by 3 | Viewed by 796
Abstract
Fusion of viral and host cell membranes is a critical step in the life cycle of enveloped viruses. In the case of influenza virus, it is mediated by subunit 2 of hemagglutinin (HA) glycoprotein whose N-terminal fragments insert into the target membrane and [...] Read more.
Fusion of viral and host cell membranes is a critical step in the life cycle of enveloped viruses. In the case of influenza virus, it is mediated by subunit 2 of hemagglutinin (HA) glycoprotein whose N-terminal fragments insert into the target membrane and initiate lipid exchange. These isolated fragments, known as fusion peptides (HAfp), already possess own fusogenic activity towards liposomes. Although they have long been studied with the hope to uncover the details of HA-mediated fusion, their actual mechanism of action remains elusive. Here, we use extensive molecular dynamics simulations combined with experimental studies of three HAfp variants to fully characterize their free energy landscape and interaction with lipid bilayer. In addition to customary assumed peptides localization at lipid–water interface, we characterize membrane-spanning configurations, which turn out to be metastable for active HAfps and unstable for the fusion inactive W14A mutant. We show that, while the degree of membrane perturbation by surface peptide configurations is relatively low and does not show any mutation-related differences, the effect of deeply inserted configurations is significant and correlates with insertion depth of the N-terminal amino group which is the highest for the wild type HAfp. Finally, we demonstrate the feasibility of spontaneous peptide transition to intramembrane location and the critical role of strictly conserved tryptofan residue 14 in this process. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Article
NO Represses the Oxygenation of Arachidonoyl PE by 15LOX/PEBP1: Mechanism and Role in Ferroptosis
Int. J. Mol. Sci. 2021, 22(10), 5253; https://doi.org/10.3390/ijms22105253 - 17 May 2021
Cited by 5 | Viewed by 1468
Abstract
We recently discovered an anti-ferroptotic mechanism inherent to M1 macrophages whereby high levels of NO suppressed ferroptosis via inhibition of hydroperoxy-eicosatetraenoyl-phosphatidylethanolamine (HpETE-PE) production by 15-lipoxygenase (15LOX) complexed with PE-binding protein 1 (PEBP1). However, the mechanism of NO interference with 15LOX/PEBP1 activity [...] Read more.
We recently discovered an anti-ferroptotic mechanism inherent to M1 macrophages whereby high levels of NO suppressed ferroptosis via inhibition of hydroperoxy-eicosatetraenoyl-phosphatidylethanolamine (HpETE-PE) production by 15-lipoxygenase (15LOX) complexed with PE-binding protein 1 (PEBP1). However, the mechanism of NO interference with 15LOX/PEBP1 activity remained unclear. Here, we use a biochemical model of recombinant 15LOX-2 complexed with PEBP1, LC-MS redox lipidomics, and structure-based modeling and simulations to uncover the mechanism through which NO suppresses ETE-PE oxidation. Our study reveals that O2 and NO use the same entry pores and channels connecting to 15LOX-2 catalytic site, resulting in a competition for the catalytic site. We identified residues that direct O2 and NO to the catalytic site, as well as those stabilizing the esterified ETE-PE phospholipid tail. The functional significance of these residues is supported by in silico saturation mutagenesis. We detected nitrosylated PE species in a biochemical system consisting of 15LOX-2/PEBP1 and NO donor and in RAW264.7 M2 macrophages treated with ferroptosis-inducer RSL3 in the presence of NO, in further support of the ability of NO to diffuse to, and react at, the 15LOX-2 catalytic site. The results provide first insights into the molecular mechanism of repression of the ferroptotic Hp-ETE-PE production by NO. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Article
ANT1 Activation and Inhibition Patterns Support the Fatty Acid Cycling Mechanism for Proton Transport
Int. J. Mol. Sci. 2021, 22(5), 2490; https://doi.org/10.3390/ijms22052490 - 02 Mar 2021
Cited by 8 | Viewed by 1478
Abstract
Adenine nucleotide translocase (ANT) is a well-known mitochondrial exchanger of ATP against ADP. In contrast, few studies have shown that ANT also mediates proton transport across the inner mitochondrial membrane. The results of these studies are controversial and lead to different hypotheses about [...] Read more.
Adenine nucleotide translocase (ANT) is a well-known mitochondrial exchanger of ATP against ADP. In contrast, few studies have shown that ANT also mediates proton transport across the inner mitochondrial membrane. The results of these studies are controversial and lead to different hypotheses about molecular transport mechanisms. We hypothesized that the H+-transport mediated by ANT and uncoupling proteins (UCP) has a similar regulation pattern and can be explained by the fatty acid cycling concept. The reconstitution of purified recombinant ANT1 in the planar lipid bilayers allowed us to measure the membrane current after the direct application of transmembrane potential ΔΨ, which would correspond to the mitochondrial states III and IV. Experimental results reveal that ANT1 does not contribute to a basal proton leak. Instead, it mediates H+ transport only in the presence of long-chain fatty acids (FA), as already known for UCPs. It depends on FA chain length and saturation, implying that FA’s transport is confined to the lipid-protein interface. Purine nucleotides with the preference for ATP and ADP inhibited H+ transport. Specific inhibitors of ATP/ADP transport, carboxyatractyloside or bongkrekic acid, also decreased proton transport. The H+ turnover number was calculated based on ANT1 concentration determined by fluorescence correlation spectroscopy and is equal to 14.6 ± 2.5 s−1. Molecular dynamic simulations revealed a large positively charged area at the protein/lipid interface that might facilitate FA anion’s transport across the membrane. ANT’s dual function—ADP/ATP and H+ transport in the presence of FA—may be important for the regulation of mitochondrial membrane potential and thus for potential-dependent processes in mitochondria. Moreover, the expansion of proton-transport modulating drug targets to ANT1 may improve the therapy of obesity, cancer, steatosis, cardiovascular and neurodegenerative diseases. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Article
A Mechanistic Model of NMDA and AMPA Receptor-Mediated Synaptic Transmission in Individual Hippocampal CA3-CA1 Synapses: A Computational Multiscale Approach
Int. J. Mol. Sci. 2021, 22(4), 1536; https://doi.org/10.3390/ijms22041536 - 03 Feb 2021
Cited by 1 | Viewed by 1381
Abstract
Inside hippocampal circuits, neuroplasticity events that individual cells may undergo during synaptic transmissions occur in the form of Long-Term Potentiation (LTP) and Long-Term Depression (LTD). The high density of NMDA receptors expressed on the surface of the dendritic CA1 spines confers to hippocampal [...] Read more.
Inside hippocampal circuits, neuroplasticity events that individual cells may undergo during synaptic transmissions occur in the form of Long-Term Potentiation (LTP) and Long-Term Depression (LTD). The high density of NMDA receptors expressed on the surface of the dendritic CA1 spines confers to hippocampal CA3-CA1 synapses the ability to easily undergo NMDA-mediated LTP and LTD, which is essential for some forms of explicit learning in mammals. Providing a comprehensive kinetic model that can be used for running computer simulations of the synaptic transmission process is currently a major challenge. Here, we propose a compartmentalized kinetic model for CA3-CA1 synaptic transmission. Our major goal was to tune our model in order to predict the functional impact caused by disease associated variants of NMDA receptors related to severe cognitive impairment. Indeed, for variants Glu413Gly and Cys461Phe, our model predicts negative shifts in the glutamate affinity and changes in the kinetic behavior, consistent with experimental data. These results point to the predictive power of this multiscale viewpoint, which aims to integrate the quantitative kinetic description of large interaction networks typical of system biology approaches with a focus on the quality of a few, key, molecular interactions typical of structural biology ones. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Article
Molecular Dynamics Simulations of Mitochondrial Uncoupling Protein 2
Int. J. Mol. Sci. 2021, 22(3), 1214; https://doi.org/10.3390/ijms22031214 - 26 Jan 2021
Cited by 5 | Viewed by 1739
Abstract
Molecular dynamics (MD) simulations of uncoupling proteins (UCP), a class of transmembrane proteins relevant for proton transport across inner mitochondrial membranes, represent a complicated task due to the lack of available structural data. In this work, we use a combination of homology modelling [...] Read more.
Molecular dynamics (MD) simulations of uncoupling proteins (UCP), a class of transmembrane proteins relevant for proton transport across inner mitochondrial membranes, represent a complicated task due to the lack of available structural data. In this work, we use a combination of homology modelling and subsequent microsecond molecular dynamics simulations of UCP2 in the DOPC phospholipid bilayer, starting from the structure of the mitochondrial ATP/ADP carrier (ANT) as a template. We show that this protocol leads to a structure that is impermeable to water, in contrast to MD simulations of UCP2 structures based on the experimental NMR structure. We also show that ATP binding in the UCP2 cavity is tight in the homology modelled structure of UCP2 in agreement with experimental observations. Finally, we corroborate our results with conductance measurements in model membranes, which further suggest that the UCP2 structure modeled from ANT protein possesses additional key functional elements, such as a fatty acid-binding site at the R60 region of the protein, directly related to the proton transport mechanism across inner mitochondrial membranes. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Article
Tetraoctylammonium, a Long Chain Quaternary Ammonium Blocker, Promotes a Noncollapsed, Resting-Like Inactivated State in KcsA
Int. J. Mol. Sci. 2021, 22(2), 490; https://doi.org/10.3390/ijms22020490 - 06 Jan 2021
Cited by 2 | Viewed by 800
Abstract
Alkylammonium salts have been used extensively to study the structure and function of potassium channels. Here, we use the hydrophobic tetraoctylammonium (TOA+) to shed light on the structure of the inactivated state of KcsA, a tetrameric prokaryotic potassium channel that serves [...] Read more.
Alkylammonium salts have been used extensively to study the structure and function of potassium channels. Here, we use the hydrophobic tetraoctylammonium (TOA+) to shed light on the structure of the inactivated state of KcsA, a tetrameric prokaryotic potassium channel that serves as a model to its homologous eukaryotic counterparts. By the combined use of a thermal denaturation assay and the analysis of homo-Förster resonance energy transfer in a mutant channel containing a single tryptophan (W67) per subunit, we found that TOA+ binds the channel cavity with high affinity, either with the inner gate open or closed. Moreover, TOA+ bound at the cavity allosterically shifts the equilibrium of the channel’s selectivity filter conformation from conductive to an inactivated-like form. The inactivated TOA+–KcsA complex exhibits a loss in the affinity towards permeant K+ at pH 7.0, when the channel is in its closed state, but maintains the two sets of K+ binding sites and the W67–W67 intersubunit distances characteristic of the selectivity filter in the channel resting state. Thus, the TOA+–bound state differs clearly from the collapsed channel state described by X-ray crystallography and claimed to represent the inactivated form of KcsA. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Article
Effect of Amyloid-β Monomers on Lipid Membrane Mechanical Parameters–Potential Implications for Mechanically Driven Neurodegeneration in Alzheimer’s Disease
Int. J. Mol. Sci. 2021, 22(1), 18; https://doi.org/10.3390/ijms22010018 - 22 Dec 2020
Cited by 6 | Viewed by 1146
Abstract
Alzheimer’s disease (AD) is a neurodegenerative disease that results in memory loss and the impairment of cognitive skills. Several mechanisms of AD’s pathogenesis were proposed, such as the progressive accumulation of amyloid-β (Aβ) and τ pathology. Nevertheless, the exact neurodegenerative mechanism of the [...] Read more.
Alzheimer’s disease (AD) is a neurodegenerative disease that results in memory loss and the impairment of cognitive skills. Several mechanisms of AD’s pathogenesis were proposed, such as the progressive accumulation of amyloid-β (Aβ) and τ pathology. Nevertheless, the exact neurodegenerative mechanism of the Aβ remains complex and not fully understood. This paper proposes an alternative hypothesis of the mechanism based on maintaining the neuron membrane’s mechanical balance. The incorporation of Aβ decreases the lipid membrane’s elastic properties, which eventually leads to the impairment of membrane clustering, disruption of mechanical wave propagation, and change in gamma oscillations. The first two disrupt the neuron’s ability to function correctly while the last one decreases sensory encoding and perception enabling. To begin discussing this mechanical-balance hypothesis, we measured the effect of two selected peptides, Aβ-40 and Aβ-42, as well as their fluorescently labeled modification, on membrane mechanical properties. The decrease of bending rigidity, consistent for all investigated peptides, was observed using molecular dynamic studies and experimental flicker-noise techniques. Additionally, wave propagation was investigated with molecular dynamic studies in membranes with and without incorporated neurodegenerative peptides. A change in membrane behavior was observed in the membrane system with incorporated Aβ. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Article
Syk Inhibitors: New Computational Insights into Their Intraerythrocytic Action in Plasmodium falciparum Malaria
Int. J. Mol. Sci. 2020, 21(19), 7009; https://doi.org/10.3390/ijms21197009 - 23 Sep 2020
Cited by 3 | Viewed by 1023
Abstract
Resistance to antimalarial drugs has spread rapidly over the past few decades. The WHO recommends artemisinin-based combination therapies for the treatment of uncomplicated malaria, but unfortunately these approaches are losing their efficacy in large areas of Southeast Asia. In 2016, artemisinin resistance was [...] Read more.
Resistance to antimalarial drugs has spread rapidly over the past few decades. The WHO recommends artemisinin-based combination therapies for the treatment of uncomplicated malaria, but unfortunately these approaches are losing their efficacy in large areas of Southeast Asia. In 2016, artemisinin resistance was confirmed in 5 countries of the Greater Mekong subregion. We focused our study on Syk inhibitors as antimalarial drugs. The Syk protein is present in human erythrocytes, and the membrane of protein band 3 is its major target following activation by oxidant stress. Tyr phosphorylation of band 3 occurs during P. falciparum growth, leading to the release of microparticles containing hemicromes and structural weakening of the host cell membrane, simplifying merozoite reinfection. Syk inhibitors block these events by interacting with the Syk protein’s catalytic site. We performed in vitro proteomics and in silico studies and compared the results. In vitro studies were based on treatment of the parasite’s cellular cultures with different concentrations of Syk inhibitors, while proteomics studies were focused on the Tyr phosphorylation of band 3 by Syk protein with the same concentrations of drugs. In silico studies were based on different molecular modeling approaches in order to analyze and optimize the ligand–protein interactions and obtain the highest efficacy in vitro. In the presence of Syk inhibitors, we observed a marked decrease of band 3 Tyr phosphorylation according to the increase of the drug’s concentration. Our studies could be useful for the structural optimization of these compounds and for the design of novel Syk inhibitors in the future. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Review

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Review
The Tiny Companion Matters: The Important Role of Protons in Active Transports in Plants
Int. J. Mol. Sci. 2022, 23(5), 2824; https://doi.org/10.3390/ijms23052824 - 04 Mar 2022
Viewed by 663
Abstract
In plants, the translocation of molecules, such as ions, metabolites, and hormones, between different subcellular compartments or different cells is achieved by transmembrane transporters, which play important roles in growth, development, and adaptation to the environment. To facilitate transport in a specific direction, [...] Read more.
In plants, the translocation of molecules, such as ions, metabolites, and hormones, between different subcellular compartments or different cells is achieved by transmembrane transporters, which play important roles in growth, development, and adaptation to the environment. To facilitate transport in a specific direction, active transporters that can translocate their substrates against the concentration gradient are needed. Examples of major active transporters in plants include ATP-binding cassette (ABC) transporters, multidrug and toxic compound extrusion (MATE) transporters, monosaccharide transporters (MSTs), sucrose transporters (SUTs), and amino acid transporters. Transport via ABC transporters is driven by ATP. The electrochemical gradient across the membrane energizes these secondary transporters. The pH in each cell and subcellular compartment is tightly regulated and yet highly dynamic, especially when under stress. Here, the effects of cellular and subcellular pH on the activities of ABC transporters, MATE transporters, MSTs, SUTs, and amino acid transporters will be discussed to enhance our understanding of their mechanics. The relation of the altered transporter activities to various biological processes of plants will also be addressed. Although most molecular transport research has focused on the substrate, the role of protons, the tiny counterparts of the substrate, should also not be ignored. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Review
New Insights into the Chloroplast Outer Membrane Proteome and Associated Targeting Pathways
Int. J. Mol. Sci. 2022, 23(3), 1571; https://doi.org/10.3390/ijms23031571 - 29 Jan 2022
Viewed by 1362
Abstract
Plastids are a dynamic class of organelle in plant cells that arose from an ancient cyanobacterial endosymbiont. Over the course of evolution, most genes encoding plastid proteins were transferred to the nuclear genome. In parallel, eukaryotic cells evolved a series of targeting pathways [...] Read more.
Plastids are a dynamic class of organelle in plant cells that arose from an ancient cyanobacterial endosymbiont. Over the course of evolution, most genes encoding plastid proteins were transferred to the nuclear genome. In parallel, eukaryotic cells evolved a series of targeting pathways and complex proteinaceous machinery at the plastid surface to direct these proteins back to their target organelle. Chloroplasts are the most well-characterized plastids, responsible for photosynthesis and other important metabolic functions. The biogenesis and function of chloroplasts rely heavily on the fidelity of intracellular protein trafficking pathways. Therefore, understanding these pathways and their regulation is essential. Furthermore, the chloroplast outer membrane proteome remains relatively uncharted territory in our understanding of protein targeting. Many key players in the cytosol, receptors at the organelle surface, and insertases that facilitate insertion into the chloroplast outer membrane remain elusive for this group of proteins. In this review, we summarize recent advances in the understanding of well-characterized chloroplast outer membrane protein targeting pathways as well as provide new insights into novel targeting signals and pathways more recently identified using a bioinformatic approach. As a result of our analyses, we expand the known number of chloroplast outer membrane proteins from 117 to 138. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Review
Uncoupling Proteins and Regulated Proton Leak in Mitochondria
Int. J. Mol. Sci. 2022, 23(3), 1528; https://doi.org/10.3390/ijms23031528 - 28 Jan 2022
Cited by 2 | Viewed by 925
Abstract
Higher concentration of protons in the mitochondrial intermembrane space compared to the matrix results in an electrochemical potential causing the back flux of protons to the matrix. This proton transport can take place through ATP synthase complex (leading to formation of ATP) or [...] Read more.
Higher concentration of protons in the mitochondrial intermembrane space compared to the matrix results in an electrochemical potential causing the back flux of protons to the matrix. This proton transport can take place through ATP synthase complex (leading to formation of ATP) or can occur via proton transporters of the mitochondrial carrier superfamily and/or membrane lipids. Some mitochondrial proton transporters, such as uncoupling proteins (UCPs), transport protons as their general regulating function; while others are symporters or antiporters, which use the proton gradient as a driving force to co-transport other substrates across the mitochondrial inner membrane (such as phosphate carrier, a symporter; or aspartate/glutamate transporter, an antiporter). Passage (or leakage) of protons across the inner membrane to matrix from any route other than ATP synthase negatively impacts ATP synthesis. The focus of this review is on regulated proton transport by UCPs. Recent findings on the structure and function of UCPs, and the related research methodologies, are also critically reviewed. Due to structural similarity of members of the mitochondrial carrier superfamily, several of the known structural features are potentially expandable to all members. Overall, this report provides a brief, yet comprehensive, overview of the current knowledge in the field. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Review
Structural Features of Cytochrome b5–Cytochrome b5 Reductase Complex Formation and Implications for the Intramolecular Dynamics of Cytochrome b5 Reductase
Int. J. Mol. Sci. 2022, 23(1), 118; https://doi.org/10.3390/ijms23010118 - 23 Dec 2021
Viewed by 1058
Abstract
Membrane cytochrome b5 reductase is a pleiotropic oxidoreductase that uses primarily soluble reduced nicotinamide adenine dinucleotide (NADH) as an electron donor to reduce multiple biological acceptors localized in cellular membranes. Some of the biological acceptors of the reductase and coupled redox proteins [...] Read more.
Membrane cytochrome b5 reductase is a pleiotropic oxidoreductase that uses primarily soluble reduced nicotinamide adenine dinucleotide (NADH) as an electron donor to reduce multiple biological acceptors localized in cellular membranes. Some of the biological acceptors of the reductase and coupled redox proteins might eventually transfer electrons to oxygen to form reactive oxygen species. Additionally, an inefficient electron transfer to redox acceptors can lead to electron uncoupling and superoxide anion formation by the reductase. Many efforts have been made to characterize the involved catalytic domains in the electron transfer from the reduced flavoprotein to its electron acceptors, such as cytochrome b5, through a detailed description of the flavin and NADH-binding sites. This information might help to understand better the processes and modifications involved in reactive oxygen formation by the cytochrome b5 reductase. Nevertheless, more than half a century since this enzyme was first purified, the one-electron transfer process toward potential electron acceptors of the reductase is still only partially understood. New advances in computational analysis of protein structures allow predicting the intramolecular protein dynamics, identifying potential functional sites, or evaluating the effects of microenvironment changes in protein structure and dynamics. We applied this approach to characterize further the roles of amino acid domains within cytochrome b5 reductase structure, part of the catalytic domain, and several sensors and structural domains involved in the interactions with cytochrome b5 and other electron acceptors. The computational analysis results allowed us to rationalize some of the available spectroscopic data regarding ligand-induced conformational changes leading to an increase in the flavin adenine dinucleotide (FAD) solvent-exposed surface, which has been previously correlated with the formation of complexes with electron acceptors. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Review
Insights into the Role of Membrane Lipids in the Structure, Function and Regulation of Integral Membrane Proteins
Int. J. Mol. Sci. 2021, 22(16), 9026; https://doi.org/10.3390/ijms22169026 - 21 Aug 2021
Cited by 5 | Viewed by 1493
Abstract
Membrane proteins exist within the highly hydrophobic membranes surrounding cells and organelles, playing key roles in cellular function. It is becoming increasingly clear that the membrane does not just act as an appropriate environment for these proteins, but that the lipids that make [...] Read more.
Membrane proteins exist within the highly hydrophobic membranes surrounding cells and organelles, playing key roles in cellular function. It is becoming increasingly clear that the membrane does not just act as an appropriate environment for these proteins, but that the lipids that make up these membranes are essential for membrane protein structure and function. Recent technological advances in cryogenic electron microscopy and in advanced mass spectrometry methods, as well as the development of alternative membrane mimetic systems, have allowed experimental study of membrane protein–lipid complexes. These have been complemented by computational approaches, exploiting the ability of Molecular Dynamics simulations to allow exploration of membrane protein conformational changes in membranes with a defined lipid content. These studies have revealed the importance of lipids in stabilising the oligomeric forms of membrane proteins, mediating protein–protein interactions, maintaining a specific conformational state of a membrane protein and activity. Here we review some of the key recent advances in the field of membrane protein–lipid studies, with major emphasis on respiratory complexes, transporters, channels and G-protein coupled receptors. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Review
Assessing the Role of Lipids in the Molecular Mechanism of Membrane Proteins
Int. J. Mol. Sci. 2021, 22(14), 7267; https://doi.org/10.3390/ijms22147267 - 06 Jul 2021
Cited by 4 | Viewed by 1663
Abstract
Membrane proteins have evolved to work optimally within the complex environment of the biological membrane. Consequently, interactions with surrounding lipids are part of their molecular mechanism. Yet, the identification of lipid–protein interactions and the assessment of their molecular role is an experimental challenge. [...] Read more.
Membrane proteins have evolved to work optimally within the complex environment of the biological membrane. Consequently, interactions with surrounding lipids are part of their molecular mechanism. Yet, the identification of lipid–protein interactions and the assessment of their molecular role is an experimental challenge. Recently, biophysical approaches have emerged that are compatible with the study of membrane proteins in an environment closer to the biological membrane. These novel approaches revealed specific mechanisms of regulation of membrane protein function. Lipids have been shown to play a role in oligomerization, conformational transitions or allosteric coupling. In this review, we summarize the recent biophysical approaches, or combination thereof, that allow to decipher the role of lipid–protein interactions in the mechanism of membrane proteins. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Review
The FtsHi Enzymes of Arabidopsis thaliana: Pseudo-Proteases with an Important Function
Int. J. Mol. Sci. 2021, 22(11), 5917; https://doi.org/10.3390/ijms22115917 - 31 May 2021
Cited by 4 | Viewed by 1503
Abstract
FtsH metalloproteases found in eubacteria, animals, and plants are well-known for their vital role in the maintenance and proteolysis of membrane proteins. Their location is restricted to organelles of endosymbiotic origin, the chloroplasts, and mitochondria. In the model organism Arabidopsis thaliana, there [...] Read more.
FtsH metalloproteases found in eubacteria, animals, and plants are well-known for their vital role in the maintenance and proteolysis of membrane proteins. Their location is restricted to organelles of endosymbiotic origin, the chloroplasts, and mitochondria. In the model organism Arabidopsis thaliana, there are 17 membrane-bound FtsH proteases containing an AAA+ (ATPase associated with various cellular activities) and a Zn2+ metalloprotease domain. However, in five of those, the zinc-binding motif HEXXH is either mutated (FtsHi1, 2, 4, 5) or completely missing (FtsHi3), rendering these enzymes presumably inactive in proteolysis. Still, homozygous null mutants of the pseudo-proteases FtsHi1, 2, 4, 5 are embryo-lethal. Homozygous ftshi3 or a weak point mutant in FTSHi1 are affected in overall plant growth and development. This review will focus on the findings concerning the FtsHi pseudo-proteases and their involvement in protein import, leading to consequences in embryogenesis, seed growth, chloroplast, and leaf development and oxidative stress management. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Review
The SARS-Coronavirus Infection Cycle: A Survey of Viral Membrane Proteins, Their Functional Interactions and Pathogenesis
Int. J. Mol. Sci. 2021, 22(3), 1308; https://doi.org/10.3390/ijms22031308 - 28 Jan 2021
Cited by 40 | Viewed by 5102
Abstract
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is a novel epidemic strain of Betacoronavirus that is responsible for the current viral pandemic, coronavirus disease 2019 (COVID-19), a global health crisis. Other epidemic Betacoronaviruses include the 2003 SARS-CoV-1 and the 2009 Middle East Respiratory Syndrome [...] Read more.
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is a novel epidemic strain of Betacoronavirus that is responsible for the current viral pandemic, coronavirus disease 2019 (COVID-19), a global health crisis. Other epidemic Betacoronaviruses include the 2003 SARS-CoV-1 and the 2009 Middle East Respiratory Syndrome Coronavirus (MERS-CoV), the genomes of which, particularly that of SARS-CoV-1, are similar to that of the 2019 SARS-CoV-2. In this extensive review, we document the most recent information on Coronavirus proteins, with emphasis on the membrane proteins in the Coronaviridae family. We include information on their structures, functions, and participation in pathogenesis. While the shared proteins among the different coronaviruses may vary in structure and function, they all seem to be multifunctional, a common theme interconnecting these viruses. Many transmembrane proteins encoded within the SARS-CoV-2 genome play important roles in the infection cycle while others have functions yet to be understood. We compare the various structural and nonstructural proteins within the Coronaviridae family to elucidate potential overlaps and parallels in function, focusing primarily on the transmembrane proteins and their influences on host membrane arrangements, secretory pathways, cellular growth inhibition, cell death and immune responses during the viral replication cycle. We also offer bioinformatic analyses of potential viroporin activities of the membrane proteins and their sequence similarities to the Envelope (E) protein. In the last major part of the review, we discuss complement, stimulation of inflammation, and immune evasion/suppression that leads to CoV-derived severe disease and mortality. The overall pathogenesis and disease progression of CoVs is put into perspective by indicating several stages in the resulting infection process in which both host and antiviral therapies could be targeted to block the viral cycle. Lastly, we discuss the development of adaptive immunity against various structural proteins, indicating specific vulnerable regions in the proteins. We discuss current CoV vaccine development approaches with purified proteins, attenuated viruses and DNA vaccines. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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Review
Fake It ‘Till You Make It—The Pursuit of Suitable Membrane Mimetics for Membrane Protein Biophysics
Int. J. Mol. Sci. 2021, 22(1), 50; https://doi.org/10.3390/ijms22010050 - 23 Dec 2020
Cited by 8 | Viewed by 1937
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Membrane Proteins: Structure, Function and Motion)
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