Function Of Membrane Proteins Research Articles

A New Preparation Method of Covalent Annular Nanodiscs Based on MTGase

The preservation of the native conformation and functionality of membrane proteins has posed considerable challenges. While detergents and liposome reconstitution have been traditional approaches, nanodiscs (NDs) offer a promising solution by embedding membrane proteins in phospholipids encircled by an amphipathic helical protein MSP belt. Nevertheless, a drawback of commonly used NDs is their limited homogeneity and stability. In this study, we present a novel approach to construct covalent annular nanodiscs (cNDs) by leveraging microbial transglutaminase (MTGase) to catalyze isopeptide bond formation between the side chains of terminal amino acids, specifically Lysine (K) and Glutamine (Q). This methodology significantly enhances the homogeneity and stability of NDs. Characterization of cNDs and the assembly of membrane proteins within them validate the successful reconstitution of membrane proteins with improved homogeneity and stability. Our findings suggest that cNDs represent a more suitable tool for investigating interactions between membrane proteins and lipids, as well as for analyzing membrane protein structures.

Phospholipid Membranes as Chemically and Functionally Tunable Materials.

The sheet-like lipid bilayer is the fundamental structural component of all cell membranes. Its building blocks are phospholipids and cholesterol. Their amphiphilic structure spontaneously leads to the formation of a bilayer in aqueous environment. Lipids are not just structural elements. Individual lipid species, the lipid membrane structure, and lipid dynamics influence and regulate membrane protein function. An exciting field is emerging where the membrane-associated material properties of different bilayer systems are used in designing innovative solutions for widespread applications across various fields, such as the food industry, cosmetics, nano- and biomedicine, drug storage and delivery, biotechnology, nano- and biosensors, and computing. Here, the authors summarize what is known about how lipids determine the properties and functions of biological membranes and how this has been or can be translated into innovative applications. Based on recent progress in the understanding of membrane structure, dynamics, and physical properties, a perspective is provided on how membrane-controlled regulation of protein functions can extend current applications and even offer new applications.

Capture of endogenous lipids in peptidiscs and effect on protein stability and activity

Compared to protein-protein and protein-nucleic acid interactions, our knowledge of protein-lipid interactions remains limited. This is primarily due to the inherent insolubility of membrane proteins (MPs) in aqueous solution. The traditional use of detergents to overcome the solubility barrier destabilizes MPs and strips away certain lipids that are increasingly recognized as crucial for protein function. Recently, membrane mimetics have been developed to circumvent the limitations. In this study, using the peptidisc, we find that MPs in different lipid states can be isolated based on protein purification and reconstitution methods, leading to observable effects on MP activity and stability. Peptidisc also enables re-incorporating specific lipids to fine-tune the protein microenvironment and assess the impact on downstream protein associations. This study offers a first look at the illusive protein-lipid interaction specificity, laying the path for a systematic evaluation of lipid identity and contributions to membrane protein function.

Functional regulation of aquaporin dynamics by lipid bilayer composition

With the diversity of lipid-protein interactions, any observed membrane protein dynamics or functions directly depend on the lipid bilayer selection. However, the implications of lipid bilayer choice are seldom considered unless characteristic lipid-protein interactions have been previously reported. Using molecular dynamics simulation, we characterize the effects of membrane embedding on plant aquaporin SoPIP2;1, which has no reported high-affinity lipid interactions. The regulatory impacts of a realistic lipid bilayer, and nine different homogeneous bilayers, on varying SoPIP2;1 dynamics are examined. We demonstrate that SoPIP2;1’s structure, thermodynamics, kinetics, and water transport are altered as a function of each membrane construct’s ensemble properties. Notably, the realistic bilayer provides stabilization of non-functional SoPIP2;1 metastable states. Hydrophobic mismatch and lipid order parameter calculations further explain how lipid ensemble properties manipulate SoPIP2;1 behavior. Our results illustrate the importance of careful bilayer selection when studying membrane proteins. To this end, we advise cautionary measures when performing membrane protein molecular dynamics simulations.

Белки мембраны молочной жировой глобулы. 8. Белок, связывающий жирные кислоты (FABP)

This article continues a series of reviews about the structure and functions of milk fat globule membrane (MFGM) proteins in cow’s milk. This installment introduces publications on the biochemistry of fatty acid binding protein (FABP), which is a polypeptide component of MFGM. The article gives a brief history of its discovery, as well as profiles the proteins that bind hydrophobic ligands and describes the methods of FABP identification in MFGM preparations. Other aspects of FABP studies include: isoforms and modern nomenclature; amino acid sequence and reactions to post-translational modification; spatial organization; mechanisms for binding fatty acids to lipids; well-investigated and hypothesized physiological functions; the effect of haplotypes on milk fatty acids, yield, and protein content.

Direct observation of autoubiquitination for an integral membrane ubiquitin ligase in ERAD

The endoplasmic reticulum associated degradation (ERAD) pathway regulates protein quality control at the endoplasmic reticulum. ERAD of lumenal and membrane proteins requires a conserved E3 ubiquitin ligase, called Hrd1. We do not understand the molecular configurations of Hrd1 that enable autoubiquitination and the subsequent retrotranslocation of misfolded protein substrates from the ER to the cytosol. Here, we have established a generalizable, single-molecule platform that enables high-efficiency labeling, stoichiometry determination, and functional assays for any integral membrane protein. Using this approach, we directly count Hrd1 proteins reconstituted into individual proteoliposomes. We report that Hrd1 assembles in different oligomeric configurations with mostly monomers and dimers detected at limiting dilution. By correlating oligomeric states with ubiquitination in vitro, we conclude that Hrd1 monomers are inefficient in autoubiquitination while dimers efficiently assemble polyubiquitin chains. Therefore, our results reveal the minimal composition of a Hrd1 oligomer that is capable of autoubiquitination. Our methods are broadly applicable to studying other complex membrane protein functions using reconstituted bilayer systems.

Quantifying a light-induced energetic change in bacteriorhodopsin by force spectroscopy

Ligand-induced conformational changes are critical to the function of many membrane proteins and arise from numerous intramolecular interactions. In the photocycle of the model membrane protein bacteriorhodopsin (bR), absorption of a photon by retinal triggers a conformational cascade that results in pumping a proton across the cell membrane. While decades of spectroscopy and structural studies have probed this photocycle in intricate detail, changes in intramolecular energetics that underlie protein motions have remained elusive to experimental quantification. Here, we measured these energetics on the millisecond time scale using atomic-force-microscopy-based single-molecule force spectroscopy. Precisely, timed light pulses triggered the bR photocycle while we measured the equilibrium unfolding and refolding of the terminal 8-amino-acid region of bR's G-helix. These dynamics changed when the EF-helix pair moved ~9 Å away from this end of the G helix during the "open" portion of bR's photocycle. In ~60% of the data, we observed abrupt light-induced destabilization of 3.4 ± 0.3 kcal/mol, lasting 38 ± 3 ms. The kinetics and pH-dependence of this destabilization were consistent with prior measurements of bR's open phase. The frequency of light-induced destabilization increased with the duration of illumination and was dramatically reduced in the triple mutant (D96G/F171C/F219L) thought to trap bR in its open phase. In the other ~40% of the data, photoexcitation unexpectedly stabilized a longer-lived putative misfolded state. Through this work, we establish a general single-molecule force spectroscopy approach for measuring ligand-induced energetics and lifetimes in membrane proteins.

EncoMPASS: An encyclopedia of membrane proteins analyzed by structure and symmetry

Protein structure determination and prediction, active site detection, and protein sequence alignment techniques all exploit information about protein structure and structural relationships. For membrane proteins, however, there is limited agreement among available online tools for highlighting and mapping such structural similarities. Moreover, no available resource provides a systematic overview of quaternary and internal symmetries, and their orientation relative to the membrane, despite the fact that these properties can provide key insights into membrane protein function and evolution. Here, we describe the Encyclopedia of Membrane Proteins Analyzed by Structure and Symmetry (EncoMPASS), a database for relating integral membrane proteins of known structure from the points of view of sequence, structure, and symmetry. EncoMPASS is accessible through a web interface, and its contents can be easily downloaded. This allows the user not only to focus on specific proteins, but also to study general properties of the structure and evolution of membrane proteins.

N-glycan on N262 of FGFR3 regulates the intracellular localization and phosphorylation of the receptor

N-glycosylation and proper processing of N-glycans are required for the function of membrane proteins including cell surface receptors. Fibroblast growth factor receptor (FGFR) is involved in a wide variety of biological processes including embryonic development, osteogenesis, angiogenesis, and cell proliferation. Human FGFR3 contains six potential N-glycosylation sites, however, the roles of glycosylation have not been elucidated. The site-specific profiles of N-glycans of the FGFR3 extracellular domain expressed and secreted by CHO-K1 cells were examined, and glycan occupancies and structures of four sites were determined. The results indicated that most sites were fully occupied by glycans, and the dominant populations were the complex type. By examining single N-glycan deletion mutants of FGFR3, it was found that N262Q mutation significantly increased the population with oligomannose-type N-glycans, which was localized in the endoplasmic reticulum. Protein stability assay suggested that fraction with oligomannose-type N-glycans in the N262Q mutant is more stable than those in the wild type and other mutants. Furthermore, it was found that ligand-independent phosphorylation was significantly upregulated in N262Q mutants with complex type N-glycans. The findings suggest that N-glycans on N262 of FGFR3 affect the intracellular localization and phosphorylation status of the receptor.

C-terminal amino acids in the type I transmembrane domain of L-type lectin VIP36 affect γ-secretase susceptibility

Regulated intramembrane proteolysis (RIP) is a two-step processing mechanism for transmembrane proteins consisting of ectodomain shedding (shedding), which removes the extracellular domain through juxtamembrane processing and intramembrane proteolysis, which processes membrane-anchored shedding products within the transmembrane domain. RIP irreversibly converts one transmembrane protein into multiple soluble proteins that perform various physiological functions. The only requirement for the substrate of γ-secretase, the major enzyme responsible for intramembrane proteolysis of type I transmembrane proteins, is the absence of a large extracellular domain, and it is thought that γ-secretase can process any type I membrane protein as long as it is shed. In the present study, we showed that the shedding susceptible type I membrane protein VIP36 (36 kDa vesicular integral membrane protein) and its homolog, VIPL, have different γ-secretase susceptibilities in their transmembrane domains. Analysis of the substitution mutants suggested that γ-secretase susceptibility is regulated by C-terminal amino acids in the transmembrane domain. We also compared the transmembrane domains of several shedding susceptible membrane proteins and found that each had a different γ-secretase susceptibility. These results suggest that the transmembrane domain is not simply a stretch of hydrophobic amino acids but is an important element that regulates membrane protein function by controlling the lifetime of the membrane-anchored shedding product.

The intricate link between membrane lipid structure and composition and membrane structural properties in bacterial membranes.

It is now evident that the cell manipulates lipid composition to regulate different processes such as membrane protein insertion, assembly and function. Moreover, changes in membrane structure and properties, lipid homeostasis during growth and differentiation with associated changes in cell size and shape, and responses to external stress have been related to drug resistance across mammalian species and a range of microorganisms. While it is well known that the biomembrane is a fluid self-assembled nanostructure, the link between the lipid components and the structural properties of the lipid bilayer are not well understood. This perspective aims to address this topic with a view to a more detailed understanding of the factors that regulate bilayer structure and flexibility. We describe a selection of recent studies that address the dynamic nature of bacterial lipid diversity and membrane properties in response to stress conditions. This emerging area has important implications for a broad range of cellular processes and may open new avenues of drug design for selective cell targeting.

Biomimetic nanotherapeutics for homotypic-targeting photothermal/chemotherapy of oral cancer

Given the inherent complexity of cancer treatment and the limitations of singular therapeutic modalities, the development of an optimal nanocarrier system capable of facilitating synergistic organic therapy remains a profound challenge. Herein, a synergetic chemo/photothermal therapy nanoplatform was exploited to specifically tailor for the augmented treatment of oral cancer. A cancer cell membrane-camouflaged nanocarrier was developed with a polymeric core encapsulating doxorubicin (DOX). The designed nanoparticles (CC@DOXNPs) inherited the functional membrane proteins from the source cancer cells, endowing their remarkable ability to selectively target cancer cells delivery both in vitro and in vivo. Moreover, indocyanine green (ICG), modified with the phospholipid polymer DSPE-PEG2000, was introduced into the cancer cell membrane to enable photothermal therapy. Remarkably, as evaluated in a preclinical subcutaneous and orthotopic mice model of oral cancer, biomimetic composite nanotherapeutics (lip-CC@DOXNPs) could significantly accumulate into tumor lesion and effectively suppress tumor growth under the near-infrared (NIR, 808 nm) irradiation, without causing the undesirable systematic toxicity. Moreover, RNA sequence analyses indicated that chemo/photothermal therapy triggers the intrinsic mitochondria-mediated apoptosis through the p53 signaling pathway. Combined with gene expression results, this intrinsic mitochondria-mediated apoptosis pathway was further demonstrated. Collectively, this multifaceted nanoplatforms possess a remarkable capability for tumor-targeting drug delivery, and the proficient photothermal conversion ability, rendering them an ideal therapeutic approach for oral cancer treatment.

Secondary Ion Mass Spectrometry of Single Giant Unilamellar Vesicles Reveals Compositional Variability.

Giant unilamellar vesicles (GUVs) are a widely used model system to interrogate lipid phase behavior, study biomembrane mechanics, reconstitute membrane proteins, and provide a chassis for synthetic cells. It is generally assumed that the composition of individual GUVs is the same as the nominal stock composition; however, there may be significant compositional variability between individual GUVs. Although this compositional heterogeneity likely impacts phase behavior, the function and incorporation of membrane proteins, and the encapsulation of biochemical reactions, it has yet to be directly quantified. To assess heterogeneity, we use secondary ion mass spectrometry (SIMS) to probe the composition of individual GUVs using non-perturbing isotopic labels. Both 13C- and 2H-labeled lipids are incorporated into a ternary mixture, which is then used to produce GUVs via gentle hydration or electroformation. Simultaneous detection of seven different ion species via SIMS allows for the concentration of 13C- and 2H-labeled lipids in single GUVs to be quantified using calibration curves, which correlate ion intensity to composition. Additionally, the relative concentration of 13C- and 2H-labeled lipids is assessed for each GUV via the ion ratio 2H-/13C-, which is highly sensitive to compositional differences between individual GUVs and circumvents the need for calibration by using standards. Both quantification methods suggest that gentle hydration produces GUVs with greater compositional variability than those formed by electroformation. However, both gentle hydration and electroformation display standard deviations in composition (n = 30 GUVs) on the order of 1-4 mol %, consistent with variability seen in previous indirect measurements.

Corrigendum to: β-Barrel Membrane Bacterial Proteins: Structure, Function, Assembly and Interaction with Lipids.

The authors declare after the publication of the article entitled ‘β-Barrel Membrane Bacterial Proteins: Structure, Function, Assembly and Interaction with Lipids’’, published in Current Protein and Peptide Science, 2007, 8, 63-82 [1], that a reference by Koebnik was inadvertently omitted. The missing reference has now been included as: Original: [1] Rosenbusch, J.P. (1988) Zentralbl. Bakteriol., 17, 259-266. Corrected: [1] (a) Rosenbusch, J.P. (1988) Zentralbl. Bakteriol., 17, 259-266. (b) Koebnik, R.; Locher, K.P.; Gelder, P.V. Structure and function of bacterial outer membrane proteins: Barrels in a nutshell. Mol. Biol., 2000, 37(2), 239-53. The original article can be found online at: https://www.eurekaselect.com/article/22780

Physiological Functions of the Ubiquitin Ligases Nedd4-1 and Nedd4-2.

The Nedd4 family of E3 ubiquitin ligases, consisting of a C2-WW(n)-HECT domain architecture, includes the closely related Nedd4/Nedd4-1 and Nedd4L/Nedd4-2, which play critical roles in human physiology and pathophysiology.This review focuses on the regulation of enzymatic activity of these Nedd4 proteins, as well as on their roles in regulating stability and function of membrane and other signaling proteins, such as ion channels, ion transporters, and growth factor receptors. The diseases caused by impairment of such regulation are discussed, as well as opportunities and challenges for targeting these enzymes for therapy.

Inner leaflet cationic lipid increases nucleic acid loading independently of outer leaflet lipid charge in asymmetric liposomes.

Use of cationic lipid vesicles (liposomes) can yield large amounts of nucleic acid entrapped inside the vesicles and/or bound to the external surface of the vesicles. To show a method to prepare asymmetric lipid vesicles (liposomes) with high amounts of entrapped nucleic acid is possible, symmetric and asymmetric lipid vesicles composed of mixtures of neutral (zwitterionic), anionic, and/or cationic phospholipids were formed in the presence of oligo DNA. For symmetric large unilamellar vesicles nucleic acid association with vesicles was roughly 100 times greater for vesicles with a net cationic charge than for vesicles having a net neutral or anionic net charge. A high degree of association between nucleic acid and lipid was also achieved using asymmetric large unilamellar vesicles with a net cationic charge in their inner leaflet, even when they had an anionic charge in their outer leaflet. In contrast, asymmetric vesicles in which only the outer leaflet had a net cationic charge had only low amounts of vesicle-associated nucleic acid, similar in amount to the amount of nucleic acid associated with asymmetric vesicles with an outer leaflet having a net anionic charge. These results indicate that in asymmetric vesicles with cationic lipid enriched inner leaflets nucleic acid is largely entrapped inside the vesicle lumen rather than bound to their external surface, and that asymmetric vesicles can be used to trap high amounts of nucleic acid even when using a lipid composition in the outer leaflet of a lipid vesicle that does not associate with nucleic acids. Such asymmetrically charged vesicles should have applications in studies of membrane protein-nucleic acid interactions as well as in studies of how membrane charge asymmetry can influence membrane protein structure, orientation, and function.

Recent advances in features generation for membrane protein sequences: From multiple sequence alignment to pre-trained language models.

Membrane proteins play a crucial role in various cellular processes and are essential components of cell membranes. Computational methods have emerged as a powerful tool for studying membrane proteins due to their complex structures and properties that make them difficult to analyze experimentally. Traditional features for protein sequence analysis based on amino acid types, composition, and pair composition have limitations in capturing higher-order sequence patterns. Recently, multiple sequence alignment (MSA) and pre-trained language models (PLMs) have been used to generate features from protein sequences. However, the significant computational resources required for MSA-based features generation can be a major bottleneck for many applications. Several methods and tools have been developed to accelerate the generation of MSAs and reduce their computational cost, including heuristics and approximate algorithms. Additionally, the use of PLMs such as BERT has shown great potential in generating informative embeddings for protein sequence analysis. In this review, we provide an overview of traditional and more recent methods for generating features from protein sequences, with a particular focus on MSAs and PLMs. We highlight the advantages and limitations of these approaches and discuss the methods and tools developed to address the computational challenges associated with features generation. Overall, the advancements in computational methods and tools provide a promising avenue for gaining deeper insights into the function and properties of membrane proteins, which can have significant implications in drug discovery and personalized medicine.

Exploring the World of Membrane Proteins: Techniques and Methods for Understanding Structure, Function, and Dynamics.

In eukaryotic cells, membrane proteins play a crucial role. They fall into three categories: intrinsic proteins, extrinsic proteins, and proteins that are essential to the human genome (30% of which is devoted to encoding them). Hydrophobic interactions inside the membrane serve to stabilize integral proteins, which span the lipid bilayer. This review investigates a number of computational and experimental methods used to study membrane proteins. It encompasses a variety of technologies, including electrophoresis, X-ray crystallography, cryogenic electron microscopy (cryo-EM), nuclear magnetic resonance spectroscopy (NMR), biophysical methods, computational methods, and artificial intelligence. The link between structure and function of membrane proteins has been better understood thanks to these approaches, which also hold great promise for future study in the field. The significance of fusing artificial intelligence with experimental data to improve our comprehension of membrane protein biology is also covered in this paper. This effort aims to shed light on the complexity of membrane protein biology by investigating a variety of experimental and computational methods. Overall, the goal of this review is to emphasize how crucial it is to understand the functions of membrane proteins in eukaryotic cells. It gives a general review of the numerous methods used to look into these crucial elements and highlights the demand for multidisciplinary approaches to advance our understanding.

S-acylation: an orchestrator of the life cycle and function of membrane proteins.

S-acylation is a covalent post-translational modification of proteins with fatty acids, achieved by enzymatic attachment via a labile thioester bond. This modification allows for dynamic control of protein properties and functions in association with cell membranes. This lipid modification regulates a substantial portion of the human proteome and plays an increasingly recognized role throughout the lifespan of affected proteins. Recent technical advancements have propelled the S-acylation field into a 'molecular era', unveiling new insights into its mechanistic intricacies and far-reaching implications. With a striking increase in the number of studies on this modification, new concepts are indeed emerging on the roles of S-acylation in specific cell biology processes and features. After a brief overview of the enzymes involved in S-acylation, this viewpoint focuses on the importance of S-acylation in the homeostasis, function, and coordination of integral membrane proteins. In particular, we put forward the hypotheses that S-acylation is a gatekeeper of membrane protein folding and turnover and a regulator of the formation and dynamics of membrane contact sites.

Applications of genetic code expansion and photosensitive UAAs in studying membrane proteins.

Membrane proteins are the targets for most drugs and play essential roles in many life activities in organisms. In recent years, unnatural amino acids (UAAs) encoded by genetic code expansion (GCE) technology have been widely used, which endow proteins with different biochemical properties. A class of photosensitive UAAs has been widely used to study protein structure and function. Combined with photochemical control with high temporal and spatial resolution, these UAAs have shown broad applicability to solve the problems of natural ion channels and receptor biology. This review will focus on several application examples of light-controlled methods to integrate GCE technology to study membrane protein function in recent years. We will summarize the typical research methods utilizing some photosensitive UAAs to provide common strategies and further new ideas for studying protein function and advancing biological processes.