In biological and biomedical research, the focus progressively moves towards difficult human proteins, which often can only be expressed in higher eukaryotic cells. Nuclear magnetic resonance (NMR) could contribute significantly to the understanding of important proteins as it is one of the most information-rich methods. However, to exploit the full potential of NMR, proteins must be isotope labeled. Although expression protocols in, for example, HEK293 cells are often established, isotope labeling is difficult and very expensive, and published protocols are mostly limited to adherent cells in plates. To resolve this disparity, we have developed a comprehensive suite of protocols for isotope labeling in HEK293 cells in suspension. We demonstrate uniform 15N and 13C labeling, as well as specific labeling, with special focus on methyl bearing amino acids, including the popular ILV 13C-methyl labeling pattern. In addition, several innovative practices are introduced to deal with special cases. Labeling is achieved with a simple laboratory setup and affordable labeling media. These are based on either labeled amino acids, their precursors or amino extracts from microorganisms, like yeast, algae or bacteria. We demonstrate how these protocols enable NMR studies of proteins that are considered to be especially difficult to produce, for example, different human membrane proteins and nuclear receptors. We therefore expect that these new methods make many highly important proteins accessible to NMR studies and allow exploiting the high information content of this method for accelerating biological and pharmaceutical research.

Membrane protein-protein interactions (memPPIs) underlie critical biological processes including mediating signal transduction, transport, cell communication, and membrane organization. Despite their importance, systematic and quantitative measurement of protein transmembrane (TM) domains laterally interacting in mammalian cells remains challenging. Conventional fusion protein assays directed at memPPIs suffer from low dynamic range due to the crowded membrane environment and interference from overexpression, trafficking, or self-complementation artifacts. Here, we assessed and further developed the split luciferase complementation NanoBit system to establish suitable expression constructs and working conditions for robust quantification of TM PPIs within human cells. We benchmarked the platform using a panel of natural and synthetic memPPI pairs with diverse architectures, insertion topologies, and trafficking patterns, finding assay settings that robustly distinguish true positive interactions from co-expressed non-interacting pairs. Adding a novel procedure measuring and normalizing protein expression levels in situ in parallel, we drastically improve assay dynamic range and consistency in discerning relative interaction propensities across broad expression levels-which also enhances compatibility with protein engineering screens. To extend measurements to versatile protein contexts, we developed membrane expression constructs and validated tagging strategies to improve trafficking with defined TM orientations. By incorporating topological control and accounting for variations in expression levels, this memPPI platform becomes a viable approach for high-throughput interaction analyses and experimental screens for membrane protein design such as TM binders targeting human membrane proteins. Owing to its sensitivity, scalability, and adaptability, we expect this assay platform to have broad cross-disciplinary utility in protein science, design, and drug discovery.

SUMMARYNearly half of the ~5,000 human membrane proteins need to assemble into stoichiometric complexes as part of their biogenesis at the endoplasmic reticulum (ER) membrane. How ER resident biogenesis factors coordinate membrane insertion, folding and assembly is not well understood. Here, we demonstrate that the ER membrane protein complex (EMC) insertase additionally acts as a chaperone to facilitate the assembly of heterotrimeric voltage-gated calcium channels (Cav). Using function-separating mutations and inhibitory nanobodies we show that nascent Cav channels are degraded prematurely when EMC’s chaperone function is selectively perturbed. Blocking EMC’s chaperone function strongly impaired Cav-dependent cardiomyocyte contraction. EMC engagement of the pore-forming Cavα-subunit occurred co-translationally and required Cavα’s first transmembrane domain bundle to protrude from the nascent ribosome•Sec61•multipass translocon complex. Our findings establish a chaperone function for the EMC and reveal that biogenesis of multi-bundle membrane proteins requires a highly orchestrated, co-translational interplay between ER biogenesis factors.

Comparative analysis of Tibetan human milk fat globule membrane proteins across lactation stages.

Amphiphilic copolymers capable of extracting membrane proteins directly from cellular membranes into"native nanodiscs" offer a simplified approach for preparing membrane proteins in lipid nanodiscs compared to approaches that rely on detergent. Copolymer amphiphilicity, length, and composition influence their performance, in addition to the protein itself and the purification conditions used. Here, we report a copolymer composed of methacrylic acid and styrene, which we term MAASTY, leveraging the inherent monomer reactivity ratios to create an anionic copolymer with a statistical distribution of monomers. We show that MAASTY can be used for high-resolution structural determination of a human membrane protein by single particle cryo-electron microscopy, preserving endogenous lipids including cholesterol and exhibiting an enrichment of phosphatidylinositol. Moreover, MAASTY copolymers effectively solubilize a broad range of lipid species and a wide range of different, eukaryotic membrane proteins from mammalian cells. We find that MAASTY copolymers are promising as effective solubilizers of membrane proteins and offer a chemical platform for structural and functional characterization of membrane proteins in native nanodiscs.

G protein-coupled receptors (GPCRs), the largest superfamily of human membrane proteins with >800 members, are primary targets for ~1/3 of all marketed drugs. Recent fluorescence experiments underscored the pivotal role of GPCR-G protein complex lifetime in their coupling efficiency and selectivity. However, these experiments are often expensive, time-consuming, and limited to a small number of GPCR-G protein systems. On the other hand, it is challenging to simulate GPCR-G protein dissociation using molecular dynamics (MD) methods. Here, we have employed Protein-Protein Interaction Gaussian accelerated MD (PPI-GaMD) simulations and experiments to probe the kinetics and pathways of G protein dissociation from GPCRs. For five systems with published experimental kinetic data, PPI-GaMD simulations successfully captured G protein dissociation from the GPCRs, including the adrenergic, adenosine, and muscarinic receptors. The simulations allowed identification of two distinct dissociation pathways and calculation of the G protein dissociation rates, which were in good agreement with experimental data. Additionally, we simulated the effect of positive allosteric modulators (PAMs) of the adenosine A1 receptor (A1R) in Gi protein dissociation and supported simulation findings with bioluminescence resonance energy transfer biosensor experiments evaluating Gβγ kinetics following A1R activation. A1R PAMs were found to strengthen the agonist-receptor and receptor-G protein interactions and significantly reduce dissociation rates of the Gi protein. In summary, complementary PPI-GaMD simulations and kinetic assays have enabled detailed characterization of the kinetics and pathways of G protein dissociation, a critical event in the GPCR signaling cascade, and the effects of GPCR allosteric modulators.

The magnetotactic bacterium Magnetospirillum magneticum produces biogenic magnetic nanoparticles called magnetosomes, each comprising a magnetite core surrounded by a lipid bilayer. By expressing the human neurotrophin receptor TrkA, which is implicated in Alzheimer's disease, cancer, and other pathologies, on magnetosomes, we created a functional human transmembrane receptor platform that enables magnetic recovery and ligand screening. However, TrkA has limited activity within the native bacterial membrane, likely due to the absence of key eukaryotic lipids such as phosphatidylcholine (PC) and cholesterol (Chol). To overcome this limitation, we combined in vivo and in vitro membrane engineering to remodel the magnetosome envelope and closely mimic the human cell membrane. Specifically, PC was biosynthesized in vivo by co-expressing phosphatidylcholine synthase, and purified magnetosomes were subsequently enriched in vitro with Chol using methyl-β-cyclodextrin. This dual-lipid strategy significantly enhanced TrkA-ligand-binding and autophosphorylation. These findings show that orthogonal membrane engineering can precisely tailor the lipid composition of bacterial magnetosomes to recreate mammalian-like bilayers, rescuing the function of complex human receptors in a microbial environment. This approach expands the metabolic and synthetic biology toolkit for producing functional membrane proteins on scalable magnetic supports and establishes a cost-effective platform for the discovery of TrkA and other membrane receptors.IMPORTANCEMembrane receptors drive essential signaling in health and disease; however, they are difficult to study and screen because most platforms fail to reproduce human-like membranes at scale. Magnetosomes from the bacterium Magnetospirillum magneticum AMB-1 offer a simple alternative: lipid-bounded, magnetic nanoparticles that can be purified in one step. This work establishes human-like remodeling of magnetosome membranes by combining in vivo phosphatidylcholine synthesis with the first in vitro cholesterol loading of these particles. Displaying human tropomyosin receptor kinase A on the remodeled membranes preserved and enhanced the receptor function without complex purification or reconstitution. Because magnetosomes can be produced inexpensively and recovered magnetically, this approach enables practical, high-throughput assays for ligand discovery and inhibitor testing. The strategy is broadly applicable to other human membrane proteins, linking microbial biotechnology with human membrane biology to accelerate translational research.

The TEMPI syndrome: Unique patient specific monoclonal antibodies as tools for target antigen discovery

Structural and functional effects of disease mutations in the interface of human membrane protein complexes.

The oligomerization of the transmembrane helices of single-pass membrane proteins is crucial to biological function and its misregulation can lead to many diseases. The study of transmembrane helix oligomerization is facilitated by the availability of genetic reporter assays, which are essential tools for understanding the organization and biology of single-pass systems. In particular, reporter assays are crucial for mapping the oligomerization interfaces of transmembrane helices through scanning mutagenesis but their application is limited by the need to clone and measure each construct individually. Here, we present “TOXGREEN sort-seq,” a high-throughput version of the TOXGREEN assay that enables the direct measurement of transmembrane helix oligomerization in large libraries using fluorescence-activated cell sorting and next-generation sequencing. We show that TOXGREEN sort-seq is robust and accurately reproduces the direct measurements of individual constructs. The method produced high-quality mutational profiles from a library of 17,400 constructs designed to probe the interface of 100 potential GASright dimers predicted from sequences of human single-pass membrane proteins. We report the validated structural model of twelve dimers involved in a variety of biological functions, including immune response (interleukin-22 receptor subunit alpha-1, butyrophilin-like protein 3, hepatitis A virus cellular receptor 2), transport (transferrin receptor protein 1), and cell-surface signaling and proliferation (syndecan-3; semaphorins 5A, 6B, and 6D). Remarkably, all semaphorins in the dataset formed strong dimers and produced mutational profiles consistent with the computational structure. These findings suggest that dimerization may be relevant to these proteins' activity and provide a validated interface for assessing their biological role.

Many physiological processes are dependent on G protein-coupled receptors (GPCRs), the biggest family of human membrane proteins and a significant class of therapeutic targets. Once activated by external stimuli, GPCRs use G proteins and arrestins as transducers to generate second messengers and trigger downstream signaling, leading to diverse signaling profiles. The G protein-coupled bile acid receptor 1 (GPBAR1, also known as Takeda G protein-coupled receptor 5, TGR5) is a class A bile acid membrane receptor that regulates energy homeostasis and glucose and lipid metabolism. GPBAR1/Gs protein interactions are implicated in the prevention of diabetes and the reduction of inflammatory responses, making GPBAR1 a potential therapeutic target for metabolic disorders. Here, we present combined hydrogen/deuterium exchange mass spectrometry (HDX-MS) and cryo-electron microscopy (cryo-EM) to identify the molecular determinants of GPBAR1 conformational dynamics upon G protein binding. Thanks to extensive optimization, we achieved over 75% sequence coverage by HDX-MS of a complete GPCR complex and a 2.5 Å resolution structure by cryo-EM, both of which are state-of-the-art. Altogether, our results provide information on the under-investigated GPBAR1 binding mode to its cognate G protein, pinpointing the synergic and powerful combination of higher cryo-EM and lower HDX-MS resolution techniques to dissect GPCR/G protein binding characteristics.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-16529-w.

Background: The search for potential therapeutic agents against the most common coronaviruses, which can pose a threat to human and animal life, is an urgent issue of modern biomedicine. Objective of the work was to evaluate in silico the ability of C60 fullerene to interact with the membrane protein ACE2, thereby preventing the formation of the "coronavirus-ACE2" complex and its further penetration into the host cell, as well as the effectiveness of the anticoronavirus action of these carbon nanoparticles in in vitro systems. Methods: The Protein Data Bank was used to study the structural organization of the human ACE2 membrane protein. The CHARMM-GUI and SwissParam web resources were used to construct the membrane and C60 fullerene, respectively. Potential binding pockets for C60 fullerene in the ACE2 structure were determined using the Caver software package. The system molecular docking algorithm (sdock+) was used to study the interaction between C60 fullerene and ACE2. Molecular dynamics (MD) calculations were performed using the Gromacs 2020 software package. Cytotoxicological and virological methods were used in in vitro experiments. Statistical processing of experimental results was carried out using the Statistica 13.3 program. Results: It was found three potential binding sites between the groove of the peptidase domain of the ACE2 protein and C60 fullerene. According to the results of molecular docking and MD, it follows that C60 fullerene forms two stable complexes with the ACE2 protein, thus blocking its potential interaction with coronaviruses. According to the results of in vitro studies, it follows that C60 fullerenes at a maximum allowable concentration of 37.5 μg/ml act on the coronaviruses of swine (α-coronavirus) and cattle (β-coronavirus) at the early stage of replication (1 h) in sensitive cellular systems, significantly reducing their infectious activity by 2.00 TCID50/ml and ≥2,28 TCID50/ml, respectively. Conclusions: C60 fullerene has been shown to form two stable complexes with the membrane protein ACE2, thereby inhibiting its functional activity and blocking potential interaction with coronaviruses. It was established that the C60 fullerenes show antiviral activity against coronaviruses of two groups at the initial stage of infection when interacting with sensitive host cells.

While SARS-CoV-2 primarily infects the respiratory tract, clinical evidence indicates that cells from diverse cell types and organs are also susceptible to infection. Using the CRISPR activation (CRISPRa) approach, we systematically targeted human membrane proteins in cells with and without overexpression of ACE2, thus identifying unrecognized host factors that may facilitate viral entry. Validation experiments with replication-competent SARS-CoV-2 confirmed the role of newly identified host factors, particularly the endo-lysosomal protease legumain (LGMN) and the potassium channel KCNA6, upon exogenous overexpression. In orthogonal experiments, we show that disruption of endogenous LGMN or KCNA6 decreases viral infection and that inhibitors of candidate factors can reduce viral entry. Additionally, using clinical data, we find possible associations between expression of either LGMN or KCNA6 and SARS-CoV-2 infection in human tissues. Our results identify potentially druggable host factors involved in SARS-CoV-2 entry, and demonstrate the utility of focused, membrane-wide CRISPRa screens in uncovering tissue-specific entry factors of emerging pathogens.

We report here transport of full-length epidermal growth factor receptor (EGFR), Insulin Receptor, 7-pass transmembrane receptor Smoothened, and 13-pass Sodium-iodide symporter to extracellular vesicles (EVs) for structural and functional studies. Mass spectrometry confirmed the transported proteins are the most abundant in EV membranes, and the presence of many receptor-interacting proteins in EVs demonstrates their utility for characterizing membrane protein interactomes. Cryo-electron tomography of EGFR-containing EVs reveals that EGFR forms clusters in both the presence and absence of EGF with a ~3 nm gap between the inner membrane and cytoplasmic density. EGFR extracellular region (ECR) dimers do not form regular arrays in these clusters. Subtomogram averaging of the 150 kDa EGF-bound EGFR ECR dimer yielded a 15 Å map into which the crystal structure of the ligand-bound EGFR ECR dimer fits well. These findings refine our understanding of EGFR activation, clustering, and signaling and establish EVs as a versatile platform for structural and functional characterization of human membrane proteins in cell-derived membranes.

G protein-coupled receptors (GPCRs) constitute the largest family of human membrane proteins. GPCRs recognize diverse extracellular stimuli and activate intracellular signaling cascades that regulate key physiological processes such as neurotransmission and cardiovascular function. The controlled transport of nascent GPCRs from the endoplasmic reticulum (ER) via the Golgi apparatus to the cell surface critically determines the cellular responsiveness to incoming ligands. Here, we present a comprehensive overview of the cellular mechanisms and motif-driven interactions with regulatory proteins that orchestrate GPCR folding, post-translational modifications, and vesicular transport along the secretory pathway. We highlight signaling cues that can modulate the anterograde transport and specialized mechanisms that deliver biosynthetic GPCRs to dendrites and axons in neurons. Furthermore, we discuss that many disease-causing GPCR mutants exhibit aberrant intracellular retention, which can be rescued by pharmacological strategies that stabilize misfolded GPCRs. Finally, we highlight insights into the agonist-driven signaling of biosynthetic GPCRs in secretory organelles. This review covers the complex roles of anterograde transport in controlling GPCR function and emerging possibilities to target the underlying mechanisms in disease.

The oligomerization of the transmembrane helices of single-pass membrane proteins is crucial to biological function and its misregulation can lead to many diseases. The study of transmembrane helix oligomerization is facilitated by the availability of genetic reporter assays, which are essential tools for understanding the organization and biology of single-pass systems. In particular, reporter assays are crucial for mapping the oligomerization interfaces of transmembrane helices through scanning mutagenesis but their application is limited by the need to clone and measure each construct individually. Here, we present “TOXGREEN sort-seq”, a high-throughput version of the TOXGREEN assay that enables the direct measurement of transmembrane helix oligomerization in large libraries using fluorescence-activated cell sorting and next-generation sequencing. We show that TOXGREEN sort-seq is robust and reproduce the direct measurements of individual constructs with good accuracy and sensitivity. The method produced high-quality mutational profiles from a library of 17,400 constructs designed to probe the interface of 100 potential GASright dimers predicted from sequences of human single-pass membrane proteins. We report the validated structural model of twelve dimers involved in a variety of biological functions, including immune response (interleukin-22 receptor subunit alpha-1, butyrophilin-like protein 3, hepatitis A virus cellular receptor 2), transport (transferrin receptor protein 1), and cell-surface signaling and proliferation (syndecan-3; semaphorins 5A, 6B and 6D). Remarkably, all three semaphorins in the dataset formed strong dimers and produced mutational profiles consistent with the computational structure. These findings open the possibility that dimerization may be relevant to these proteins’ activity and provide a validated interface for assessing their biological role.

Abstract Antibody drug development is lengthy and complex, often hindered by various challenges in clinical advancement. Reports indicate that off-target binding of antibodies may be a significant issue, with approximately 25% of preclinical candidates potentially experiencing these off-target problems. With the rise of new modalities such as CD3 bispecific antibodies, ADCs, and CAR-T therapies, preliminary off-target screening of antibodies has become increasingly important. Despite the use of techniques like immunohistochemistry, ELISA, and flow cytometry (FACS) for off-target testing of antibodies, these methods still exhibit limitations in sensitivity, specificity, and throughput. To address the need for antibody target deconvolution and receptor identification, we developed a novel membrane protein screening array (MPSA) AB5000. MPSA-AB5000 is a high-throughput, cell-based platform for identifying antibody targets and off-targets, enhancing antibody specificity screening and reducing development risks. The MPSA-AB5000 covers diverse human membrane proteins, expressed in live cells to preserve native conformations. C-terminal epitope-tagged clones allow for expression confirmation and cellular localization. To provide the highest level of sensitivity, MPSA-AB5000 uses luciferase reporter cell line detection. The wash-free strategy reduces false negatives, and signal amplification makes remarkable discrimination. Compared to similar technologies, we possess significant advantages in the following aspects: a. High sensitivity and sigh throughput: our technology platform offers not only high sensitivity, capable of detecting subtle interactions but also supports high-throughput screening, allowing for the evaluation of a large number of samples in a short timeframe. b. Avoidance of false negative signals: by eliminating washing steps, our method reduces the risk of false negative signals, ensuring reliable detection of non-specific binding. c. Comprehensive validation approaches: we employ various validation methods such as FACS and ELISA to eliminate false positive signals, enhancing the accuracy and credibility of our results. All the advantages ensure that MPSA-AB5000 is more rapid, simple, and highly sensitive than traditional assays. Citation Format: Liping Wang, Yuanyuan Sun, Guoqian Wang, Jinying Ning, Feng Hao. MPSA-AB5000: a high-throughput membrane proteins screening array for therapeutic biologics specificity profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5680.

Chagas disease, caused by the protozoan Trypanosoma cruzi, affects millions globally, leading to severe cardiac and gastrointestinal complications in its chronic phase. The invasion of host cells by T. cruzi is mediated by the interaction between the parasite's glycoprotein gp82 and the human receptor lysosome-associated membrane protein 2 (LAMP2). While experimental studies have identified a few residues involved in this interaction, a comprehensive molecular-level understanding has been lacking. In this study, we present a 1.44-million-atom computational model of the gp82 complex, including over 3300 lipids, glycosylation sites, and full molecular representations of gp82 and LAMP2, making it the most complete model of a parasite-host interaction to date. Using microsecond-long molecular dynamics simulations and dynamic network analysis, we identified critical residue interactions, including novel regions of contact that were previously uncharacterized. Our findings also highlight the significance of the transmembrane domain of LAMP2 in stabilizing the complex. These insights extend beyond traditional hydrogen bond interactions, revealing a complex network of cooperative motions that facilitate T. cruzi invasion. This study not only confirms key experimental observations but also uncovers new molecular targets for therapeutic intervention, offering a potential pathway to disrupt T. cruzi infection and combat Chagas disease.

This review examines the complex relationship between gestational diabetes mellitus (GDM) and breastfeeding outcomes, integrating recent evidence on maternal health benefits, milk composition, and clinical support strategies. Understanding these relationships is important as GDM affects approximately 14% of pregnancies worldwide, with rates continuing to rise alongside increasing obesity and maternal age. Women who breastfeed for longer periods after GDM show significant improvements in metabolic health, including reduced weight retention and better cardiometabolic profiles. While macronutrient content of breast milk appears preserved, significant differences exist in human milk oligosaccharides and milk fat globule membrane proteins. A previous history of predominant breastfeeding shows a 47% reduction in abnormal fasting glucose odds in subsequent pregnancies. Initial positive indications of personalized support programs, particularly during pregnancy, are emerging however evaluation in comparison to current evidence-based interventions is yet to be carried out. Lifestyle factors are known to reduce subsequent diabetes after a GDM and recent evidence suggests these are important in pregnancy and may improve breastfeeding outcomes. Despite the challenges of delayed secretory activation and reduced milk supply in women with GDM, successful breastfeeding offers substantial health benefits. Healthcare providers could implement comprehensive, individualized support strategies beginning in pregnancy and extending through the postpartum period to optimize outcomes for both mother and infant.

G protein-coupled receptors (GPCRs) are the largest human membrane protein family that transduce extracellular signals into cellular responses. They are major pharmacological targets, with approximately 26% of marketed drugs targeting GPCRs, primarily at their orthosteric binding site. Despite their prominence, predicting the pharmacological effects of novel GPCR-targeting drugs remains challenging due to the complex functional dynamics of these receptors. Recent methodological advances, including X-ray, Cryo-EM, spectroscopic techniques, and molecular simulations, have facilitated studies on how ligands influence the GPCR conformational ensemble. This knowledge is crucial for understanding their interaction with intracellular effector proteins, such as G proteins and β-arrestins, which ultimately modulate downstream cell signaling. In this review, we highlight such aspects in recently discovered small-molecule drugs and drug candidates targeting GPCRs, focusing on three categories: i) allosteric modulators; ii) biased ligands; and iii) bivalent and bitopic compounds. Integrating structural data on ligand-induced receptor functional dynamics into the drug discovery pipeline has the potential to revolutionize GPCR drug design, moving beyond traditional chemical scaffolds towards compounds with tailored pharmacological response.