MP
CryoEM structure of an MHC-I/TAPBPR peptide-bound intermediate reveals the mechanism of antigen proofreading.
Sun Y, Pumroy RA, Mallik L, Chaudhuri A, Wang C, Hwang D, Danon JN, Dasteh Goli K, Moiseenkova-Bell VY, Sgourakis NG.
Proc Natl Acad Sci U S A. 2025 Jan 14;122(2):e2416992122.
doi: 10.1073/pnas.2416992122. Epub 2025 Jan 9.
PMID: 39786927.
Class I major histocompatibility complex (MHC-I) proteins play a pivotal role in adaptive immunity by displaying epitopic peptides to CD8+ T cells. The chaperones tapasin and TAPBPR promote the selection of immunogenic antigens from a large pool of intracellular peptides. Interactions of chaperoned MHC-I molecules with incoming peptides are transient in nature, and as a result, the precise antigen proofreading mechanism remains elusive. Here, we leverage a high-fidelity TAPBPR variant and conformationally stabilized MHC-I, to determine the solution structure of the human antigen editing complex bound to a peptide decoy by cryogenic electron microscopy (cryo-EM) at an average resolution of 3.0 Å. Antigen proofreading is mediated by transient interactions formed between the nascent peptide binding groove with the P2/P3 peptide anchors, where conserved MHC-I residues stabilize incoming peptides through backbone-focused contacts. Finally, using our high-fidelity chaperone, we demonstrate robust peptide exchange on the cell surface across multiple clinically relevant human MHC-I allomorphs. Our work has important ramifications for understanding the selection of immunogenic epitopes for T cell screening and vaccine design applications.
Cryo-EM structure and oligomerization of the human planar cell polarity core protein Vangl1.
Zhang F, Li S, Wu H, Chen S.
Nat Commun. 2025 Jan 3;16(1):135.
doi: 10.1038/s41467-024-55397-2.
PMID: 39753546.
Vangl is a planar cell polarity (PCP) core protein essential for aligned cell orientation along the epithelial plane perpendicular to the apical-basal direction, which is important for tissue morphogenesis, development and collective cell behavior. Mutations in Vangl are associated with developmental defects, including neural tube defects (NTDs), according to human cohort studies of sporadic and familial cases. The complex mechanisms underlying Vangl-mediated PCP signaling or Vangl-associated human congenital diseases have been hampered by the lack of molecular characterizations of Vangl. Here, we show biochemical and structural evidence that human Vangl1 oligomerizes as dimers of trimers, and that the dimerization of trimers promotes binding to the PCP effector Prickle1 (Pk1) in vitro. Mapping of human disease-associated point mutations suggests potential pathological mechanisms and paves the way for future studies on the importance of lipid binding, central vestibule and oligomerization of Vangl, thereby providing insights into the molecular mechanisms of the PCP signaling pathway.
Structural basis of human VANGL-PRICKLE interaction.
Song Y, Jian S, Teng J, Zheng P, Zhang Z.
Nat Commun. 2025 Jan 3;16(1):132.
doi: 10.1038/s41467-024-55396-3.
PMID: 39753555.
Planar cell polarity (PCP) is an evolutionarily conserved process for development and morphogenesis in metazoans. The well-organized polarity pattern in cells is established by the asymmetric distribution of two core protein complexes on opposite sides of the cell membrane. The Van Gogh-like (VANGL)-PRICKLE (PK) pair is one of these two key regulators; however, their structural information and detailed functions have been unclear. Here, we present five cryo-electron microscopy structures of human VANGL1, VANGL2, and their complexes with PK1 at resolutions of 2.2-3.0 Å. Through biochemical and cell imaging experiments, we decipher the molecular details of the VANGL-PK interaction. Furthermore, we reveal that PK1 can target VANGL-containing intracellular vesicles to the peripheral cell membrane. These findings provide a solid foundation to understand the explicit interaction between VANGL and PK while opening new avenues for subsequent studies of the PCP pathway.
Structural basis of THC analog activity at the Cannabinoid 1 receptor.
Thorsen TS, Kulkarni Y, Sykes DA, Bøggild A, Drace T, Hompluem P, Iliopoulos-Tsoutsouvas C, Nikas SP, Daver H, Makriyannis A, Nissen P, Gajhede M, Veprintsev DB, Boesen T, Kastrup JS, Gloriam DE.
Nat Commun. 2025 Jan 8;16(1):486.
doi: 10.1038/s41467-024-55808-4.
PMID: 39779700.
Tetrahydrocannabinol (THC) is the principal psychoactive compound derived from the cannabis plant Cannabis sativa and approved for emetic conditions, appetite stimulation and sleep apnea relief. THC’s psychoactive actions are mediated primarily by the cannabinoid receptor CB1. Here, we determine the cryo-EM structure of HU210, a THC analog and widely used tool compound, bound to CB1 and its primary transducer, Gi1. We leverage this structure for docking and 1000 ns molecular dynamics simulations of THC and 10 structural analogs delineating their spatiotemporal interactions at the molecular level. Furthermore, we pharmacologically profile their recruitment of Gi and β-arrestins and reversibility of binding from an active complex. By combining detailed CB1 structural information with molecular models and signaling data we uncover the differential spatiotemporal interactions these ligands make to receptors governing potency, efficacy, bias and kinetics. This may help explain the actions of abused substances, advance fundamental receptor activation studies and design better medicines.
AFM observation of protein translocation mediated by one unit of SecYEG-SecA complex.
Kanaoka Y, Mori T, Nagaike W, Itaya S, Nonaka Y, Kohga H, Haruyama T, Sugano Y, Miyazaki R, Ichikawa M, Uchihashi T, Tsukazaki T.
Nat Commun. 2025 Jan 8;16(1):225.
doi: 10.1038/s41467-024-54875-x.
PMID: 39779699.
Protein translocation across cellular membranes is an essential and nano-scale dynamic process. In the bacterial cytoplasmic membrane, the core proteins in this process are a membrane protein complex, SecYEG, corresponding to the eukaryotic Sec61 complex, and a cytoplasmic protein, SecA ATPase. Despite more than three decades of extensive research on Sec proteins, from genetic experiments to cutting-edge single-molecule analyses, no study has visually demonstrated protein translocation. Here, we visualize the translocation, via one unit of a SecYEG-SecA-embedded nanodisc, of an unfolded substrate protein by high-speed atomic force microscopy (HS-AFM). Additionally, the uniform unidirectional distribution of nanodiscs on a mica substrate enables the HS-AFM image data analysis, revealing dynamic structural changes in the polypeptide-crosslinking domain of SecA between wide-open and closed states depending on nucleotides. The nanodisc-AFM approach will allow us to execute detailed analyses of Sec proteins as well as visualize nano-scale events of other membrane proteins.
Mutation experiments with AcrB and MexB D408A mutants indicate the efflux liability of antibiotics.
Laura JV Piddock, Vito Ricci, and Maria Laura Ciusa
bioRxiv posted 6 January 2025
doi:10.1101/2025.01.06.631490
We showed that exposure of an AcrB D408A mutant to efflux inhibitors applied evolutionary pressure to select bacteria with the wild type acrB sequence. This suggested that reversion to wild type can differentiate between efflux inhibitors. Thus, we hypothesized that this experiment could identify inhibitors of the primary RND pump, AcrB or its homologues in other species.
To construct three mutants, Escherichia coli AcrB D408A, Klebsiella pneumoniae AcrB D408A and Pseudomonas aeruginosa MexB D408A and expose the mutants to substrates and non-substrates of AcrB and/or MexB and determine the rate of reversion to wildtype acrB/mexB sequence.
Mutant Escherichia coli AcrB D408A, Klebsiella pneumoniae AcrB D408A and Pseudomonas aeruginosa MexB D408A were constructed with site-directed mutagenesis of the relevant nucleotide in the acrB/mexB gene. Mutants were exposed on agar to substrates and the mutation frequency and mutation rate determined. The MIC of antibiotics and the presence/absence of the D408A substitution was determined for mutants.
Exposure to the AcrB substrates chlorpromazine and minocycline reverted the D408A genotypes to wild type in a species-dependent manner. Exposure to a non-AcrB substrate, spectinomycin, did not select wild type acrB. Chlorpromazine selected for wild type acrB K. pneumoniae as it had for S. Typhimurium, whereas minocycline selected for wild type E. coli acrB. None of the antibiotics selected wild type mexB, including the tested MexB substrates.
Evolutionary paths depend upon the genetic background of the species and availability of alternative routes/genetic pathways that can confer resistance/decreased susceptibility to antibiotics.
Structure of WzxE the lipid III flippase for Enterobacterial Common Antigen polysaccharide.
Le Bas A, Clarke BR, Teelucksingh T, Lee M, El Omari K, Giltrap AM, McMahon SA, Liu H, Beale JH, Mykhaylyk V, Duman R, Paterson NG, Ward PN, Harrison PJ, Weckener M, Pardon E, Steyaert J, Liu H, Quigley A, Davis BG, Wagner A, Whitfield C, Naismith JH.
Open Biol. 2025 Jan;15(1):240310.
doi: 10.1098/rsob.240310. Epub 2025 Jan 8.
PMID: 39772807.
The enterobacterial common antigen (ECA) is conserved in Gram-negative bacteria of the Enterobacterales order although its function is debated. ECA biogenesis depends on the Wzx/Wzy-dependent strategy whereby the newly synthesized lipid-linked repeat units, lipid III, are transferred across the inner membrane by the lipid III flippase WzxE. WzxE is part of the Wzx family and required in many glycan assembly systems, but an understanding of its molecular mechanism is hindered due to a lack of structural evidence. Here, we present the first X-ray structures of WzxE from Escherichia coli in complex with nanobodies. Both inward- and outward-facing conformations highlight two pairs of arginine residues that move in a reciprocal fashion, enabling flipping. One of the arginine pairs coordinated to a glutamate residue is essential for activity along with the C-terminal arginine rich tail located close to the entrance of the lumen. This work helps understand the translocation mechanism of the Wzx flippase family.
Protein translocation through α-helical channels and insertases.
Chen J, Zhou X, Yang Y, Li L.
Structure. 2025 Jan 2;33(1):15-28.
doi: 10.1016/j.str.2024.10.032. Epub 2024 Nov 25.
PMID: 39591975.
Protein translocation systems are essential for distributing proteins across various lipid membranes in cells. Cellular membranes, such as the endoplasmic reticulum (ER) membrane and mitochondrial inner membrane, require highly regulated protein translocation machineries that specifically allow the passage of protein polypeptides while blocking smaller molecules like ions and water. Key translocation systems include the Sec translocation channel, the protein insertases of the Oxa1 superfamily, and the translocases of the mitochondrial inner membrane (TIM). These machineries utilize different mechanisms to create pathways for proteins to move across membranes while preventing ion leakage during the dynamic translocation processes. In this review, we highlight recent advances in our understanding of these α-helical translocation machineries and examine their structures, mechanisms, and regulation. We also discuss the therapeutic potential of these translocation pathways and summarize the progress in drug development targeting these systems for treating diseases.
Structural basis of a microbial trimethylamine transporter.
Gao C, Ding H-T, Li K, Cao H-Y, Wang N, Gu Z-T, Wang Q, Sun M-L, Chen X-L, Chen Y, Zhang Y-Z, Fu H-H, Li C-Y.
mBio. 2025 Jan 8;16(1):e0191424.
doi: 10.1128/mbio.01914-24. Epub 2024 Nov 22.
PMID: 39576113.
The volatile trimethylamine (TMA) plays an important role in promoting cardiovascular diseases and depolarizing olfactory sensory neurons in humans and serves as a key nutrient source for a variety of ubiquitous marine microbes. While the TMA transporter TmaT has been identified from a marine bacterium, the structure of TmaT and the molecular mechanism involved in TMA transport remain unclear. In this study, we elucidated the high-resolution cryo-EM structures of TmaT and TmaT-TMA complexes and revealed the TMA binding and transport mechanisms by structural and biochemical analyses. The results advance our understanding of the TMA transport processes across biological membranes.
Cryo-EM structure and complementary drug efflux activity of the Acinetobacter baumannii multidrug efflux pump AdeG.
Ouyang Z, He W, Wu D, An H, Duan L, Jiao M, He X, Yu Q, Zhang J, Qin Q, Wang R, Zheng F, Hwang PM, Hua X, Zhu L, Wen Y.
Structure. 2024 Dec 31:S0969-2126(24)00544-6.
doi: 10.1016/j.str.2024.12.009. Epub ahead of print.
PMID: 39798571.
Multidrug-resistant Acinetobacter baumannii has emerged as one of the most antibiotic-resistant bacterial pathogens associated with nosocomial infection, with its resistance highly depending on multiple multidrug efflux pumps. Here, we report the cryoelectron microscopy (cryo-EM) structure of Acinetobacter drug efflux G (AdeG), the inner membrane component of one of three important resistance-nodulation-cell division (RND) pump family members in A. baumannii, which is involved in drug resistance to chloramphenicol, trimethoprim, ciprofloxacin, and clindamycin. We systematically compare the structures and substrate binding specificities of AdeG, AdeB, and AdeJ multidrug efflux pumps via molecular docking, revealing potential determinants for drug binding. Knockout experiments demonstrate a functional complementarity between AdeABC, AdeFGH, and AdeIJK. Our study provides a structural understanding of A. baumannii multidrug efflux pump AdeG and reveals complementary drug efflux activity between AdeG and other RND efflux pumps, which may promote further rational drug discovery efforts targeting multidrug efflux pumps.
Membrane
Its own architect: Flipping cardiolipin synthase.
Sawasato K, Dowhan W, Bogdanov M.
Sci Adv. 2025 Jan 3;11(1):eads0244.
doi: 10.1126/sciadv.ads0244. Epub 2025 Jan 3.
PMID: 39752486.
Current dogma assumes that lipid asymmetry in biological membranes is actively maintained and dispensable for cell viability. The inner (cytoplasmic) membrane (IM) of Escherichia coli is asymmetric. However, the molecular mechanism that maintains this uneven distribution is unknown. We engineered a conditionally lethal phosphatidylethanolamine (PE)-deficient mutant in which the presence of cardiolipin (CL) on the periplasmic leaflet of the IM is essential for viability, revealing a mechanism that provides CL on the desired leaflet of the IM. CL synthase (ClsA) flips its catalytic cytoplasmic domain upon depletion of PE to supply nonbilayer-prone CL in the periplasmic leaflet of the IM for cell viability. In the presence of a physiological amount of PE, osmotic down-shock induces a topological inversion of ClsA, establishing the biological relevance of membrane protein reorientations in wild-type cells. These findings support a flippase-less mechanism for maintaining membrane lipid asymmetry in biogenic membranes by self-organization of a lipid-synthesizing enzyme.
Structural Dynamics of Neutral Amino Acid Transporter SLC6A19 in Simple and Complex Lipid Bilayers.
Dehury B, Mishra S, Panda S, Singh MK, Simha NL, Pati S.
J Cell Biochem. 2025 Jan;126(1):e30693.
doi: 10.1002/jcb.30693.
PMID: 39749651.
B0AT1 (SLC6A19) is a major sodium-coupled neutral amino acid transporter that relies on angiotensin converting enzyme 2 (ACE2) or collectrin for membrane trafficking. Despite its significant role in disorders associated with amino acid metabolism, there is a deficit of comprehensive structure-function understanding of B0AT1 in lipid environment. Herein, we have employed molecular dynamics (MD) simulations to explore the architectural characteristics of B0AT1 in two distinct environments: a simplified POPC bilayer and a complex lipid system replicating the native membrane composition. Notably, our B0AT1 analysis in terms of structural stability and regions of maximum flexibility shows consistency in both the systems with enhanced structural features in the case of complex lipid system. Our findings suggest that diacylglycerol phospholipids significantly alter the pore radius, hydrophobic index, and surface charge distribution of B0AT1, thereby affecting the flexibility of transmembrane helices TM7, TM12, and loop connecting TM7-TM8, crucial for ACE2-B0AT1 interaction. Pro41, Ser190, Arg214, Arg240, Ser413, Pro414, Cys463, and Val582 are among the most prominent lipid binding residues that might influence B0AT1 functionality. We also perceive notable lipid mediated deviation in the degree of tilt and loss of helicity in TM1 and TM6 which might affect the substrate binding sites S1 and S2 in B0AT1. Considerably, destabilization in the structure of B0AT1 in lipid environment was evident upon mutation in TM domain, associated with Hartnup disorder through various structure-based protein stability tools. Our two-tiered approach allowed us to validate the use of POPC as a baseline for initial analyses of SLC transporters. Altogether, our all-atoms MD study provides a platform for future investigations into the structure-function mechanism of B0AT1 in realistic lipid mimetic bilayers and offers a framework for developing new therapeutic agents targeting this transporter.
Toxic Effects of Butanol in the Plane of the Cell Membrane.
Tan L, Scott HL, Smith MD, Pingali SV, Cheng X, O’Neill HM, Katsaras J, Smith JC, Elkins JG, Davison BH, Nickels JD.
Langmuir. 2025 Jan 8.
doi: 10.1021/acs.langmuir.4c03677. Epub ahead of print.
PMID: 39772768.
Solvent toxicity limits n-butanol fermentation titer, increasing the cost and energy consumption for subsequent separation processes and making biobased production more expensive and energy-intensive than petrochemical approaches. Amphiphilic solvents such as n-butanol partition into the cell membrane of fermenting microorganisms, thinning the transverse structure, and eventually causing a loss of membrane potential and cell death. In this work, we demonstrate the deleterious effects of n-butanol partitioning upon the lateral dimension of the membrane structure, called membrane domains or lipid rafts. Lipid rafts are regions of the cell membrane enriched with certain lipids, providing a reservoir of high melting temperature lipids and a platform for membrane protein partitioning and oligomerization. Neutron scattering experiments and molecular dynamics simulations revealed that n-butanol increased the size of the lipid domains in a model membrane system. The data showed that n-butanol partitions more into the disordered lipid regions than into the raft-like phase, leading to a differential thinning of these coexisting phases in the plane of the membrane and increasing the hydrophobic mismatch. The resulting increase in line tension at the interface favors domain coalescence to minimize the ratio of the interfacial length to domain area. A detailed computational investigation of the lipid domain interface identifies the boundary as a site of membrane disorder and thinning due to an accumulation of n-butanol. Solvent-induced changes to domain morphology and membrane instability at the domain interface are unrecognized modes of solvent-induced stress to fermenting microbes, representing targets for new solvent tolerance strategies to increase the n-butanol titer.
The influence of phosphoinositide lipids in the molecular biology of membrane proteins: recent insights from simulations.
Hedger G, Yen HY.
J Mol Biol. 2025 Jan 8:168937.
doi: 10.1016/j.jmb.2025.168937. Epub ahead of print.
PMID: 39793883.
The phosphoinositide family of membrane lipids play diverse and critical roles in eukaryotic molecular biology. Much of this biological activity derives from interactions of phosphoinositide lipids with integral and peripheral membrane proteins, leading to modulation of protein structure, function, and cellular distribution. Since the discovery of phosphoinositides in the 1940s, combined molecular biology, biophysical, and structural approaches have made enormous progress in untangling this vast and diverse cellular network of interactions. More recently, in silico approaches such as molecular dynamics simulations have proven to be an asset in prospectively identifying, characterising, explaining the structural basis of these interactions, and in the best cases providing atomic level testable hypotheses on how such interactions control the function of a given membrane protein. This review details a number of recent seminal discoveries in phosphoinositide biology, enabled by advanced biomolecular simulation, and its integration with molecular biology, biophysical, and structural biology approaches. The results of the simulation studies agree well with experimental work, and in a number of notable cases have arrived at the key conclusion several years in advance of the experimental structures. Condensed title:Simulations of phosphoinositides and membrane proteins SUMMARY: Hedger and Yen review developments in simulations of phosphoinositides and membrane proteins.
Molecules
Unsymmetric triazine-based triglucoside detergents for membrane protein stability.
Ehsan M, Ghani L, Lan B, Katsube S, Poulsen IH, Zhang X, Arslan M, Byrne B, Loland CJ, Guan L, Liu X, Chae PS.
Chembiochem. 2025 Jan 8:e202400958.
doi: 10.1002/cbic.202400958. Epub ahead of print.
PMID: 39779472.
Membrane proteins play a crucial role in a variety of biological processes and are key targets for pharmaceutical development. Structural studies of membrane proteins provide molecular insights into the mechanisms of these processes and are essential for effective drug discovery. Historically, these studies have relied on solubilization of the target protein using detergents, but conventional detergents often fail to maintain the stability of challenging membrane proteins. To address this issue, there is a need to develop novel detergents with enhanced protein stabilization properties. In this study, we synthesized unsymmetric variants of recently reported tris(hydroxymethyl)aminomethane(TRIS)-linker-bearing triazine-based triglucosides (TTGs) by incorporating two different alkyl chains (long and short) into the detergent structure. When tested with model membrane proteins, including a G protein-coupled receptor, TTG-8,12 demonstrated superior efficacy in stabilizing membrane proteins compared to the original TTGs and the gold standard detergent DDM/LMNG. These results suggest that detergent unsymmetry is an important concept for improving detergent performance and unsymmetric detergents such as TTG-8,12 hold significant potential for advancing membrane protein structural studies.
Peptide-Scaffolded Detergents for Membrane Protein Studies.
Yang M, Dai Y, Zhou F, Zhou X, Qiu Y, Tan Y, Zhao S, Xue D, Zhao F, Tao H.
Chemistry. 2025 Jan 7:e202404520.
doi: 10.1002/chem.202404520. Epub ahead of print.
PMID: 39777805.
Detergents are essential for preserving the structural integrity and functionality of membrane proteins (MPs) outside the biological membrane or in aqueous solution, and thus ensuring accurate biochemical and structural analyses. Here, we introduce peptide-scaffolded detergents, a novel class of hybrid molecules formed by preassembling detergent monomers with peptides of varying lengths, mediated via Click chemistry. These detergents are characterized by scalable, straightforward synthesis and enhanced solubility. Among the variants, A4B2 emerged as the optimal detergent, demonstrating superior thermal stabilization across a range of G protein-coupled receptors, including A2AAR, SMO and GLP-1R. Additionally, A4B2 exhibits a low critical micelle concentration and small micelle size, together making it particularly effective for electron microscopy studies of A2AAR. This innovative design leverages the benefits of peptide-based and traditional detergents, offering new insights for the development of advanced detergents in MP research.
Cell-free expression and SMA copolymer encapsulation of a functional receptor tyrosine kinase disease variant, FGFR3-TACC3.
Alexander J D Snow, Tharushi Wijesiriwardena, Benjamin J Lane, Brendan Farrell, Polly C Dowdle, Matilda Katan, Stephen P Muench, and Alexander Breeze.
bioRxiv posted 8 January 2025.
doi:10.1101/2024.06.04.596442.
Despite their high clinical relevance, obtaining structural and biophysical data on transmembrane proteins has been bottlenecked by challenges involved in their expression. The inherent enzymatic activity of receptor tyrosine kinases (RTK) presents an additional hurdle to producing functional protein. The oncogenic fusion of proteins to such RTKs creates a particularly difficult-to-express protein subtype due to their high flexibility, lack of stability, and propensity for aggregation. One such protein is the fibroblast growth factor receptor 3 fused with transforming acidic coiled-coil-containing protein 3 (FGFR3-TACC3), which has failed to express to sufficient quality or functionality in traditional expression systems. Cell-free protein expression (CFPE) is a burgeoning arm of synthetic biology, enabling the rapid and efficient generation of recombinant proteins. This platform is characterised by utilising an optimised solution of cellular machinery to facilitate protein synthesis in vitro. In doing so, CFPE can act as a surrogate system for a range of proteins that are otherwise difficult to express through traditional host cell-based approaches. Here, functional FGFR3-TACC3 was expressed through a novel cell-free expression system in under 48 hours. The resultant protein can be reconstituted using SMA copolymers. Functionally, the protein demonstrated significant kinase domain phosphorylation (t<0.0001). Currently, there is no published, high-resolution structure of any full-length RTK. These findings form a promising foundation for future research on oncogenic RTKs and the application of cell-free systems for synthesising functional membrane proteins.
Assembly of oleosin during efficient extraction: Altering the sequence of defatting solvents.
Li Y, Qiao Y, Zhu Y, Shen W, Jin W, Peng D, Huang Q.
Food Chem X. 2024 Nov 20;25:102022.
doi: 10.1016/j.fochx.2024.102022.
PMID: 39758061.
During the extraction of membrane proteins from oil bodies (OBs), organic solvents dissolve the lipid core and precipitate proteins through solvent stress. Here the effects of solvent type and defatting sequence on the composition and structure of membrane proteins were investigated via SDS-PAGE, FTIR, and SEM-EDS. High purity oleosin (86 %) was obtained by treatment first with a Floch solution and then with cold acetone and petroleum ether after twice washing OBs with urea. The 3D spatial structure of oleosin was predicted using AlphaFold 2, revealing that the secondary structure of oleosin was dominated by α-helices (>60 %). Oleosin consisted of two district types, with oleosin-H (16-17 kDa) being the part of the molecule with limited water solubility, while oleosin-L (13-14 kDa) constituted the non-soluble part. The results provided a technical means of efficient extraction of Camellia oleosins and selective separation of oleosin-L and oleosin-H.
Comparison of lipid dynamics and permeability in styrene-maleic acid and diisobutylene-maleic acid copolymer lipid nanodiscs by electron paramagnetic resonance spectroscopy.
Morris AK, McCarrick RM, Lorigan GA.
J Biol Inorg Chem. 2025 Jan 11.
doi: 10.1007/s00775-024-02091-9. Epub ahead of print.
PMID: 39797925.
Lipid nanoparticles formed with copolymers are a new and increasingly powerful tool for studying membrane proteins, but the extent to which these systems affect the physical properties of the membrane is not completely understood. This is critical to understanding the caveats of these new systems and screening for structural and functional artifacts that might be caused in the membrane proteins they are used to study. To better understand these potential effects, the fluid properties of dipalmitoylphosphatidylcholine lipid bilayers were examined by electron paramagnetic resonance (EPR) spectroscopy with spin-labeled reporter lipids in either liposomes or incorporated into nanoparticles with the copolymers diisobutylene-maleic acid or styrene maleic acid. Lineshape analysis at varying temperatures reveal a major change in the phase transition behavior of the lipids from a sharp melting curve in liposomes to a more gradual transition in nanoparticles. Electron spin echo envelope modulation (ESEEM) spectroscopy reveals changes in water permeability between mimetic systems, which is further supported by power-saturation measurements showing increased dequenching of spin lipids in diisobutylene-maleic acid nanoparticles compared to maleic acid nanoparticles. These results suggest that diisobutylene-maleic acid nanoparticles may have more physiological fluid properties than styrene-maleic acid nanoparticles when incorporated with saturated phospholipids.
Methods
MPicker: visualizing and picking membrane proteins for cryo-electron tomography.
Yan X, Li S, Huang W, Wang H, Zhao T, Huang M, Zhou N, Shen Y, Li X.
Nat Commun. 2025 Jan 8;16(1):472.
doi: 10.1038/s41467-024-55767-w.
PMID: 39774981.
Advancements in cryo-electron tomography (cryoET) allow the structure of macromolecules to be determined in situ, which is crucial for studying membrane protein structures and their interactions in the cellular environment. However, membranes are often highly curved and have a strong contrast in cryoET tomograms, which masks the signals from membrane proteins. These factors pose difficulties in observing and revealing the structures of membrane proteins in situ. Here, we report a membrane-flattening method and the corresponding software, MPicker, designed for the visualization, localization, and orientation determination of membrane proteins in cryoET tomograms. This method improves the visualization of proteins on and around membranes by generating a flattened tomogram that eliminates membrane curvature and reduces the spatial complexity of membrane protein analysis. In MPicker, we integrated approaches for automated particle picking and coarse alignment of membrane proteins for sub-tomogram averaging. MPicker was tested on tomograms of various cells to evaluate the method for visualizing, picking, and analyzing membrane proteins.
A fiducial-assisted strategy compatible with resolving small MFS transporter structures in multiple conformations using cryo-EM.
Xie P, Li Y, Lamon G, Kuang H, Wang DN, Traaseth NJ.
Nat Commun. 2025 Jan 2;16(1):7.
doi: 10.1038/s41467-024-54986-5.
PMID: 39746942.
Advancements in cryo-EM have stimulated a revolution in structural biology. Yet, for membrane proteins near the cryo-EM size threshold of approximately 40 kDa, including transporters and G-protein coupled receptors, the absence of distinguishable structural features makes image alignment and structure determination a significant challenge. Furthermore, resolving more than one protein conformation within a sample, a major advantage of cryo-EM, represents an even greater degree of difficulty. Here, we describe a strategy for introducing a rigid fiducial marker (BRIL domain) at the C-terminus of membrane transporters from the Major Facilitator Superfamily (MFS) with AlphaFold2. This approach involves fusion of the last transmembrane domain helix of the target protein with the first helix of BRIL through a short poly-alanine linker to promote helicity. Combining this strategy with a BRIL-specific Fab, we elucidated four cryo-EM structures of the 42 kDa Staphylococcus aureus transporter NorA, three of which were derived from a single sample corresponding to inward-open, inward-occluded, and occluded conformations. Hence, this fusion construct facilitated experiments to characterize the conformational landscape of NorA and validated our design to position the BRIL/antibody pair in an orientation that avoids steric clash with the transporter. The latter was enabled through AlphaFold2 predictions, which minimized guesswork and reduced the need for screening several constructs. We further validated the suitability of the method to three additional MFS transporters (GlpT, Bmr, and Blt), results that supported a rigid linker between the transporter and BRIL. The successful application to four MFS proteins, the largest family of secondary transporters in nature, and analysis of predicted structures for the family indicates this strategy will be a valuable tool for studying other MFS members using cryo-EM.
Cell-Free Systems and Their Importance in the Study of Membrane Proteins.
González-Ponce KS, Celaya-Herrera S, Mendoza-Acosta MF, Casados-Vázquez LE.
J Membr Biol. 2025 Jan 6.
doi: 10.1007/s00232-024-00333-0. Epub ahead of print.
PMID: 39760767.
The Cell-Free Protein Synthesis (CFPS) is an innovative technique used to produce various proteins. It has several advantages, including short expression times, no strain engineering is required, and toxic proteins such as membrane proteins can be produced. However, the most important advantage is that it eliminates the need for a living cell as a production system. Membrane proteins (MPs) are difficult to express in heterologous strains such as Escherichia coli. Modified strains must be used, and sometimes the strain produces them as inclusion bodies, which makes purification difficult. CFPS can avoid the problem of toxicity and, with the use of additives, allows the production of folded and functional membrane proteins. In this review, we focus on describing what cell-free systems are. We address the advantages and disadvantages of the different organisms that can be used to obtain cell extracts, including PURE systems, where the components are obtained recombinantly, and the methodologies that allow the synthesis of membrane proteins in cell-free systems, which, given their hydrophobic nature, require additives for their correct folding.
Real-time analysis of nanoscale dynamics in membrane protein insertion via single-molecule imaging.
Yang C, Ma D, Hu S, Li M, Lu Y.
Biophys Rep. 2024 Dec 31;10(6):369-376.
doi: 10.52601/bpr.2024.240024.
PMID: 39758427.
Membrane proteins often need to be inserted into or attached to the cell membrane to perform their functions. Understanding their transmembrane topology and conformational dynamics during insertion is crucial for elucidating their roles. However, it remains challenging to monitor nanoscale changes in the insertion depth of individual proteins in membranes. Here, we introduce two single-molecule imaging methods, SIFA and LipoFRET, designed for in vitro observation of the nanoscale architecture of membrane proteins within membranes. These methods have demonstrated their efficacy in studying biomolecules interacting with bio-membranes with sub-nanometer precision.
Robust visualization of membrane protein by aptamer mediated proximity ligation assay and Förster resonance energy transfer.
Li Y, Qian M, Cheng Y, Qiu X.
Colloids Surf B Biointerfaces. 2024 Dec 30;248:114486.
doi: 10.1016/j.colsurfb.2024.114486. Epub ahead of print.
PMID: 39756158.
In situ cell imaging plays a crucial role in studying physiological and pathological processes of cells. Proximity ligation assay (PLA) and rolling circle amplification (RCA) are commonly used to study the abundance and interactions of biological macromolecules. The most frequently applied strategy to visualize the RCA products is with single-fluorophore probe, however, cellular auto-fluorescence and unbound fluorescent probes could interfere with RCA products, leading to non-specific signals. Here, we present a novel approach combining aptamer mediated PLA, RCA, and Förster Resonance Energy Transfer (FRET), namely Apt-PLA-RCA-FRET, for sensitive in situ imaging and analysis of the abundances and interactions of membrane proteins such as tetraspanin CD63 and human epidermal growth factor receptor 2 (HER2). Apt-RCA-FRET was initially designed to show its ability to assess the abundance of target proteins on different cells. Dual functional oligonucleotides served as both the aptamer for recognizing specific membrane proteins and the primer of circular DNA for following RCA process, and the resulting RCA products were subsequently imaged by FRET signals from Cy3 to Cy5 probes which hybridized sequentially on them. FRET was demonstrated to show its great potential to resist the interferences of nonspecific fluorescence compared to single-fluorophore strategies. PLA was then introduced to Apt-RCA-FRET to investigate the spatial localization of different proteins on cell membrane and their interactions. Our approach utilizing aptamer as membrane proteins recognition element simply converted the abundance of proteins into nucleic acid signals and facilitated the following signal amplification, thus it serves as an important alternative to methods typically based on antibody and presents a more robust and sensitive method for analyzing the abundances of different cell membrane proteins and their spatial localization, which offers valuable insights into physiological and pathological processes of cells.
Lighting Up Dual-Aptamer-Based DNA Logic-Gated Series Lamp Probes with Specific Membrane Proteins for Sensitive and Accurate Cancer Cell Identification.
Zhou X, Yu C, Wei X, Jia H, Zheng L, Shen Z, Wu R, Xue C.
Anal Chem. 2025 Jan 9.
doi: 10.1021/acs.analchem.4c05505. Epub ahead of print.
PMID: 39786914.
Accurate identification of cancer cells under complex physiological environments holds great promise for noninvasive diagnosis and personalized medicine. Herein, we developed dual-aptamer-based DNA logic-gated series lamp probes (DApt-SLP) by coupling a DNA cell-classifier (DCC) with a self-powered signal-amplifier (SSA), enabling rapid and sensitive identification of cancer cells in a blood sample. DCC is endowed with two extended-aptamer based modules for recognizing the two cascade cell membrane receptors and serves as a DNA logic gate to pinpoint a particular and narrow subpopulation of cells from a larger population of similar cells. DCC leverages a dual-receptor co-recognition strategy for enhanced specificity of cell identification by performing the matching operation between aptamer and receptor twice on cell membranes. SSA is a signal converter attached at the end of DCC that changes the cell identification process into detectable signals, as well as a signal amplifier to output amplified signals by using a simple and efficient hybridization chain reaction. Unique from those who are multicomponent systems, DApt-SLP is an all-in-one compact DNA nanodevice, exhibiting an enhanced nuclease-degradation resistance and targeting ability. In vitro feasibility, cell imaging, and flow cytometry analysis showed that the DApt-SLP system successfully operated under buffered solution and physiological environment and precisely differentiated the target cell from large populations of similar cells. Benefiting from its integrated design and single-step cancer cell identification with high sensitivity and accuracy, the DApt-SLP system is a practical tool in personalized medicine and biomedical engineering.
Anisotropic interactions for continuum modeling of protein-membrane systems.
Oppelstrup T, Stanton LG, Tempkin JOB, Ozturk TN, Ingólfsson HI, Carpenter TS.
J Chem Phys. 2024 Dec 28;161(24):244908.
doi: 10.1063/5.0237408.
PMID: 39786911.
In this work, a model for anisotropic interactions between proteins and cellular membranes is proposed for large-scale continuum simulations. The framework of the model is based on dynamic density functional theory, which provides a formalism to describe the lipid densities within the membrane as continuum fields while still maintaining the fidelity of the underlying molecular interactions. Within this framework, we extend recent results to include the anisotropic effects of protein-lipid interactions. As applications, we consider two membrane proteins of biological interest: a RAS-RAF complex tethered to the membrane and a membrane embedded G protein-coupled receptor. A strong qualitative and quantitative agreement is found between the numerical results and the corresponding molecular dynamics simulations. Combining the scope of continuum level simulations with the details from molecular level particle simulations enables research into protein-membrane behaviors at a more biologically relevant scale, which crucially can also be accessed via experiment.
Vesicle-Encapsulated Chemosensing Ensembles Allow Monitoring of Transmembrane Uptake Coupled with Enzymatic Reactions.
Jiang R, Nilam M, Piselli C, Winterhalter M, Guo DS, Yu SY, Hennig A, Nau WM.
Angew Chem Int Ed Engl. 2025 Jan 9:e202425157.
doi: 10.1002/anie.202425157. Epub ahead of print.
PMID: 39785152.
Compartmentalized models with coupled catalytic networks are considered as “protocells” in the context of research related to the origin of life. To model the kinetics of a simple cellular uptake-metabolism process, we use a compartmentalized protocell system that combines liposome-encapsulated intravesicular reporter pairs with co-encapsulated enzymes to monitor the membrane transport of a substrate (analyte uptake) and its subsequent enzymatic reaction inside the vesicles (metabolism to the product). The intravesicular chemosensing ensembles consist of the macrocycles cucurbit[7]uril or p-sulfonatocalix[4]arene and matching fluorescent dyes to set up suitable reporter pairs. When these macrocycle/dye reporter pairs are co-encapsulated with enzymes (trypsin, protein kinase A, or butyrylcholinesterase), it is possible to monitor first the transport of different substrates (polylysine, protamine, H-LRRWSLG-OH, or butyrylcholine) through added pores (outer membrane proteins F and C), with synthetic carriers (amphiphilic calixarenes), or by direct permeation (only for butyrylcholine). The subsequent enzymatic conversions of the substrates after they have entered the corresponding protocells can be monitored as consecutive reactions. The new type of in vitro assays can be applied to different enzymes and analytes, affording a comprehensive chemosensing system of high chemical complexity.
Probing ligand-induced conformational changes in an MFS transporter in vivo using site-directed PEGylation.
Booncherm V, Gill H, Anderson E, Mostafa S, Mercado C, Jiang X.
J Mol Biol. 2025 Jan 10:168941.
doi: 10.1016/j.jmb.2025.168941. Epub ahead of print.
PMID: 39799991.
So far, site-directed alkylation (SDA) studies on transporters in the Major Facilitator Superfamily (MFS) are mostly performed at conditions different from the native cellular environment. In this study, using GFP-based site-directed PEGylation, ligand-induced conformational changes in the lactose permease of Escherichia coli (LacY), were examined in vivo for the first time. Accessibility/reactivity of single-Cys replacements in a Cys-less LacY-eGFP fusion background was tested using methoxy polyethylene glycol-maleimide-5K (mPEG-Mal-5K) in the absence or presence of a ligand, and the band-shift of the fusion upon PEGylation was detected by in-gel fluorescence. Ligand binding increases the rate of PEGylation at five out of eight tested positions on the periplasmic side in vivo, while decreasing the rate of PEGylation at both positions tested on the cytoplasmic side in situ. Upon ligand binding, the rate of PEGylation at two periplasmic positions, K42 and Q242, slightly decreases in vivo, but increases in situ, indicating the conformational behavior of these two residues in living cells may not be identical to that in isolated cell membranes. Furthermore, abolishing the electrochemical H+ gradient (Δµ̃H+) reduces the rate of PEGylation at all tested positions on the periplasmic side. We also found that, unlike the linear form, the branched (Y-shape) mPEG-Mal-5K cannot pass the outer membrane. This work characterizes the alternating access of LacY in the context of a living cell and demonstrates that this methodology is feasible and effective for dynamical studies of MFS transporters.
Microbio
Role of the two-component system AmgRS in early resistance of Pseudomonas aeruginosa to cinnamaldehyde.
Dubois E, Spasovski V, Plésiat P, Llanes C.
Microbiol Spectr. 2025 Jan 7;13(1):e0169924.
doi: 10.1128/spectrum.01699-24. Epub 2024 Dec 10.
PMID: 39656006.
Exposure of Pseudomonas aeruginosa to cinnamaldehyde (CNA), a natural electrophilic antimicrobial often used as self-medication to treat mild infections, triggers overproduction of the MexAB-OprM efflux system, leading to multidrug resistance. In this study, we demonstrate that CNA exposure induces expression of genes regulated by the two-component system AmgRS. AmgRS activates MexAB-OprM production, independent of repressors MexR and NalD. In addition to the essential role played by the NalC-ArmR pathway in this adaptive process, AmgRS is critical for the survival of P. aeruginosa challenged with CNA. Altogether, these data suggest that efflux-dependent and -independent mechanisms are activated in the early phase of CNA exposure, allowing for progressive enzymatic reduction of the biocide to non-toxic cinnamic alcohol.IMPORTANCEExposure of Pseudomonas aeruginosa to cinnamaldehyde (CNA), an antimicrobial used in self-medication, induces overproduction of the MexAB-OprM efflux system, leading to multidrug resistance. Our study demonstrates that the AmgRS two-component system aids in the survival of P. aeruginosa strain PA14 under CNA exposure through both MexAB-OprM-dependent and -independent mechanisms until the enzymatic reduction of CNA into the less toxic cinnamic alcohol. This discovery highlights the pivotal role of AmgRS in mediating defense against aldehyde biocides, emphasizing its significance in the persistence of P. aeruginosa, a pathogen associated with hospital-acquired infections and cystic fibrosis, and underscores the potential impact on clinical treatment strategies.
A Conjugated Oligomer with Drug Efflux Pump Inhibition and Photodynamic Therapy for Synergistically Combating Resistant Bacteria.
Li M, Li L, Zhang X, Yuan Q, Bao B, Tang Y.
ACS Appl Mater Interfaces. 2025 Jan 9.
doi: 10.1021/acsami.4c20278. Epub ahead of print.
PMID: 39787568.
High expression of drug efflux pump makes antibiotics ineffective against bacteria, leading to drug-resistant strains and even the emergence of “superbugs”. Herein, we design and synthesize a dual functional o-nitrobenzene (NB)-modified conjugated oligo-polyfluorene vinylene (OPFV) photosensitizer, OPFV-NB, which can depress efflux pump activity and also possesses photodynamic therapy (PDT) for synergistically overcoming drug-resistant bacteria. Upon light irradiation, the OPFV-NB can produce aldehyde active groups to covalently bind outer membrane proteins, such as tolerant colicin (TolC), blocking drug efflux of bacteria. The minimum inhibitory concentration of antibiotic model chloramphenicol (CHL) is reduced about 64 times, significantly resensitizing drug-resistant bacteria to antibiotics. Also, the probe can produce highly efficient reactive oxygen species (ROS) under light irradiation. Consequently, the unimolecular OPFV-NB-based system demonstrates insusceptibility to antibiotic resistance while maintaining significant antimicrobial effects (100%) against drug-resistant bacteria. More importantly, in vivo assays corroborate that the combined system greatly accelerates wound healing by eradicating the bacterial population, dampening inflammation, and promoting angiogenesis. Overall, the OPFV-NB not only counteracts antibiotic resistance but also holds tremendous PDT efficiency, which provides a promising therapeutic strategy for combating drug-resistant bacteria and treating bacteria-infected wounds.
Deciphering bacterial protein functions with innovative computational methods.
Cheskis S, Akerman A, Levy A.
Trends Microbiol. 2024 Dec 29:S0966-842X(24)00316-0.
doi: 10.1016/j.tim.2024.11.013. Epub ahead of print.
PMID: 39736484.
Bacteria colonize every niche on Earth and play key roles in many environmental and host-associated processes. The sequencing revolution revealed the remarkable bacterial genetic and proteomic diversity and the genomic content of cultured and uncultured bacteria. However, deciphering functions of novel proteins remains a high barrier, often preventing the deep understanding of microbial life and its interaction with the surrounding environment. In recent years, exciting new bioinformatic tools, many of which are based on machine learning, facilitate the challenging task of gene and protein function discovery in the era of big genomics data, leading to the generation of testable hypotheses for bacterial protein functions. The new tools allow prediction of protein structures and interactions and allow sensitive and efficient sequence- and structure-based searching and clustering. Here, we summarize some of these recent tools which revolutionize modern microbiology research, along with examples for their usage, emphasizing the user-friendly, web-based ones. Adoption of these capabilities by experimentalists and computational biologists could save resources and accelerate microbiology research.
Miscellaneous
We need to talk about human genome editing.
Nature. 2025 Jan;637(8045):252.
doi: 10.1038/d41586-025-00015-4. PMID: 39780015.
Editorial: In a few decades, gene-editing technologies could reduce the likelihood of common human diseases. Societies must use this time to prepare for their arrival.
Pan-European atmospheric lead pollution, enhanced blood lead levels, and cognitive decline from Roman-era mining and smelting.
McConnell JR, Chellman NJ, Plach A, Wensman SM, Plunkett G, Stohl A, Smith NK, Møllesøe Vinther B, Dahl-Jensen D, Steffensen JP, Fritzsche D, Camara-Brugger SO, McDonald BT, Wilson AI.
Proc Natl Acad Sci U S A. 2025 Jan 21;122(3):e2419630121.
doi: 10.1073/pnas.2419630121. Epub 2025 Jan 6.
PMID: 39761387.
There was so much lead in the air during the ancient Pax Romana period that it might have caused cognitive decline in people across Europe. The smelting of silver from the mineral galena a widespread industry at the time of the Roman Empire releases lead as a gas. By combining data from ice cores which can act as records of pollution over time with historical climate information researchers worked out that the amount of lead Roman people might have had in their blood in childhood taken in from air pollution could have caused an average drop of 2.5-3 IQ points.
Should offensive species names be changed? The organisms that honour dictators, racists and criminals.
Thompson B.
Nature. 2024 Dec 16. doi: 10.1038/d41586-024-04200-9.
Epub ahead of print.
PMID: 39690231.
In episode 1 of What’s in a name we look at how species are named, and whether the current system needs to evolve in the face of societal pressure.
Nanopasta: electrospinning nanofibers of white flour.
Britton B, Zhang F, Anthony DB, Reyes CIDL, Pawlus M, Williams GR, Clancy AJ.
Nanoscale Adv. 2024 Oct 30;6(24):6129-6133.
doi: 10.1039/d4na00601a.
PMID: 39583133.
Scientists have created the world’s thinnest “spaghetti,” : less than a thousandth of a millimeter thick !
However, it is too delicate to be used as edible pasta. Instead, this “nanopasta” has promising applications in medicine, such as wound dressings, tissue scaffolds, and drug delivery systems, due to its porosity and ability to mimic the extracellular matrix that supports cell growth.
What’s going wrong with tennis balls
Giri Nathan
https://defector.com/has-the-tennis-ball-gotten-worse
Professional players say that there’s trouble in the state of tennis: the balls are no good — and getting worse. Complaints have led ball-makers to reveal the specifics of engineering and testing that goes into producing tennis balls. As well as manufacturing challenges that arose from the COVID-19 pandemic, companies point to environmental variations that can change ball behaviour and rough courts that can strip the spheres’ fuzz. Meanwhile the game gets ever faster and more powerful, leaving balls struggling to bounce back as they once did.
Les Folies Moléculaires
Where science is a fun and creative adventure !!!
Many thanks Marc Baaden for sharing this cool site …