Synthetic cells aim to emulate living systems by reconstituting essential cellular processes within lipid-bound architectures. However, their functional complexity remains constrained by a key challenge: the synthesis and correct integration of hydrophobic membrane proteins via cell-free approaches. Here, inspired by natural cells, we developed a spatially regulated translation strategy in which membrane-anchored mRNAs recruit ribosomes to drive the cotranslational insertion of membrane proteins into lipid bilayers. This design enables efficient in situ synthesis and integration of multiple transmembrane proteins within giant unilamellar vesicles, supporting selective small-molecule transport across membranes. Importantly, the method allows for precise stoichiometric control of membrane protein composition. Together, this work establishes a minimal yet versatile framework for the direct synthesis and integration of membrane proteins, advancing the construction of functional synthetic cells.

DNA polymerases (DNAPs) catalyze DNA replication by coordinating the polymerase and exonuclease activities to enhance the fidelity through exonuclease proofreading at the cost of additional dNTP consumption. Whether and how DNAPs achieve an optimal or even a robust optimal fidelity-cost trade-off remains elusive. By building a ladder-like model to describe the synthesis-excision kinetics and adopting an objective function Q to explicitly quantify the trade-off, we revealed by Q maximization that DNAPs, having long-range mismatch sensing and complex excision modes, could fine-tune key parameters that are intrinsic to excision to achieve an optimal fidelity-cost trade-off, which is robust to large fluctuations in synthesis-specific parameters. Part of the predictions on the key parameters were validated by high-resolution single-molecule assays on two DNAPs (KF and gp5). Our work reveals a new and potentially universal optimization strategy of DNAPs, and also offers a new perspective to understand the architecture of molecular machines with distinctly separated multiple domains, i.e., the structural modularity may enable robust functional optimization of the machine if part of its domains are subject to fluctuating parameters.

Cryo-electron microscopy (cryo-EM) has emerged as a very powerful tool for high-resolution structure determination of macromolecule complexes. However, sample preparation remains a major bottleneck in cryo-EM workflows. Most structures were resolved not in a native solution environment, but rather in the non-physiological interfaces such as the air-water interface (AWI), where the folding/unfolding energy landscape of macromolecules may be significantly altered, leading to artifact, preferential orientation, particle disassembly or even denaturation. To address this challenge, we developed a robust, sample-independent method utilizing liposome encapsulation that preserve macromolecules in a solution-like and near native environment throughout sample preparation. Using equine spleen apoferritin and the Escherichia coli ( E. coli) ribosome as model systems, we demonstrate efficient particle incorporation into liposomes and successful high-resolution structure determination. Notably, our method yields significantly reduced particle disassembly, denaturation, and preferential orientation compared to samples on holey carbon and graphene grids. We anticipate that this approach will facilitate structural studies of other challenging macromolecular complexes that are sensitive to interfacial effects. ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, T2221001

Nucleosomes are the fundamental unit of chromatin. Chromatin remodeler plays a crucial role in the regulation of gene expression in eukaryotes. It is involved in important physiological processes, such as development, immune response, and metabolic regulation. During gene expression regulation, chromatin remodelers slide nucleosomes along genomic DNA and play a major role in chromatin organization. Chd1 senses the extranucleosomal linker DNA and controls nucleosome spacing in cells. However, the mechanism of linker DNA sensing by Chd1 is not completely understood. Here, we report the cryo-electron microscope (cryoEM) structures of Chd1 engaging nucleosomes in different states. Chd1 induces two exit-DNA conformations, either fully wrapped or partially unwrapped states. Notably, in the unwrapped conformation, the exit DNA interacts with a positively charged loop of the motor, named the exit-DNA binding loop, and traps Chd1 in the closed state in the ATPase cycle, suggesting attenuation of its remodeling activity. Explored single-molecule fluorescence resonance energy transfer (smFRET) and biochemical data supported the regulation of Chd1 remodeling activity by the exit-DNA conformations, which is important for the linker DNA sensitivity. Mutants of the Chd1 exit-DNA binding loop compromised nucleosome organization in yeast cells. Together, our findings provide valuable insights into Chd1 regulation by exit DNA unwrapping. These results provide a new perspective for the study of cell development and metabolism.

Understanding rapid, nonequilibrium dynamics of single proteins in lipid membranes is crucial but challenging. This study advances fluorescence lifetime analysis by developing a computationally efficient variational Bayesian framework for photon-by-photon hidden Markov modeling. It enables robust and accurate model selection, facilitating real-time tracking of state evolution of a molecule within a brief time frame. We applied the method to investigate nonequilibrium membrane insertion of a model peptide, revealing that unsaturated bonds in acyl chains not only merely modulate the fluidity of lipid membranes but also directly interact with transmembrane proteins, answering a long-standing question about unsaturated bonds' roles in membrane-protein interactions.

DNA polymerases achieved high fidelity via kinetic proofreading. However, due to the lack of proper enzymatic reaction models, the mechanism has not yet been successfully applied to quantify the fidelity of polymerases. Here, we examine the multistep conformational transitions of DNA polymerase I in both binary and ternary complexes, each having three conformations: open, partially closed (ajar), and closed. The transition patterns and dwell time distributions reveal a hidden transition pathway connecting the ternary ajar state to the binary ajar state, reflecting the second rejection point of an incorrect nucleotide, in addition to the first one upon initial binding. Once the ternary complex reaches the closed state, the incorrect nucleotide can hardly be rejected directly. However, the increased closed-to-ajar reverse transition enables it to be rejected at the ternary ajar state. We constructed a reaction network connecting the dynamics of the binary and ternary complexes. It allows for quantifying the contribution of each checkpoint to the fidelity under various conditions. Our results emphasize the critical role of the ajar state in enhancing the fidelity of the polymerase.

Channel proteins act as precise molecular regulators of transmembrane transport,which is a fundamental process essential for maintaining cellular homeostasis.These proteins dynamically modulate their functional states through conformational changes,thereby forming the structural basis for complex physiological processes such as signal transduction and energy metabolism.Single-molecule fluorescence spectroscopy and single-channel patch-clamp electrophysiology represent two cornerstone techniques in modern biophysics:the former enables molecular-resolution analysis of structural dynamics,while the latter provides direct functional characterization of ion channel activity.Despite their complementary capabilities,integrating these techniques to simultaneously monitor protein conformational dynamics and functional states remains technically challenging,primarily due to the strong autofluorescence background inherent in single-molecule imaging in cellular environments.To address this limitation,we develop a spatially selective optical excitation system capable of localized illumination.By integrating tunable optical modules,we generate a dynamically adjustable excitation field on living cell membranes,achieving precise spatial registration between the excitation volume and the patch-clamp recording site.This system achieves submicron-scale alignment between the excitation zone and the micropipette contact area,enabling simultaneous electrophysiological recording and background-suppressed fluorescence detection within the clamped membrane domain.Experimental validation demonstrates that the systemcan perform single-molecule fluorescence imaging and trajectory analysis within a specified observation areas,with imaging resolution inversely related to the size of the illuminated region.Optimized optical design allows for precise excitation targeting while minimizing background illumination,thereby achieving high signal-to-noise ratio single-molecule imaging and significantly reducing photodamage.Integration with cell-attached patch-clamp configurations establishes a dual-modality platform for synchronized acquisition of single-molecule fluorescence images and single-channel recordings.The validation using mechanosensitive mPiezo1 channels confirms the system's compatibility with single-channel recording,indicating that optical imaging induces no detectable interference to electrophysiological signal acquisition.This method overcomes longstanding challenges in the simultaneous application of single-molecule imaging and electrophysiological techniques in live-cell environments.It establishes a novel experimental framework for investigating the structure-function relationships of channel proteins and membrane-related molecular machines through spatially coordinated optoelectronic measurements on live-cell membranes,which has broad applicability in molecular biophysics and transmembrane transport mechanism research.

The intrinsically disordered C-terminal domain (CTD) serves as a critical regulatory element in GPCR[1][1]–[3][2]. However, directly interrogating the CTD responses to different ligands is challenging due to its high flexibility, which renders it invisible to conventional structural biology techniques. To address the challenge, we developed a live-cell fluorescence imaging strategy that enables real-time visualization of CTD conformational transitions under physiological conditions. Our dual-mode approach integrates single-cell fluorescence lifetime imaging microscopy (FLIM) with single-molecule total internal reflection fluorescence microscopy (TIRFM), facilitating multi-scale analysis. Our data revealed that the dynamics of both full-length and truncated CTDs in the β2-adrenergic receptors are ligand-specific. By bridging single-molecule dynamics with ensemble cellular responses, our method uncovered previously inaccessible molecular mechanisms underlying receptor activation. This advance not only elucidates how GPCRs transduce ligand binding into functional outcomes but also establishes a versatile platform for drug discovery, enabling rapid assessment of ligand efficacy and receptor activity in physiological contexts. ### Competing Interest Statement The authors have declared no competing interest. National Key Research and Development Program of China, 2019YFA0709304 [1]: #ref-1 [2]: #ref-3

The pH-triggered structural transition and translocation of the pH low-insertion peptide (pHLIP) across cell membranes, facilitated by its distinct protonation property, render it a valuable model for investigating the membrane insertion mechanism of molecules. This capability also holds significant promise for advancements in cancer diagnosis and transmembrane transport. In this study, we investigated the dynamics of membrane insertion of wild-type pHLIP and its three variants using real-time tracking of single-peptide translocation kinetics. We identified three distinct metastable molecular conformations of pHLIPs within the bilayer, referred to as ″kinetic intermediate states″ at varying depths within the bilayer. These metastable conformations were observed during both the pH-triggered membrane insertion process and at intervening pH levels (between 7.4 and 5.0). Over time following a decrease in pH, these molecular conformations gradually transitioned with an increasing number of peptides shifting from a horizontally bound state to an inserted state, with a gradual deepening of their depth until equilibrium was reached around 10 min. Additionally, all individual peptides within the membrane experienced subsecond level kinetic fluctuations. Modifications such as P20G increased penetration depth without affecting the insertion process, whereas truncating residues D and E from the C-terminal accelerated membrane insertion speed but reduced penetration depth. Our findings elucidate how residue protonation-driven conformational changes influence peptide dynamics during membrane insertion, thereby providing insights for designing advanced drug delivery systems.

Lipid nanoparticles (LNPs) are an effective delivery system for gene therapeutics. By optimizing their formulation, the physiochemical properties of LNPs can be tailored to improve tissue penetration, cellular uptake, and precise targeting. The application of these targeted delivery strategies within the LNP framework ensures efficient delivery of therapeutic agents to specific organs or cell types, thereby maximizing therapeutic efficacy. In the realm of genome editing, LNPs have emerged as a potent vehicle for delivering CRISPR/Cas components, offering significant advantages such as high in vivo efficacy. The incorporation of machine learning into the optimization of LNP platforms for gene therapeutics represents a significant advancement, harnessing its predictive capabilities to substantially accelerate the research and development process. This review highlights the dynamic evolution of LNP technology, which is expected to drive transformative progress in the field of gene therapy.

Channel proteins act as precise molecular regulators of transmembrane transport, a fundamental process essential for maintaining cellular homeostasis. These proteins dynamically modulate their functional states through conformational changes, forming the structural basis for complex physiological processes such as signal transduction and energy metabolism. Single-molecule fluorescence spectroscopy and single-channel patch-clamp electrophysiology represent two cornerstone techniques in modern biophysics: the former enables molecular-resolution analysis of structural dynamics, while the latter provides direct functional characterization of ion channel activity. Despite their complementary capabilities, integrating these techniques to simultaneously monitor protein conformational dynamics and functional states remains technically challenging, primarily due to the strong autofluorescence background inherent to single-molecule imaging in cellular environments. To address this limitation, we developed a spatially selective optical excitation system capable of localized illumination. By integrating tunable optical modules, we generated a dynamically adjustable excitation field on living cell membranes, achieving precise spatial registration between the excitation volume and the patch-clamp recording site. This system achieved submicron-scale alignment between the excitation zone and the micropipette contact area, enabling simultaneous electrophysiological recording and background-suppressed fluorescence detection within the patched membrane domain. Experimental validation demonstrated the system’s ability to perform single-molecule fluorescence imaging and trajectory analysis within designated observation areas, with imaging resolution inversely correlated with the size of the illuminated region. Optimized optical design allowed for precise excitation targeting while minimizing background illumination, resulting in high signal-to-noise ratio single-molecule imaging with significantly reduced photodamage. Integration with cell-attached patch-clamp configurations established a dual-modality platform for synchronized acquisition of single-molecule fluorescence images and single-channel recordings. Validation using mechanosensitive mPiezo1 channels confirmed the system’s compatibility with single-channel recordings, demonstrating that optical imaging induces no detectable interference with electrophysiological signal acquisition. This methodology overcomes longstanding challenges in the concurrent application of single-molecule imaging and electrophysiological techniques in live-cell environments. It establishes a novel experimental framework for investigating structure–function relationships in channel proteins and membrane-associated molecular machines through spatially coordinated optoelectronic measurements on live-cell membranes, with broad applicability in molecular biophysics and studies of transmembrane transport mechanisms.

Insufficient infiltration of cytotoxic T cells into solid tumors remains a critical obstacle in cancer immunotherapy. Despite extensive efforts to comprehend the mechanisms governing this limited infiltration, few studies have focused on the evolution of T cell motility behavior after co-culture. In this study, we combined quantitative cell trajectory analysis, computational modeling, and bulk/single-cell RNA sequencing to systematically characterize the impact of cell interactions. We reveal that in a 2.5D co-culture system with multiple cancer-cell clusters, cancer-specific T cells exhibit increased directional persistence, which facilitates their efficient searching of cancer-cell clusters. Additionally, these T cells form prolonged interactions with cancer cells, which is the most crucial factor for their accumulation on cancer-cell clusters. Furthermore, post-interaction, a cancer-cell subpopulation displays immunosuppressive traits, reducing T cell attractant expression, and undergoing epithelial-to-mesenchymal transition. These findings offer valuable insights into improving immunotherapy efficacy and tackling T cell infiltration challenges in solid tumors.

Homologous recombination is a central mechanism for maintaining genome stability and biodiversity. RecA, as the first discovered homologous recombinase, plays a crucial role in homologous recombination strand exchange. In recent years, with the development of structural biology, significant breakthroughs have been made in understanding the static structure of the RecA nucleoprotein filament. However, research on the kinetic process of homologous recombination strand exchange mediated by RecA continues to encounter significant challenges. Research into the dynamic process has been ongoing for decades. In recent years, the use of single-molecule techniques has resulted in significant breakthroughs in this field. Among these techniques, single-molecule fluorescence resonance energy transfer (FRET) technology is widely used due to its ultra-high temporal and spatial resolution, making it well suitable for studying RecA-mediated homologous recombination strand exchange. However, the fluorescent labels required for FRET experiments may affect the RecA-mediated strand exchange process, which is often overlooked by researchers. Most of related articles focus on the effect of fluorescent labels on local structure. This paper primarily examines the effect of DNA fluorescent labeling on protein function, focusing on its effects on strand exchange from two perspectives: strand specificity and conformational sensitivity of the fluorescent labeling. Using experiments such as double-strand binding, single-strand invasion, and strand exchange, we develop a labeling scheme with the minimal effect—9 bp spaced C-strand double-base labeling in triplet— that can effectively improve the efficiency of studying the homologous recombination process. This result enhances the understanding of the effect of fluorescent labeling, allowing researchers to rapidly optimize the position and method of fluorescent labeling, and reduce its negative effects on the strand exchange process. Moreover, it provides some inspirations for other fluorescent labeling experiments.

This study introduces a perylene diimide (PDI) small molecule in the photocatalytic degradation of antibiotics for the first time. Initially, we optimized its photoelectric performance through self-assembly to obtain an n-type photocatalyst (SA-PDI). Subsequently, WO3 and Cu2O were incorporated using a facile one-step method to prepare n-n type and n-p type organic-inorganic hybrid photocatalysts (PDI-WO3 and PDI-Cu2O). This approach simultaneously achieved self-assembly, metal ion doping, and inorganic hybridization, further modulating the photocatalytic properties of the materials. Studies indicated that PDI-WO3 operates via a type-II heterojunction mechanism, whereas PDI-Cu2O follows an S-scheme heterojunction mechanism. SA-PDI demonstrated superior degradation efficiency toward tetracycline hydrochloride (TCH), while PDI-WO3 exhibited enhanced activity for both TCH and doxycycline hydrochloride (DOX). In contrast, PDI-Cu2O showed higher efficacy for ciprofloxacin (CIP) and norfloxacin (NOF). These differences in photocatalytic performance are attributed to the distinct active species generated by each catalyst, aligning with the degradation requirements of specific antibiotics. Moreover, since all three materials are based on the PDI molecule, they are inherently compatible. Consequently, we directly mixed these materials to prepare composite photocatalysts for the simultaneous photocatalytic degradation of multiple antibiotics. In a simulated wastewater system, we systematically investigated the effects of inorganic ions, humic acid, and pH on the effectiveness of the composite photocatalysts in treating mixed antibiotic solutions. Ultimately, this study provides novel design strategies for organic-inorganic hybrid materials and demonstrates promising potential for practical applications in mitigating antibiotic contamination in real wastewater systems.

In this work, we present a room temperature (RT, 25 +/- 3 degrees C) nitrogen dioxide (NO2) sensor based on antimony doped gallium nitride (Sb-GaN) thin film, fabricated using a multi-target co-sputtering method with a physical vapor deposition (PVD). The effects of Sb incorporation and sputtering power on gas-sensitive properties of the fabricated sensor were investigated. Intriguingly, the optimized Sb-GaN thin film demonstrated a substantial response (S = 5.8) to 10 ppm NO2 gas at 25 degrees C in darkness, with response/recovery times of 185/750 s and a detection limit (DL) as low as 1.64 ppb. Additionally, the sensor exhibited excellent selectivity, reproducibility and good stability in high humidity environment. The XPS study showed that the sensor's stability deteriorates in 10 days due to the residual adsorption of oxygen ions on the film's surface. In addition, the sensor's response-recovery characteristics under six different light irradiations were compared, and red light was found to significantly accelerate the recovery process of the device. The sensing enhancement (4.5 times) of the Sb-GaN sensor is ascribed to the structure induced by Sb-doping and the augmented active sites. Importantly, these investigations offer a valuable reference for synthesis of reliable, eco-friendly, and energy-efficient sensing materials for gas detection in darkness.

African swine fever virus (ASFV) is a large and structurally complex DNA virus encoding more than 160 proteins, including more than 68 structural proteins. A protein library covering recombinant ASFV proteins is fundamentally important for studies on protein function, antigenicity, vaccine development, and virus-host interactions. Here, to construct an ASFV protein library, we add a glutathione S-transferase (GST) tag at the N-terminus of each ASFV protein to facilitate solubilization and purification and express the recombinant proteins in the yeast host. By optimizing codons, expression vectors and strains and conditions of expression and purification, we achieve satisfactory protein yields for analytical applications and maximized access to the whole proteome of ASFV, with coverage of ca. 95%. Using the library, a protein chip is constructed and used to screen for interactions between ASFV and swine proteins ( e.g., IRF3, p65, and IκBα). The ASFV protein library lays the groundwork for understanding and combatting ASFV. The methods for constructing the library are instructive for generating other protein libraries for high-throughput applications.

The mitochondrion is a highly dynamic organelle, constantly undergoing fusion and fission, which are critical processes for the health of cells. Fusion of the outer mitochondrial membrane (OMM) is mediated by the mitofusins belonging to the dynamin superfamily of GTPases. Most eukaryotic organisms possess two cooperatively functioning mitofusins, but yeast has only one mitofusin (Fzo1). How Fzo1 solely catalyzes OMM fusion is unclear. Here, we present crystal structures of truncated Fzo1 (Fzo1IM) in different nucleotide-loading states and report a special mechanistic feature of Fzo1 through systematic functional studies. Differing from mammalian mitofusins, Fzo1 contains an extra latch bulge (LB) that is essential for the viability of yeast. Upon GTP loading, Fzo1IM dimerizes via the GTPase domain and prefers the closed conformation. This state is then locked by the subsequent trans interaction mediated by the LB of each protomer, so that Fzo1IM remains dimerized in the closed conformation even after GTP hydrolysis. This special mechanistic feature may be relevant to the previous observation that degradation of Fzo1 by the ubiquitin-proteasome system is required for mitochondrial fusion. Our study reveals how mitochondrial fusion in yeast is efficiently ensured with limited GTP consumption, which broadens current understanding of this fundamental biological process.

NuA4 is the only essential acetyltransferase in yeast that can catalyze the acetylation of the histones H2A, H2A.Z, and H4, thereby affecting gene transcription. However, the acetylation process of NuA4, such as how NuA4 acetylates H4 and H2A.Z differently, remains largely elusive. Here, using cryoelectron microscopy (cryo-EM) single particle analysis, we present seven cryo-EM structures of piccolo NuA4 (pNuA4) in complex with wild-type H2A.Z or H2A.Z-mutant-containing nucleosomes in the absence or presence of acetyl coenzyme A (Ac-CoA). We revealed that, in the absence of Ac-CoA, pNuA4 adopts multiple conformations to search for its substrates. After adding Ac-CoA, the single-molecule Förster resonance energy transfer (smFRET) and cryo-EM data indicated that pNuA4 prefers to bind H4 and undergoes a dynamic conformational change to complete the acetylation. We also obtained previously unseen structures in states associated with the acetylation of H2A.Z. These cryo-EM structures and smFRET results suggest a complex acetylation process on H4 and H2A.Z by pNuA4. The results provide a comprehensive picture of the mechanism by which pNuA4 acetylates its substrates within an H2A.Z-containing nucleosome.

Ninjurin1 (NINJ1) serves as a pivotal mediator of plasma membrane rupture (PMR) during pyroptosis, post-apoptotic lysis, necrosis, and related cell death pathways. While recent structural studies revealed near-atomic resolution architectures of NINJ1 in both inactive and active states, its activation mechanisms and membrane remodeling dynamics remain elusive. Through combined methodologies employing atomic force microscopy (AFM), proteoliposome systems, and cellular experiments, we identified negatively charged phospholipids—particularly PS—as critical cofactors in promoting NINJ1 activation. Using membrane-reconstituted NINJ1 with high-speed AFM (HS-AFM) real-time imaging, we captured the PMR stages: Membrane-bound NINJ1 transitions from metastable monomers/dimers/tetramers to arc-shaped oligomers, nucleating ~34-nm pores. Subsequent curvature-driven pore expansion generates paired filaments and irregular membrane fragments. Furthermore, our work integrates previous structural models with dynamic assembly data to propose a unified mechanistic framework that reconciles multiple aspects of NINJ1 function in membrane disruption.