Enhancing structural control in covalent organic frameworks through steric interaction-driven linker design.

Covalent Organic Frameworks (COFs) exhibiting kagome () structures are promising crystalline porous materials with two distinct pores. However, there are no reliable synthetic methods to exclusively target the over the polymorphic square-lattice () structure. To address this, we introduce a linker design strategy featuring bulky functional groups, which through steric interactions can hinder the net formation, thereby leading to a structure.

Disorder-to-order active site capping regulates the rate-limiting step of the inositol pathway.

Myo-inositol-1-phosphate synthase (MIPS) catalyzes the NAD-dependent isomerization of glucose-6-phosphate (G6P) into inositol-1-phosphate (IMP), controlling the rate-limiting step of the inositol pathway. Previous structural studies focused on the detailed molecular mechanism, neglecting large-scale conformational changes that drive the function of this 240 kDa homotetrameric complex. In this study, we identified the active, endogenous MIPS in cell extracts from the thermophilic fungus .

Delineating organizational principles of the endogenous L-A virus by cryo-EM and computational analysis of native cell extracts.

The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus.

Protein-Rich Rafts in Hybrid Polymer/Lipid Giant Unilamellar Vesicles.

Considerable attention has been dedicated to lipid rafts due to their importance in numerous cell functions such as membrane trafficking, polarization, and signaling. Next to studies in living cells, artificial micrometer-sized vesicles with a minimal set of components are established as a major tool to understand the phase separation dynamics and their intimate interplay with membrane proteins. In parallel, mixtures of phospholipids and certain amphiphilic polymers simultaneously offer an interface for proteins and mimic this segregation behavior, presenting a tangible synthetic alternative for fundamental studies and bottom-up design of cellular mimics.

Cryo-EM of a heterogeneous biochemical fraction elucidates multiple protein complexes from a multicellular thermophilic eukaryote.

Biomolecular complexes and their interactions govern cellular structure and function. Understanding their architecture is a prerequisite for dissecting the cell's inner workings, but their higher-order assembly is often transient and challenging for structural analysis. Here, we performed cryo-EM on a single, highly heterogeneous biochemical fraction derived from cell extracts to visualize the biomolecular content of the multicellular eukaryote.

Cryo-EM structure of a plant photosystem II supercomplex with light-harvesting protein Lhcb8 and α-tocopherol.

The heart of oxygenic photosynthesis is the water-splitting photosystem II (PSII), which forms supercomplexes with a variable amount of peripheral trimeric light-harvesting complexes (LHCII). Our knowledge of the structure of green plant PSII supercomplex is based on findings obtained from several representatives of green algae and flowering plants; however, data from a non-flowering plant are currently missing. Here we report a cryo-electron microscopy structure of PSII supercomplex from spruce, a representative of non-flowering land plants, at 2.

Structural analysis of an endogenous 4-megadalton succinyl-CoA-generating metabolon.

The oxoglutarate dehydrogenase complex (OGDHc) participates in the tricarboxylic acid cycle and, in a multi-step reaction, decarboxylates α-ketoglutarate, transfers succinyl to CoA, and reduces NAD+. Due to its pivotal role in metabolism, OGDHc enzymatic components have been studied in isolation; however, their interactions within the endogenous OGDHc remain elusive. Here, we discern the organization of a thermophilic, eukaryotic, native OGDHc in its active state.

Encapsulation of cannabidiol in oil-in-water nanoemulsions and nanoemulsion-filled hydrogels: A structure and biological assessment study.

Hypothesis: Lipophilic cannabidiol can be solubilized in oil-in water nanoemulsions, which can then be impregnated into chitosan hydrogels forming another colloidal system that will facilitate cannabidiol's release. The delivery from both systems was compared, alongside structural and biological studies, to clarify the effect of the two carriers' structure on the release and toxicity of the systems.

Experiments: Oil-in-water nanoemulsions (NEs) and the respective nanoemulsion-filled chitosan hydrogels (NE/HGs) were formulated as carriers of cannabidiol (CBD).

Cryo-Electron Microscopy Snapshots of Eukaryotic Membrane Proteins in Native Lipid-Bilayer Nanodiscs.

New technologies for purifying membrane-bound protein complexes in combination with cryo-electron microscopy (EM) have recently allowed the exploration of such complexes under near-native conditions. In particular, polymer-encapsulated nanodiscs enable the study of membrane proteins at high resolution while retaining protein-protein and protein-lipid interactions within a lipid bilayer. However, this powerful technology has not been exploited to address the important question of how endogenous─as opposed to overexpressed─membrane proteins are organized within a lipid environment.

Cryo-EM structure of the SEA complex.

The SEA complex (SEAC) is a growth regulator that acts as a GTPase-activating protein (GAP) towards Gtr1, a Rag GTPase that relays nutrient status to the Target of Rapamycin Complex 1 (TORC1) in yeast. Functionally, the SEAC has been divided into two subcomplexes: SEACIT, which has GAP activity and inhibits TORC1, and SEACAT, which regulates SEACIT. This system is conserved in mammals: the GATOR complex, consisting of GATOR1 (SEACIT) and GATOR2 (SEACAT), transmits amino acid and glucose signals to mTORC1.

Filling the Gap with Long -Alkanes: Incorporation of C20 and C30 into Phospholipid Membranes.

Investigating how hydrophobic molecules mix with phospholipid bilayers and how they affect membrane properties is commonplace in biophysics. Despite this, a molecular-level empirical description of a membrane model as simple as a phospholipid bilayer with long linear hydrophobic chains incorporated is still missing. Here, we present an unprecedented molecular characterization of the incorporation of two long -alkanes, -eicosane (C20) and -triacontane (C30) with 20 and 30 carbons, respectively, in phosphatidylcholine (PC) bilayers using a combination of experimental techniques (H NMR, P NMR, H-C dipolar recoupling solid-state NMR, X-ray scattering, and cryogenic electron microscopy) and atomistic molecular dynamics (MD) simulations.

Increased efficiency of charge-mediated fusion in polymer/lipid hybrid membranes.

Due to their augmented properties, biomimetic polymer/lipid hybrid compartments are a promising substitute for natural liposomes in multiple applications, but the protein-free fusion of those semisynthetic membranes is unexplored to date. Here, we study the charge-mediated fusion of hybrid vesicles composed of poly(dimethylsiloxane)-graft-poly(ethylene oxide) and different lipids and analyze the process by size distribution and the mixing of membrane species at μm and nano scales. Remarkably, the membrane mixing of oppositely charged hybrids surpasses by far the degree in liposomes, which we correlate with properties like membrane disorder, rigidity, and ability of amphiphiles for flip-flop.

Investigating bolalipids as solubilizing agents for poorly soluble drugs: Effects of alkyl chain length on solubilization and cytotoxicity.

Synthetic single-chain bolalipids with symmetrical headgroups have shown potential in various pharmaceutical applications, such as the stabilization of liposome bilayers. Despite their amphiphilic character, synthetic bolalipids have not yet been investigated for their suitability as solubilizing agents for poorly soluble drug compounds. In this study, three synthetic single-chain bolalipids with increasing alkyl chain lengths (C22, C24 and C26) were investigated.

Cryo-EM and artificial intelligence visualize endogenous protein community members.

Cellular function is underlined by megadalton assemblies organizing in proximity, forming communities. Metabolons are protein communities involving metabolic pathways such as protein, fatty acid, and thioesters of coenzyme-A synthesis. Metabolons are highly heterogeneous due to their function, making their analysis particularly challenging.

Cryo-EM snapshots of a native lysate provide structural insights into a metabolon-embedded transacetylase reaction.

Found across all kingdoms of life, 2-keto acid dehydrogenase complexes possess prominent metabolic roles and form major regulatory sites. Although their component structures are known, their higher-order organization is highly heterogeneous, not only across species or tissues but also even within a single cell. Here, we report a cryo-EM structure of the fully active Chaetomium thermophilum pyruvate dehydrogenase complex (PDHc) core scaffold at 3.

En route to dynamic life processes by SNARE-mediated fusion of polymer and hybrid membranes.

A variety of artificial cells springs from the functionalization of liposomes with proteins. However, these models suffer from low durability without repair and replenishment mechanisms, which can be partly addressed by replacing the lipids with polymers. Yet natural membranes are also dynamically remodeled in multiple cellular processes.

Solubilization of artificial mitochondrial membranes by amphiphilic copolymers of different charge.

Certain amphiphilic copolymers form lipid-bilayer nanodiscs from artificial and natural membranes, thereby rendering incorporated membrane proteins optimal for structural analysis. Recent studies have shown that the amphiphilicity of a copolymer strongly determines its solubilization efficiency. This is especially true for highly negatively charged membranes, which experience pronounced Coulombic repulsion with polyanionic polymers.

Integrative structure of a 10-megadalton eukaryotic pyruvate dehydrogenase complex from native cell extracts.

The pyruvate dehydrogenase complex (PDHc) is a giant enzymatic assembly involved in pyruvate oxidation. PDHc components have been characterized in isolation, but the complex's quaternary structure has remained elusive due to sheer size, heterogeneity, and plasticity. Here, we identify fully assembled Chaetomium thermophilum α-keto acid dehydrogenase complexes in native cell extracts and characterize their domain arrangements utilizing mass spectrometry, activity assays, crosslinking, electron microscopy (EM), and computational modeling.

Enhanced optical imaging properties of lipid nanocapsules as vehicles for fluorescent conjugated polymers.

Conjugated polymer nanoparticles (CPNs) have emerged as highly photostable probes for optical and photoacoustic imaging. However, the aggregation of conjugated polymer (CP) molecules upon nanoparticle formation is associated with fluorescence quenching, poor yields and mutable particle sizes. This study investigated whether the CP encapsulation within the liquid midchain triglyceride (MCT) core of lipid nanocapsules (LNCs) may achieve reduced packing of CP chains leading to a stable system with enhanced optical features.

Constructing artificial respiratory chain in polymer compartments: Insights into the interplay between oxidase and the membrane.

Cytochrome ubiquinol oxidase is a transmembrane protein, which oxidizes ubiquinone and reduces oxygen, while pumping protons. Apart from its combination with FF-ATPase to assemble a minimal ATP regeneration module, the utility of the proton pump can be extended to other applications in the context of synthetic cells such as transport, signaling, and control of enzymatic reactions. In parallel, polymers have been speculated to be phospholipid mimics with respect to their ability to self-assemble in compartments with increased stability.

2.7 Å cryo-EM structure of vitrified M. musculus H-chain apoferritin from a compact 200 keV cryo-microscope.

Here we present the structure of mouse H-chain apoferritin at 2.7 Å (FSC = 0.143) solved by single particle cryogenic electron microscopy (cryo-EM) using a 200 kV device, the Thermo Fisher Glacios®.