Abstract Lanthanide-doped NaYF4 nanocrystals (NCs) are widely used for their exceptional photoluminescence properties. Their performance and processability are critically dependent on surface-bound ligands such as oleic acid. While these ligands stabilize NCs during synthesis and in nonpolar solvents, applications often require post-synthetic ligand exchange. Here, we investigate the protonation state, surface ion binding, and exchange dynamics of oleic acid on NaYF4 NCs using a variety of analysis techniques, including infrared spectroscopy (IR), nuclear magnetic resonance spectroscopy (NMR), and thermogravimetric analysis (TGA). Our methods distinguish bound and free ligand populations, quantify surface coverage, and detail how common purification steps alter ligand density. These findings offer key insights into NC surface chemistry and provide a foundation for rational NC purification and ligand-exchange strategies, with implications for optimization of colloidal stability and functionalization.

Abstract Cardiolipin (CL) is a very important lipid in bacteria and the mitochondria of higher cells. CL is characterized by a unique structure, featuring four acyl chains and two negative charges, exhibiting a highly negative intrinsic curvature. We investigate the influence of CL on the function of the intramembrane E. coli protease GlpG, reconstituted into E. coli-like membranes. To underline the importance of CL, we find a ∼40% accelerated substrate cleavage rate in the presence of 10 mol % CL in phosphatidylethanolamine (PE)/phosphatidylglycerol (PG) membranes. CL induces a lateral restructuring of the membrane: without CL, PE/PG in the lipid annulus show a decreased chain length to match the hydrophobic thickness of GlpG. When CL is present, PE and PG share the properties of the bulk lipids, while CL undergoes the same amount of thinning that was observed for PE/PG before. To reveal the molecular properties of CL, we collect a multitude of NMR parameters to quantitatively describe the structure and dynamics of CL in the absence and presence of GlpG. In addition to the chain disordering and reduction of membrane thickness, we observe that some CL species follow the slow rotational diffusive motions of GlpG. This implies an association with GlpG, indicating preferential GlpG-CL interactions. We suggest that the high negative charge and the negative intrinsic curvature of CL are the main determinants of this remarkable behavior.

Lipoproteins are involved in diverse cellular processes that take place in and around cell membranes. A prominent example refers to the biogenesis of outer membrane proteins mediated by the β-barrel assembly machinery (BAM) that comprises the integral membrane protein BamA and several lipoproteins (BamB-BamE). Using a combination of solid-state NMR spectroscopy in native bacterial settings and molecular dynamics (MD) simulations, we have investigated the structural conformation, dynamics, and topology of lipoproteins BamC and BamE in their native bacterial membrane at the atomic level. We found significant dynamic and topological differences in BamC and BamE when associated with their native membrane settings as compared to their conformation within the fully assembled BAM complex. The combination of these dynamic protein-protein and protein-membrane interactions may be critical to modulating the activity of BamA during BAM-mediated protein insertion.

NZ2114 is a triple-mutant of the natural peptide antibiotic plectasin that targets the peptidoglycan precursor molecule Lipid II using a supramolecular action, involving assembly of plectasin-Lipid II complexes in a large oligomeric structure, that is enhanced by calcium ions. Due to its superior potency against Staphylococcus aureus strains, NZ2114 was the candidate that was advanced to clinical trials, and it has become the standard-template for the development of improved plectasin derivatives. However, the molecular underpinning for NZ2114’s improved potency remains opaque, with biochemical data pointing to chemical modification of the Lipid II target in Staphylococci that would impair the target binding capacity of plectasin but not the one of NZ2114. Here, using an integrative structural biology approach based on solid-state NMR, high-speed atomic force microscopy, and affinity assays, we demonstrate that both NZ2114 and plectasin bind effectively to Staphylococcal Lipid II variants, which means that NZ2114’s greater potency against S. aureus does not result from a difference in target binding. Instead, we show that the three residue substitutions in NZ2114 change its N-terminal fold, markedly increasing its sensitivity to calcium ions, which results in a different supramolecular action on the membrane surface. Altogether, our study provides new insights for the design of superior drug candidates.

Polyprenyl quinones, such as ubiquinone and menaquinone, are essential membrane-embedded redox cofactors that are involved in electron transport and found across all domains of life. However, their highly hydrophobic structure, which includes a quinone head-group and long polyprenyl tail, has limited their chemical derivatization for biological studies. Here, we report a versatile synthetic approach for the chemical diversification of natural polyprenyl quinones, enabling the introduction of various reporter groups including fluorophores, quenchers, NMR-active nuclei, and photoaffinity and bioaffinity tags. These functionalized analogues retain their membrane-associating properties and enable new applications in antibiotic discovery. We show that fluorescently labelled menaquinone analogues retain their strong binding affinity to the menaquinone-binding peptide antibiotics lysocin E and lysomeb (MBA2). Incorporation of BODIPY-quinones into vesicles allowed visualization of the peptide-quinone interaction, revealing their effects on membrane integrity and quinone aggregation. This study expands the chemical toolbox for polyprenyl quinones, enabling targeted functionalization of these essential biomolecules and facilitating further exploration of their roles in biological systems.

The antimicrobial resistance (AMR) crisis has been associated with millions of deaths. Of particular concern is the threat of bioweapons, exemplified by anthrax. Introduction of novel antibiotics helps mitigate AMR, but does not address the threat of bioweapons with engineered resistance. We reasoned that teixobactin, an antibiotic with no detectable resistance, is uniquely suited to address the challenge of weaponized anthrax. Teixobactin binds to immutable targets, precursors of cell wall polymers. Here we show that teixobactin is highly efficacious in a rabbit model of inhalation anthrax. Inhaling spores of Bacillus anthracis causes overwhelming morbidity and mortality. Treating rabbits with teixobactin after the onset of disease rapidly eliminates the pathogen from blood and tissues, normalizes body temperature, and prevents tissue damage. Teixobactin assembles into an irreversible supramolecular structure on the surface of B. anthracis membrane, likely contributing to its unusually high potency against anthrax. Antibiotics evading resistance provide a rational solution to both AMR and engineered bioweapons.

Human cathelicidin LL-37 derivative, the 24-mer SAAP-148, is highly effective in vitro in eradicating multidrug-resistant bacteria without inducing resistance. SAAP-148 has a high cationic charge (+11) and 46% hydrophobicity, which, once the peptide folds into an alpha helix, forms a wide hydrophobic face. This highly amphipathic nature facilitates on the one hand its insertion into the membrane's fatty acyl chain region and on the other hand it´s interaction with anionic membrane components, which aids in killing bacteria. However, the contributions of the secondary and quaternary structures have not been thoroughly investigated so far. To address this, we applied circular dichroism, NMR spectroscopy, X-ray scattering, AlphaFold 3 protein folding software, and molecular dynamics simulations. Our results reveal that SAAP-148 adopts a stable hexameric bundle composed of three parallel dimers, that together form a hydrophobic core of aromatic side chain residues. The hexameric structure is retained at the membrane interface, whereby, MD simulation studies indicated the formation of a fiber-like structure in the presence of anionic membranes. This certainly seems plausible, as oligomers are stabilized by aromatic residues, and the exposure of positively charged side chains on the surface likely facilitates the transition of the peptide into fibrils on anionic membranes.

Membrane lipid oxidation is a universal process that occurs in situations of oxidative stress and is encountered in numerous physiological and pathological situations. Oxidized truncated phospholipids make up a large part of the oxidation products and alter the membrane properties in a way that can lead to cell death. However, the underlying mechanisms are not well understood nor is it clear whether environmental factors, such as pH, can modulate these effects. Using model membranes, we investigate how individual lipid aldehydes and carboxylic acids with truncated acyl chains alter the membrane structure. Our data shows that lipid aldehydes and carboxylic acids have different permeabilization efficiencies towards molecules of varying charge and size and that ΔC9 truncated lipids are usually more efficient in permeabilizing membranes than ΔC5. In terms of physical mechanisms, the ΔC9 truncated lipid carboxylic acid induces permeabilization and membrane curvature in a pH-dependent fashion. This is explained by ionization-dependent exposure of the carboxyl group to the water-bilayer interface, which increases the intrinsic molecular curvature of the oxidized lipid. Conversely, ΔC9 truncated lipid aldehydes and nonionized carboxyls do not induce curved structures but are more efficient in increasing permeability toward larger molecules. We further show that truncated lipids can escape the bilayer and accumulate at interfaces, implying that they might act on neighboring cells. This study indicates that oxidized phospholipids with truncated acyl chains disrupt membrane structure, depending on their specific molecular structure and the pH of the environment, opening a possible route for the design of lipid nanoparticles with pH-dependent drug release.

The emergence of multidrug-resistant bacteria presents a critical threat to global health. These multidrug-resistant bacteria are often protected by complex cell envelopes that many antibiotics cannot penetrate, creating an important barrier to treatment. In response, targeting bacterial envelopes has long been recognized as an effective strategy, offering potential to bypass the challenges of drug entry and efflux resistance mechanisms. Moreover, many unique bacterial envelope sites remain clinically untapped, and new compounds directed at them have the potential to diversify the space of antimicrobial mechanisms, lowering the risk for cross-resistance. Compounds that target non-proteinaceous envelope components, such as lipopolysaccharide or prenylated peptidoglycan-precursors, are particularly attractive owing to their reduced susceptibility to antimicrobial resistance development. In this Review, we explore both recently discovered compounds and established envelope-targeting antibiotics, including compounds that target Gram-positive bacteria, more complex Gram-negative bacteria and mycobacterial pathogens, shedding light on this still clinically underexplored and vital therapeutic approach. In this Review, Weingarth and colleagues discuss both recently discovered compounds and established envelope-targeting antibiotics, including compounds that target Gram-positive bacteria, more complex Gram-negative bacteria and mycobacterial pathogens, with a particular focus on their drug–target interactions.

The increasing prevalence of antibiotic resistance represents a significant public health concern, underscoring the urgent need for the development of novel therapeutic strategies. The antibiotic effects of macrolides, the second most widely used class of antibiotics, are counteracted by Erm proteins through the methylation of adenosine 2058 of the 23S ribosomal RNA (rRNA) ( 2900 nucleotides), yielding either monomethylated or dimethylated A2058. This methylation is the molecular basis for preventing macrolides from binding and leads to the development of resistance of bacteria including Staphylococcus, Streptococcus and Enterococcus. While the function of Erm proteins have been thoroughly investigated, the role of the ribosomal RNA in acquiring antibiotic resistance is frequently underestimated, given that the ribosomal RNA is the actual target for methylation. Here, we present the comprehensive 1H, 13C and 15N NMR resonance assignment for the part of the 23S rRNA that serves as the Erm substrate in antimicrobial resistance. Furthermore, we compare the chemical shift signature of the unmethylated to the monomethylated and dimethylated RNA construct and show that changes in the RNA upon methylation are locally restricted. The resonance assignments provide a starting point for investigating and targeting the molecular mechanism of the resistance-conferring Erm proteins.

In this Review, we explore natural product antibiotics that do more than simply inhibit an active site of an essential enzyme. We review these compounds to provide inspiration for the design of much-needed new antibacterial agents, and examine the complex mechanisms that have evolved to effectively target bacteria, including covalent binders, inhibitors of resistance, compounds that utilize self-promoted entry, those that evade resistance, prodrugs, target corrupters, inhibitors of 'undruggable' targets, compounds that form supramolecular complexes, and selective membrane-acting agents. These are exemplified by β-lactams that bind covalently to inhibit transpeptidases and β-lactamases, siderophore chimeras that hijack import mechanisms to smuggle antibiotics into the cell, compounds that are activated by bacterial enzymes to produce reactive molecules, and antibiotics such as aminoglycosides that corrupt, rather than merely inhibit, their targets. Some of these mechanisms are highly sophisticated, such as the preformed β-strands of darobactins that target the undruggable β-barrel chaperone BamA, or teixobactin, which binds to a precursor of peptidoglycan and then forms a supramolecular structure that damages the membrane, impeding the emergence of resistance. Many of the compounds exhibit more than one notable feature, such as resistance evasion and target corruption. Understanding the surprising complexity of the best antimicrobial compounds provides a roadmap for developing novel compounds to address the antimicrobial resistance crisis by mining for new natural products and inspiring us to design similarly sophisticated antibiotics.

Lipids adhere to membrane proteins to stimulate or suppress molecular and ionic transport and signal transduction. Yet, the molecular details of lipid-protein interaction and their functional impact are poorly characterized. Here we combine NMR, coarse-grained molecular dynamics (CGMD), and functional assays to reveal classic cooperativity in the binding and subsequent activation of a bacterial inward rectifier potassium (Kir) channel by phosphatidylglycerol (PG), a common component of many membranes. Past studies of lipid activation of Kir channels focused primarily on phosphatidylinositol bisphosphate, a relatively rare signaling lipid that is tightly regulated in space and time. We use solid-state NMR to quantify the binding of unmodified C-13-PG to the K+ channel KirBac1.1 in liposomes. This specific lipid-protein interaction has a dissociation constant (K-d) of similar to 7 mol percentage PG (Chi(PG)) with positive cooperativity (n = 3.8) and approaches saturation near 20% Chi(PG). Liposomal flux assays show that K+ flux also increases with PG in a cooperative manner with an EC50 of similar to 20% Chi(PG), within the physiological range. Further quantitative fitting of these data reveals that PG acts as a partial (80%) agonist with fivefold K+ flux amplification. Comparisons of NMR chemical shift perturbation and CGMD simulations at different Chi(PG) confirm the direct interaction of PG with key residues, several of which would not be accessible to lipid headgroups in the closed state of the channel. Allosteric regulation by a common lipid is directly relevant to the activation mechanisms of several human ion channels. This study highlights the role of concentration-dependent lipid-protein interactions and tightly controlled protein allostery in the activation and regulation of ion channels.

Inward-rectifier K+ (Kir) channels are tetrameric ligand-gated ion channels. These Kir channels conduct inward K+ current in response to this anionic lipid association, regulating the resting membrane potential in excitable cells. Noted channelopathies include forms of heart disease, psychiatric disorders, and alcoholism. The prokaryotic channel KirBac1.1 shares its fold and regulatory mechanisms with homologous human channels. Using solid-state NMR (SSNMR), K+ efflux assays, SSNMR-driven simulated annealing structure calculations, and coarse-grain molecular dynamics (CG-MD) we resolved an intricate allosteric network between the inner gate, outer gate, and intracellular Kir domain.

Antimicrobial resistance is a leading cause of mortality, calling for the development of new antibiotics. The fungal antibiotic plectasin is a eukaryotic host defence peptide that blocks bacterial cell wall synthesis. Here, using a combination of solid-state nuclear magnetic resonance, atomic force microscopy and activity assays, we show that plectasin uses a calcium-sensitive supramolecular killing mechanism. Efficient and selective binding of the target lipid II, a cell wall precursor with an irreplaceable pyrophosphate, is achieved by the oligomerization of plectasin into dense supra-structures that only form on bacterial membranes that comprise lipid II. Oligomerization and target binding of plectasin are interdependent and are enhanced by the coordination of calcium ions to plectasin's prominent anionic patch, causing allosteric changes that markedly improve the activity of the antibiotic. Structural knowledge of how host defence peptides impair cell wall synthesis will likely enable the development of superior drug candidates. Plectasin, a natural host defence peptide of fungal origin, uses a calcium-sensitive supramolecular killing mechanism to block bacterial cell wall synthesis.

Neurodegeneration in Huntington's disease (HD) is accompanied by the aggregation of fragments of the mutant huntingtin protein, a biomarker of disease progression. A particular pathogenic role has been attributed to the aggregation-prone huntingtin exon 1 (HTTex1), generated by aberrant splicing or proteolysis, and containing the expanded polyglutamine (polyQ) segment. Unlike amyloid fibrils from Parkinson's and Alzheimer's diseases, the atomic-level structure of HTTex1 fibrils has remained unknown, limiting diagnostic and treatment efforts. We present and analyze the structure of fibrils formed by polyQ peptides and polyQ-expanded HTTex1 in vitro. Atomic-resolution perspectives are enabled by an integrative analysis and unrestrained all-atom molecular dynamics (MD) simulations incorporating experimental data from electron microscopy (EM), solid-state NMR, and other techniques. Alongside the use of prior data, we report magic angle spinning NMR studies of glutamine residues of the polyQ fibril core and surface, distinguished via hydrogen-deuterium exchange (HDX). Our study provides a molecular understanding of the structure of the core as well as surface of aggregated HTTex1, including the fuzzy coat and polyQ-water interface. The obtained data are discussed in context of their implications for understanding the detection of such aggregates (diagnostics) as well as known biological properties of the fibrils.

Physical properties of biological membranes directly or indirectly govern biological processes. Yet, the interplay between membrane and integral membrane proteins is difficult to assess due to reciprocal effects between membrane proteins, individual lipids, and membrane architecture. Using solid-state NMR (SSNMR) we previously showed that KirBac1.1, a bacterial Inward-Rectifier K+ channel, nucleates bilayer ordering and microdomain formation through tethering anionic lipids. Conversely, these lipids cooperatively bind cationic residues to activate the channel and initiate K+ flux. The mechanistic details governing the relationship between cooperative lipid loading and bilayer ordering are, however unknown. To investigate, we generated KirBac1.1 samples with different concentrations of 13C-lableded phosphatidyl glycerol (PG) lipids and acquired a full suite of SSNMR 1D temperature series experiments using the ordered all-trans (AT) and disordered trans-gauche (TG) acyl conformations as markers of bilayer dynamics. We observed increased AT ordered signal, decreased TG disordered signal, and increased bilayer melting temperature with increased PG concentration. Further, we identified cooperativity between ordering and direct binding of PG lipids, indicating KirBac1.1-driven bilayer ordering and microdomain formation is a classically cooperative Hill-type process driven by and predicated upon direct binding of PG lipids. Our results provide unique mechanistic insight into how proteins and lipids in tandem contribute to supramolecular bilayer heterogeneity in the lipid membrane.