Holger A. Scheidt

Carbamazepine (CBZ) is a widely used anticonvulsant whose pharmacological effects include state- and use-dependent inhibition of voltage-gated sodium channels in neuronal membranes. Because CBZ interacts with these targets within a lipid environment, its membrane partitioning, preferred depth, orientation, and influence on bilayer organization are critical to understanding its physicochemical context of action. This study combines solid-state nuclear magnetic resonance spectroscopy and molecular dynamics simulations to investigate CBZ interactions with a POPC bilayer, a well-established model of phospholipid membranes. Through 1H MAS NOESY, 2H, and 31P NMR experiments, the location of CBZ and its effects on lipid chain order and phosphocholine headgroup dynamics were determined. Both experimental and computational approaches demonstrated that CBZ preferentially associates with the lipid–water interface. Molecular dynamics density profiles revealed that CBZ primarily localizes near the glycerol/phosphocholine region, with the amide group favoring the polar interfacial environment and the tricyclic moiety extending further into the membrane. This localization was corroborated by the highest 1H–1H NOESY cross-relaxation rates for the glycerol/phosphocholine segments. At the simulated CBZ concentration, the overall POPC bilayer structure remained largely unchanged, with no significant alterations in acyl-chain order or area per lipid. However, 31P NMR indicated a slight increase in phosphocholine headgroup mobility in the presence of CBZ. These findings suggest that CBZ accumulates strongly at the POPC membrane interface while causing only minimal disruption to overall bilayer organization, thereby providing a lipid-level framework for future studies in more complex neuronal and ion-channel-containing membranes.

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.

Progesterone is an important steroid hormone in the mammalian reproductive system, but our knowledge about its membrane properties and intracellular trafficking is scarce. This is largely due to a lack of suitable analogues, allowing for tracking progesterone in cells with minimal perturbation to its structure. Here, we present an intrinsically fluorescent progesterone derivative, which differs from progesterone only by having three additional double bonds in the ring system. Using NMR and fluorescence spectroscopy combined with molecular dynamics (MD) simulations, we show that this progesterone tetraene has nearly identical membrane properties to the endogenous molecule and is highly fluorescent in alcohols and lipid membranes. NMR experiments and MD simulations reveal that both progesterone and its analogue are rather mobile in the lipid bilayer and are largely unable to induce fatty acyl chain ordering of phospholipids. Molecular docking suggests that both steroids bind to the nuclear progesterone receptor (nPR) with comparable affinity. Electronic structure calculations predict a high two-photon cross-section of the progesterone tetraene, which was experimentally confirmed by two-photon live-cell imaging of its distribution in living cells. In breast cancer cells expressing nPR, the progesterone analogue localizes to the nucleus, the endoplasmic reticulum and the nuclear membrane, emanating into the nucleus. The latter could play a role in the regulation of gene expression by the nucleoplasmic reticulum. Together, we present a progesterone analogue, which mimics the hormone closely and will be an important tool for the analysis of steroid trafficking and function in the future.

The brain, though less than 10% of body mass, contains about 25% of total cholesterol (CHL), emphasizing CHL’s key role in neuronal function. Many CHL actions are stereospecific, as shown by differences from its 3 α -hydroxy epimer, epicholesterol (epiCHL). How this minor structural change alters membrane properties and sterol transport remains unclear. Here, we compare fluorescent analogs of CHL (cholestatrienol, CTL) and epiCHL (epicholestatrienol, epiCTL), which closely mimic their natural counterparts. Biophysical membrane properties, such as flip-flop, acyl-chain ordering, and interbilayer transfer, depend on the orientation of the 3-hydroxy group. Similarly, transport by sterol transport proteins (STPs) and intracellular trafficking of the sterols in human astrocytes are stereospecific. Treatment with 25-hydroxycholesterol increases uptake of both epimers, but only CTL shows enhanced esterification and lipid droplet storage. These findings demonstrate that subtle cholesterol structural changes affect cellular homeostasis and establish epiCTL as a useful probe of sterol stereospecificity and trafficking.

The investigation of the interactions of small lipophilic molecules (e.g., drugs) with lipid membranes represents an active field of contemporary biophysical research. The determination of their membrane insertion, their distribution within the lipid bilayer, and their effect on lipid membranes themselves hold significant pharmacological importance since the plasma membrane often represents the first contact site for the interaction of a drug with the cell. In this work, we review recent applications of solid-state NMR spectroscopy that have been conducted to study the interaction of lipid membranes with a large variety of small drug-like molecules (e.g., local anesthetics, statins, NSAIDs, kinase inhibitors). We aim to briefly highlight previous research while outlining the promising prospects using experimental and computational methods, including ²H, ³¹P, ¹H magic-angle spinning (MAS) NOESY NMR, and molecular dynamics (MD) simulations, which provide highly useful tools for a comprehensive understanding of these interactions.

Exposure of organisms to nanoplastics (NPs) is inevitable given their global abundance and environmental persistence. Polyethylene terephthalate (PET) is a common plastic used in a wide range of products, including clothing and food and beverage packaging. Recent studies suggest that NPs can cross the blood-brain barrier and cause potential neurotoxicity. It is widely known that aggregation of amyloid beta (Aβ) peptides in the brain is a pathological hallmark of Alzheimer’s disease (AD). While the impact of nanoplastics such as polystyrene (PS) on amyloid aggregation has been studied, the effects of PET NPs remain unexplored. In this study, we examined the effect of PET NPs of different sizes (PET_50nm and PET_140nm) and concentrations (0, 10, 50, and 100 ppm) on the fibrillation of Aβ_1-40. Our results showed that the presence of PET_50nm as well as PET_140nm decreased the lag phase of the fibrillation processes in a dose- and size-dependent manner from 6.7 ± 0.08 h for Aβ in the absence of PET (Aβ_control) to 3.1 ± 0.03 h for PET_50nm and 3.8 ± 0.06 h for PET_140nm. CD spectroscopy showed that PET_50nm significantly impacts the structural composition of Aβ aggregates. A significant rise in antiparallel β-sheet content and β-turn structure and a substantial reduction in other structures were observed in the presence of 100 ppm PET_50nm. These changes indicate that higher concentrations (100 ppm) of PET_50nm promote more rigid and uniform peptide aggregates. Although PET_50nm NPs influence the kinetics of aggregation and secondary structure, the overall morphology of the resulting fibrils remains largely unaltered, as seen using transmission electron microscopy. Also, the local cross-β structure of the fibrils was not affected by the presence of PET_50nm NPs during fibrillation, as confirmed using ¹³C solid-state NMR spectroscopy. Overall, these findings show that PET NPs accelerate amyloid fibril formation and alter the secondary structure of Aβ fibrils. These results also indicate that the accumulation of PET-NPs in the brain may facilitate the progression of various neurodegenerative diseases, including Alzheimer’s disease.

Exposure of organisms to nanoplastics (NPs) is inevitable given their global abundance and environmental persistence. Polyethylene terephthalate (PET) is a common plastic used in a wide range of products, including clothing and food and beverage packaging. Recent studies suggest that NPs can cross the blood-brain barrier and cause potential neurotoxicity. It is widely known that aggregation of amyloid beta (Aβ) peptides in the brain is a pathological hallmark of Alzheimer's disease (AD). While the impact of nanoplastics such as polystyrene (PS) on amyloid aggregation has been studied, the effects of PET NPs remain unexplored. In this study, we examined the effect of PET NPs of different sizes (PET50 nm and PET140 nm) and concentrations (0, 10, 50 and 100 ppm) on the fibrillation of Aβ1-40. Our results showed that the presence of PET50 nm as well as PET140 nm decreased the lag phase of the fibrillation processes in a dose- and size-dependent manner from 6.7 ± 0.08 h for Aβ in the absence of PET (Aβcontrol) to 3.1 ± 0.03 h for PET50 nm and 3.8 ± 0.06 h for PET140 nm. CD spectroscopy showed that PET50nm significantly impacts the structural composition of Aβ aggregates. A significant rise in antiparallel β-sheet content and β-turn structure and a substantial reduction in other structures were observed in presence of 100 ppm PET50 nm. These changes indicate that higher concentrations (100 ppm) of PET50 nm promote more rigid and uniform peptide aggregats. Although PET50 nm NPs influence the kinetics of aggregation and secondary structure, the overall morphology of the resulting fibrils remains largely unaltered, as seen by transmission electron microscopy. Also the local cross-β structure of the fibrils was not affected by the presence of PET50 nm NPs during fibrillation, as confirmed by 13C solid-state NMR spectroscopy. Overall, these findings show that PET NPs accelerate amyloid fibril formation and alter the secondary structure of Aβ fibrils. These results also indicate that the accumulation of PET-NPs in the brain may facilitate the progression of various neurodegenerative diseases, including Alzheimer's disease.

Allopregnanolone (AlloP) is an example of neuroactive steroids (NAS), which is a potent allosteric activator of the γ-aminobutyric acid A (GABA_A) receptor. The mechanisms underlying the biological activity of AlloP and other NAS are only partially understood. Here, we present intrinsically fluorescent analogs of AlloP (MQ-323) and its 3β-epimer, epi-allopregnanolone (E-AlloP) (YX-11), and show, by a combination of spectroscopic and computational studies, that these analogs mimic the membrane properties of AlloP and E-AlloP very well. We found stereospecific differences in the orientation and dynamics of the NAS as well as in their impact on membrane permeability. However, all NAS are unable to condense the lipid bilayer, in stark contrast to cholesterol. Using Förster resonance energy transfer (FRET) and electrophysiological measurements, we show that MQ-323 but not YX-11 binds at the intersubunit site of the ELICα₁GABA_A receptor and potentiates GABA-induced receptor currents. In aqueous solvents, YX-11 forms aggregates at much lower concentrations than MQ-323, and loading both analogs onto cyclodextrin allows for their uptake by human astrocytes, where they become enriched in lipid droplets (LDs), as shown by quantitative fluorescence microscopy. Trafficking of the NAS analogs is stereospecific, as uptake and lipid droplet targeting is more pronounced for YX-11 compared to MQ-323. In summary, we present novel minimally modified analogs of AlloP and E-AlloP, which enable us to reveal stereospecific membrane properties, allosteric receptor activation, and intracellular transport of these neurosteroids. Our fluorescence design strategy will be very useful for the analysis of other NAS in the future.

Live-cell imaging of cholesterol trafficking depends on suitable cholesterol analogs. However, existing fluorescent analogs of cholesterol either show very different physicochemical properties compared to cholesterol or demand excitation in the ultraviolet spectral region. We present a strategy to synthesize two novel intrinsically fluorescent sterol probes with a close resemblance of cholesterol. The analogs contain four conjugated double bonds in the ring system and either a keto group (probe 5) or a hydroxy group (probe 6) in the C3 position. The emission of 5 is in the visible range of the spectrum, i.e., red-shifted by 150 nm compared to the widely used dehydroergosterol. Together with its high multiphoton absorption, this allows for imaging of 5 on conventional microscopes, including multicolor 3D and time-lapse microscopy. Molecular dynamics simulations and nuclear magnetic resonance spectroscopy reveal that 5 can condense the fatty acyl chains of phospholipids in model membranes. In giant unilamellar vesicles, 5 partitions equally into the liquid-ordered and disordered phases. In contrast, 6 emits in the ultraviolet range and is unstable in solution, preventing its use in live-cell imaging applications. The good photophysical properties of 5 make it a suitable analogue for improved live-cell imaging of sterol transport.