Supramolecular host-guest systems have long served as models for understanding receptor-substrate interactions and enzyme-like behavior. In fact, they can also serve as a powerful tool to study competition for substrates in a crowded cellular milieu, where many distinct proteins can bind the same or similar classes of substrates. The fundamental challenge in the biological context is to unequivocally elaborate on substrate exchange dynamics between multiple enzymes or proteins that act as molecular hosts. Herein, using a biomimetic approach, we model the guest exchange dynamics of 1,4-dimethylnaphthalene between two geometrically distinct supramolecular cages having octahedral and square-pyramidal shapes. Guest exchange was monitored via 1H NMR spectroscopy, and the NMR spectra indicate an intermediate time scale exchange. Line-shape simulations of the experimental spectra using a two-state exchange model provide a quantitative description of the guest exchange rates on the millisecond time scale. We utilize the exchange time scales to perform a visible-light-driven hetero C-C coupling reaction between two distinct terminal aromatic alkynes in water. Our work therefore demonstrates that supramolecular photoredox catalysis using multiple cages can be controlled via the guest exchange dynamics, providing inspiration for multistep organic transformations in water.

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.

G protein-coupled receptors (GPCRs) transduce extracellular signals into the cell through binding and activation of intracellular effector proteins. Highly conserved residues such as tryptophan W6.48 of transmembrane helix 6 can play a role in GPCR activation, where W6.48 acts as a microswitch, changing its rotameric state depending on whether the receptor is bound to an agonist or antagonist. However, its exact role is not entirely clear. Here we investigate the role of W6.48 in the neuropeptide Y1 receptor (Y1R). Via NMR experiment and molecular dynamics simulations, we find that on the one hand, W6.48 exhibits multiple rotameric conformations, where simulations indicate that these are coupled to backbone structure. On the other hand, mutation of W6.48 to alanine does not prevent G-protein signaling, and agonist vs antagonist bound Y1R exhibits the same W6.48 rotameric state, calling into question whether its core function is to regulate Y1R activation. Further investigation indicates that the W6.48 rotameric state restricts microstates accessible by Y1R and impacts backbone dynamics. Using principal component analysis of multiple MD trajectories, we determine the structural similarity between Y1R for the various apo, NPY, NPY/Gi, and antagonist-bound conformational states. We propose a role for W6.48 in regulating Y1R binding, in which rotameric changes for W6.48 influence ligand binding by favoring backbone structures in apo Y1R that are similar to those observed for the active protein. Mutation of W6.48 then does not eliminate these structures but reduces their prevalence. Therefore, W6.48 provides "fine-tuning" of Y1R signaling; mutation of the 6.48 position leaves Y1R active but "detuned" such that signaling is still possible, but proper functioning is inhibited by a decrease in ligand binding rate.

The neurotransmitter serotonin is involved in physiological processes such as appetite, sleep, and mood and diseases such as anxiety and depression. Traditionally, the effects of serotonin were thought to be initiated by binding to its target transmembrane receptors. It is also known that serotonin can bind directly to the membrane with high affinity and modulate lipid dynamics, lateral segregation of lipids, vesicular association, and membrane protein activity. We investigated if other small molecules in the serotonin metabolic pathway, some of which are known to be signaling molecules while some others are not, have similar membrane modulating effects. Therefore, we examined serotonin and several of its metabolites: 5-hydroxytryptophan (5-HTP), serotonin, N-acetylserotonin (NAS), and melatonin in model membranes mimicking synaptic membranes. Using 2H NMR spectroscopy of deuterated 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), we observed that all metabolites disorder the synaptic membrane-mimicking model membranes. The largest disordering effect was observed for NAS and the smallest for tryptophan. Using fluorescence correlation spectroscopy, it was found that only NAS promotes vesicular association similar to that of serotonin, while the others did not. Furthermore, we found that the serotonin metabolites differed in their membrane distribution by employing solid state 1H magic angle spinning nuclear Overhauser enhancement spectroscopy (NOESY) experiments in simple POPC membranes. Similar results were obtained in synaptic membrane mimics using molecular dynamics simulations. In conclusion, while the causal correlation between membrane modulation effects and membrane distribution for the serotonin metabolites remains elusive, this study suggests that small-molecule metabolites and drugs can have drastic biological effects mediated through the membrane. The finding that small changes in structure lead to very different membrane modulation and distributions suggests the possibility of developing membrane modulating drugs in the future.

The interaction of the finger loop sequence of arrestin-3 with the neuropeptide Y1 and Y2 G protein-coupled receptors was studied. In cell assays, the finger loop part of arrestin was shown to be essential for Y2R internalization, but had no relevance for the interaction with Y1R. Here, we combined experimental data from nanoBRET, EPR, and NMR spectroscopy studies using a small library of NMR- and EPR-labeled finger loop peptides. Computer models of arrestin interacting with both receptors were built using AlphaFold2 and filtered by experimental constraints. Overall, Y1 and Y2 receptors show different binding modalities with the interacting finger loop region. In the interaction with Y1 receptor, the finger loop features a tail conformation critical for binding but also optional interactions. The Y2 receptor showed fewer contacts with the finger loop and two different conformations were found, which allow for higher intrinsic flexibility of this arrestin-3 segment.

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 (PET50nm and PET140nm) and concentrations (0, 10, 50, and 100 ppm) on the fibrillation of Aβ1-40. Our results showed that the presence of PET50nm as well as PET140nm 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 PET50nm and 3.8 ± 0.06 h for PET140nm. 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 the presence of 100 ppm PET50nm. These changes indicate that higher concentrations (100 ppm) of PET50nm promote more rigid and uniform peptide aggregates. Although PET50nm 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 PET50nm 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.

We studied the structure and dynamics of asymmetric POPCout/(POPE/POPG)in and POPSout/(POPE/POPG)in lipid membranes. To this end, the outer layer of multilamellar POPE/POPG (molar ratio 9 : 1) vesicles was exchanged (using methyl-β-cyclodextrin) by either chain deuterated POPC-d31 or POPS-d31, for which 2H NMR order parameters were measured. As controls, we prepared symmetric POPC-d31/POPE/POPG and POPS-d31/POPE/POPG membranes of the composition of just the outer membrane of the asymmetric multilamellar vesicles and pure POPC-d31 or POPS-d31 multilamellar vesicles. Compared to symmetric membranes of the same lipid composition, chain order parameters (S) of the asymmetric preparations were higher in the upper half of the chain and lower in the lower half. This reshuffling of acyl chain order is also expressed in higher 2H NMR Zeeman order relaxation rates (R1Z) of the chain segments in asymmetric membranes indicating alterations in the elastic properties of asymmetric bilayers as inferred from plots of R1Zvs. S2. Asymmetric membranes showed increased stiffness and rigidity although the lipid acyl chain composition between the inner and outer leaflets were identical. There were no indications for chain interdigitation between the two leaflets in the NMR spectra, which led us to speculate that the interleaflet coupling could be accomplished by sensing the differences in lipid packing densities between the two leaflets. These alterations in leaflet properties should have consequences for lipid protein interaction and ultimately protein function.

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.

The sheet-like lipid bilayer is the fundamental structural component of all cell membranes. Its building blocks are phospholipids and cholesterol. Their amphiphilic structure spontaneously leads to the formation of a bilayer in aqueous environment. Lipids are not just structural elements. Individual lipid species, the lipid membrane structure, and lipid dynamics influence and regulate membrane protein function. An exciting field is emerging where the membrane-associated material properties of different bilayer systems are used in designing innovative solutions for widespread applications across various fields, such as the food industry, cosmetics, nano- and biomedicine, drug storage and delivery, biotechnology, nano- and biosensors, and computing. Here, the authors summarize what is known about how lipids determine the properties and functions of biological membranes and how this has been or can be translated into innovative applications. Based on recent progress in the understanding of membrane structure, dynamics, and physical properties, a perspective is provided on how membrane-controlled regulation of protein functions can extend current applications and even offer new applications.

NMR spectroscopy techniques can provide important information about protein-ligand interactions. Here we tested an NMR approach which relies on the measurement of paramagnetic relaxation enhancements (PREs) arising from analogous cationic, anionic or neutral soluble nitroxide molecules, which distribute around the protein-ligand complex depending on near-surface electrostatic potentials. We applied this approach to two protein-ligand systems, interleukin-8 interacting with highly charged glycosaminoglycans and the SH2 domain of Grb2 interacting with less charged phospho-tyrosine tripeptides. The electrostatic potential around interleukin-8 and its changes upon binding of glycosaminoglycans could be derived from the PRE data and confirmed by theoretical predictions from Poisson-Boltzmann calculations. The ligand influence on the PREs and NMR-derived electrostatic potentials of Grb2 SH2 was localized to a narrow protein region which allowed the localization of the peptide binding pocket. Our analysis suggests that experiments with nitroxide cosolutes can be useful for investigating protein-ligand electrostatic interactions and mapping ligand binding sites.

Cellular membranes exhibit a huge diversity of lipids and membrane proteins that differ in their properties and chemical structure. Cells organize these molecules into distinct membrane compartments characterized by specific lipid profiles and hydrophobic thicknesses of the respective domains. If a hydrophobic mismatch occurs between a membrane protein and the surrounding lipids, there can be functional consequences such as reduced protein activity. This phenomenon has been extensively studied for single-pass transmembrane proteins, rhodopsin, and small polypeptides such as gramicidin. Here, we investigate the E. coli rhomboid intramembrane protease GlpG as a model to systematically explore the impact of membrane thickness on GlpG activity. We used fully saturated 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine(DMPC) model lipids and altered membrane thickness by varying the cholesterol content. Physical membrane parameters were determined by 2H and 31P NMR spectroscopy and correlated with GlpG activity measurements in the respective host membranes. Differences in bulk and annular lipids as well as alterations in protein structure in the respective host membranes were determined using molecular dynamics simulations. Our findings indicate that GlpG can influence the membrane thickness in DLPC/cholesterol membranes but not in DMPC/cholesterol membranes. Moreover, we observe that GlpG protease activity is reduced in DLPC membranes at low cholesterol content, which was not observed for DMPC. While a change in GlpG activity can already be due to smallest differences in the lipid environment, potentially enabling allosteric regulation of intramembrane proteolysis, there is no overall correlation to cholesterol-mediated lipid bilayer organization and phase behavior. Additional factors such as the influence of cholesterol on membrane bending rigidity and curvature energy need to be considered. In conclusion, the functionality of α-helical membrane proteins such as GlpG relies not only on hydrophobic matching but also on other membrane properties, specific lipid interaction, and the composition of the annular layer.