Flow-based bioconjugation of coumarin phosphatidylethanolamine probes: Optimised synthesis and membrane molecular dynamics studies.

A series of phosphatidylethanolamine fluorescent probes head-labelled with 3-carboxycoumarin was prepared by an improved bioconjugation approach through continuous flow synthesis. The established procedure, supported by a design of experiment (DoE) set-up, resulted in a significant reduction in the reaction time compared to the conventional batch method, in addition to a minor yield increase. The characterization of these probes was enhanced by an in-depth molecular dynamics (MD) study of the behaviour of a representative probe of this family, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine labelled with 3-carboxycoumarin (POPE-COUM), in bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (SLPC) 2:1, mimicking the composition of the egg yolk lecithin membranes recently used experimentally by our group to study POPE-COUM as a biomarker of the oxidation state and integrity of large unilamellar vesicles (LUVs). The MD simulations revealed that the coumarin group is oriented towards the bilayer interior, leading to a relatively internal location, in agreement with what is observed in the nitrobenzoxadiazole fluorophore of commercial head-labelled NBD-PE probes. This behaviour is consistent with the previously stated hypothesis that POPE-COUM is entirely located within the LUVs structure. Hence, the delay on the oxidation of the probe in the oxygen radical absorbance capacity (ORAC) assays performed is related with the inaccessibility of the probe until alteration of the LUV structure occurs. Furthermore, our simulations show that POPE-COUM exerts very little global and local perturbation on the host bilayer, as evaluated by key properties of the unlabelled lipids. Together, our findings establish PE-COUM as suitable fluorescent lipid analogue probes.

Interaction of MRI Contrast Agent [Gd(DOTA)]- with Lipid Membranes: A Molecular Dynamics Study.

Contrast agents are important imaging probes in clinical MRI, allowing the identification of anatomic changes that otherwise would not be possible. Intensive research on the development of new contrast agents is being made to image specific pathological markers or sense local biochemical changes. The most widely used MRI contrast agents are based on gadolinium(III) complexes. Due to their very high charge density, they have low permeability through tight biological barriers such as the blood-brain barrier, hampering their application in the diagnosis of neurological disorders. In this study, we explore the interaction between the widely used contrast agent [Gd(DOTA)]- (Dotarem) and POPC lipid bilayers by means of molecular dynamics simulations. This metal complex is a standard reference where several chemical modifications have been introduced to improve key properties such as bioavailability and targeting. The simulations unveil detailed insights into the agent's interaction with the lipid bilayer, offering perspectives beyond experimental methods. Various properties, including the impact on global and local bilayer properties, were analyzed. As expected, the results indicate a low partition coefficient (KP) and high permeation barrier for this reference compound. Nevertheless, favorable interactions are established with the membrane leading to moderately long residence times. While coordination of one inner-sphere water molecule is maintained for the membrane-associated chelate, the physical-chemical attributes of [Gd(DOTA)]- as a MRI contrast agent are affected. Namely, increases in the rotational correlation times and in the residence time of the inner-sphere water are observed, with the former expected to significantly increase the water proton relaxivity. This work establishes a reference framework for the use of simulations to guide the rational design of new contrast agents with improved relaxivity and bioavailability and for the development of liposome-based formulations for use as imaging probes or theranostic agents.

Exploring protein-protein interactions and oligomerization state of pulmonary surfactant protein C (SP-C) through FRET and fluorescence self-quenching.

Pulmonary surfactant (PS) is a lipid-protein complex that forms films reducing surface tension at the alveolar air-liquid interface. Surfactant protein C (SP-C) plays a key role in rearranging the lipids at the PS surface layers during breathing. The N-terminal segment of SP-C, a lipopeptide of 35 amino acids, contains two palmitoylated cysteines, which affect the stability and structure of the molecule. The C-terminal region comprises a transmembrane α-helix that contains a ALLMG motif, supposedly analogous to a well-studied dimerization motif in glycophorin A. Previous studies have demonstrated the potential interaction between SP-C molecules using approaches such as Bimolecular Complementation assays or computational simulations. In this work, the oligomerization state of SP-C in membrane systems has been studied using fluorescence spectroscopy techniques. We have performed self-quenching and FRET assays to analyze dimerization of native palmitoylated SP-C and a non-palmitoylated recombinant version of SP-C (rSP-C) using fluorescently labeled versions of either protein reconstituted in different lipid systems mimicking pulmonary surfactant environments. Our results reveal that doubly palmitoylated native SP-C remains primarily monomeric. In contrast, non-palmitoylated recombinant SP-C exhibits dimerization, potentiated at high concentrations, especially in membranes with lipid phase separation. Therefore, palmitoylation could play a crucial role in stabilizing the monomeric α-helical conformation of SP-C. Depalmitoylation, high protein densities as a consequence of membrane compartmentalization, and other factors may all lead to the formation of protein dimers and higher-order oligomers, which could have functional implications under certain pathological conditions and contribute to membrane transformations associated with surfactant metabolism and alveolar homeostasis.

Interaction of Hoechst 33342 with POPC Membranes at Different pH Values.

Hoechst 33342 (H33342) is a fluorescent probe that is commonly used to stain the DNA of living cells. To do so, it needs to interact with and permeate through cell membranes, despite its high overall charge at physiological pH values. In this work, we address the effect of pH in the association of H33342 with lipid bilayers using a combined experimental and computational approach. The partition of H33342 to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid membranes was experimentally quantified using fluorescence spectroscopy and isothermal titration calorimetry (ITC) measurements. Quantum chemical calculations were performed to select the most stable isomer of H33342 for the overall charges 0, +1, and +2, expected to predominate across the 5 < pH < 10 range. The interaction of these isomers with POPC bilayers was then studied by both unrestrained and umbrella sampling molecular dynamics (MD) simulations. Both experimental results and computational free energy profiles indicate that the partition coefficient of H33342 displays a small variation over a wide pH range, not exceeding one order of magnitude. The enthalpy variation upon partition to the membrane suggests efficient hydrogen bonding between the probe and the lipid, namely, for the protonated +2 form, which was confirmed in the MD simulation studies. The relatively high lipophilicity obtained for the charged species contrasts with the decrease in their general hydrophobicity as estimated from octanol/water partition. This highlights the distinction between lipophilicity and hydrophobicity, as well as the importance of considering the association with lipid bilayers when predicting the affinity for biomembranes.

Permeation of a Homologous Series of NBD-Labeled Fatty Amines through Lipid Bilayers: A Molecular Dynamics Study.

Permeation through biomembranes is ubiquitous for drugs to reach their active sites. Asymmetry of the cell plasma membrane (PM) has been described as having an important role in this process. Here we describe the interaction of a homologous series of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled amphiphiles (NBD-Cn, n = 4 to 16) with lipid bilayers of different compositions (1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine (POPC):cholesterol (1:1) and palmitoylated sphingomyelin (SpM):cholesterol (6:4)), including an asymmetric bilayer. Both unrestrained and umbrella sampling (US) simulations (at varying distances to the bilayer center) were carried out. The free energy profile of NBD-Cn at different depths in the membrane was obtained from the US simulations. The behavior of the amphiphiles during the permeation process was described regarding their orientation, chain elongation, and H-bonding to lipid and water molecules. Permeability coefficients were also calculated for the different amphiphiles of the series, using the inhomogeneous solubility-diffusion model (ISDM). Quantitative agreement with values obtained from kinetic modeling of the permeation process could not be obtained. However, for the longer, and more hydrophobic amphiphiles, the variation trend along the homologous series was qualitatively better matched by the ISDM when the equilibrium location of each amphiphile was taken as reference (ΔG = 0), compared to the usual choice of bulk water.

Fluorescent Probes cis- and trans-Parinaric Acids in Fluid and Gel Lipid Bilayers: A Molecular Dynamics Study.

Fluorescence probes are indispensable tools in biochemical and biophysical membrane studies. Most of them possess extrinsic fluorophores, which often constitute a source of uncertainty and potential perturbation to the host system. In this regard, the few available intrinsically fluorescent membrane probes acquire increased importance. Among them, cis- and trans-parinaric acids (c-PnA and t-PnA, respectively) stand out as probes of membrane order and dynamics. These two compounds are long-chained fatty acids, differing solely in the configurations of two double bonds of their conjugated tetraene fluorophore. In this work, we employed all-atom and coarse-grained molecular dynamics simulations to study the behavior of c-PnA and t-PnA in lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), representative of the liquid disordered and solid ordered lipid phases, respectively. All-atom simulations indicate that the two probes show similar location and orientation in the simulated systems, with the carboxylate facing the water/lipid interface and the tail spanning the membrane leaflet. The two probes establish interactions with the solvent and lipids to a similar degree in POPC. However, the almost linear t-PnA molecules have tighter lipid packing around them, especially in DPPC, where they also interact more with positively charged lipid choline groups. Probably for these reasons, while both probes show similar partition (assessed from computed free energy profiles across bilayers) to POPC, t-PnA clearly partitions more extensively than c-PnA to the gel phase. t-PnA also displays more hindered fluorophore rotation, especially in DPPC. Our results agree very well with experimental fluorescence data from the literature and allow deeper understanding of the behavior of these two reporters of membrane organization.

Interaction of a Homologous Series of Amphiphiles with P-glycoprotein in a Membrane Environment-Contributions of Polar and Non-Polar Interactions.

The transport of drugs by efflux transporters in biomembranes limits their bioavailability and is a major determinant of drug resistance development by cancer cells and pathogens. A large number of chemically dissimilar drugs are transported, and despite extensive studies, the molecular determinants of substrate specificity are still not well understood. In this work, we explore the role of polar and non-polar interactions on the interaction of a homologous series of fluorescent amphiphiles with the efflux transporter P-glycoprotein. The interaction of the amphiphiles with P-glycoprotein is evaluated through effects on ATPase activity, efficiency in inhibition of [125I]-IAAP binding, and partition to the whole native membranes containing the transporter. The results were complemented with partition to model membranes with a representative lipid composition, and details on the interactions established were obtained from MD simulations. We show that when the total concentration of amphiphile is considered, the binding parameters obtained are apparent and do not reflect the affinity for P-gp. A new formalism is proposed that includes sequestration of the amphiphiles in the lipid bilayer and the possible binding of several molecules in P-gp's substrate-binding pocket. The intrinsic binding affinity thus obtained is essentially independent of amphiphile hydrophobicity, highlighting the importance of polar interactions. An increase in the lipophilicity and amphiphilicity led to a more efficient association with the lipid bilayer, which maintains the non-polar groups of the amphiphiles in the bilayer, while the polar groups interact with P-gp's binding pocket. The presence of several amphiphiles in this orientation is proposed as a mechanism for inhibition of P-pg function.

Analysis of the Equilibrium Distribution of Ligands in Heterogeneous Media-Approaches and Pitfalls.

The equilibrium distribution of small molecules (ligands) between binding agents in heterogeneous media is an important property that determines their activity. Heterogeneous systems containing proteins and lipid membranes are particularly relevant due to their prevalence in biological systems, and their importance to ligand distribution, which, in turn, is crucial to ligand's availability and biological activity. In this work, we review several approaches and formalisms for the analysis of the equilibrium distribution of ligands in the presence of proteins, lipid membranes, or both. Special attention is given to common pitfalls in the analysis, with the establishment of the validity limits for the distinct approaches. Due to its widespread use, special attention is given to the characterization of ligand binding through the analysis of Stern-Volmer plots of protein fluorescence quenching. Systems of increasing complexity are considered, from proteins with single to multiple binding sites, from ligands interacting with proteins only to biomembranes containing lipid bilayers and membrane proteins. A new formalism is proposed, in which ligand binding is treated as a partition process, while considering the saturation of protein binding sites. This formalism is particularly useful for the characterization of interaction with membrane proteins.

Modeling Gd3+ Complexes for Molecular Dynamics Simulations: Toward a Rational Optimization of MRI Contrast Agents.

The correct parametrization of lanthanide complexes is of the utmost importance for their characterization using computational tools such as molecular dynamics simulations. This allows the optimization of their properties for a wide range of applications, including medical imaging. Here we present a systematic study to establish the best strategies for the correct parametrization of lanthanide complexes using [Gd(DOTA)]- as a reference, which is used as a contrast agent in MRI. We chose the bonded model to parametrize the lanthanide complexes, which is especially important when considering the study of the complex as a whole (e.g., for the study of the dynamics of its interaction with proteins or membranes). We followed two strategies: a so-called heuristic approach employing strategies already published by other authors and another based on the more recent MCPB.py tool. Adjustment of the Lennard-Jones parameters of the metal was required. The final topologies obtained with both strategies were able to reproduce the experimental ion to oxygen distance, vibrational frequencies, and other structural properties. We report a new strategy to adjust the Lennard-Jones parameters of the metal ion in order to capture dynamic properties such as the residence time of the capping water (τm). For the first time, the correct assessment of the τm value for Gd-based complexes was possible by recording the dissociative events over up to 10 µs all-atom simulations. The MCPB.py tool allowed the accurate parametrization of [Gd(DOTA)]- in a simpler procedure, and in this case, the dynamics of the water molecules in the outer hydration sphere was also characterized. This sphere was divided into the first hydration layer, an intermediate region, and an outer hydration layer, with a residence time of 18, 10 and 19 ps, respectively, independent of the nonbonded parameters chosen for Gd3+. The Lennard-Jones parameters of Gd3+ obtained here for [Gd(DOTA)]- may be used with similarly structured gadolinium MRI contrast agents. This allows the use of molecular dynamics simulations to characterize and optimize the contrast agent properties. The characterization of their interaction with membranes and proteins will permit the design of new targeted contrast agents with improved pharmacokinetics.

Molecular Dynamics Simulations: Advances and Applications.

Molecular dynamics (MD) simulations have led to great advances in many scientific disciplines, such as chemical physics, materials science, and biophysics [...].

Interactions between Rhodamine Dyes and Model Membrane Systems-Insights from Molecular Dynamics Simulations.

BACKGROUND: rhodamines are dyes widely used as fluorescent tags in cell imaging, probing of mitochondrial membrane potential, and as P-glycoprotein model substrates. In all these applications, detailed understanding of the interaction between rhodamines and biomembranes is fundamental. METHODS: we combined atomistic molecular dynamics (MD) simulations and fluorescence spectroscopy to characterize the interaction between rhodamines 123 and B (Rh123 and RhB, respectively) and POPC bilayers. RESULTS: while the xanthene moiety orients roughly parallel to the membrane plane in unrestrained MD simulations, variations on the relative position of the benzoic ring (below the xanthene for Rh123, above it for RhB) were observed, and related to the structure of the two dyes and their interactions with water and lipids. Subtle distinctions were found among different ionization forms of the probes. Experimentally, RhB displayed a lipid/water partition coefficient more than two orders of magnitude higher than Rh123, in agreement with free energy profiles obtained from umbrella sampling MD. CONCLUSIONS: this work provided detailed insights on the similarities and differences in the behavior of bilayer-inserted Rh123 and RhB, related to the structure of the probes. The much higher affinity of RhB for the membranes increases the local concentration and explains its higher apparent affinity for P-glycoprotein reconstituted in model membranes.

The Secret Lives of Fluorescent Membrane Probes as Revealed by Molecular Dynamics Simulations.

Fluorescent probes have been employed for more than half a century to study the structure and dynamics of model and biological membranes, using spectroscopic and/or microscopic experimental approaches. While their utilization has led to tremendous progress in our knowledge of membrane biophysics and physiology, in some respects the behavior of bilayer-inserted membrane probes has long remained inscrutable. The location, orientation and interaction of fluorophores with lipid and/or water molecules are often not well known, and they are crucial for understanding what the probe is actually reporting. Moreover, because the probe is an extraneous inclusion, it may perturb the properties of the host membrane system, altering the very properties it is supposed to measure. For these reasons, the need for independent methodologies to assess the behavior of bilayer-inserted fluorescence probes has been recognized for a long time. Because of recent improvements in computational tools, molecular dynamics (MD) simulations have become a popular means of obtaining this important information. The present review addresses MD studies of all major classes of fluorescent membrane probes, focusing in the period between 2011 and 2020, during which such work has undergone a dramatic surge in both the number of studies and the variety of probes and properties accessed.

Pulmonary surfactant protein SP-B nanorings induce the multilamellar organization of surfactant complexes.

Surfactant protein SP-B is absolutely required for the generation of functional pulmonary surfactant, a unique network of multilayered membranes, which stabilizes the respiratory air-liquid interface. It has been proposed that SP-B assembles into hydrophobic rings and tubes that facilitate the rapid transfer of phospholipids from membrane stores into the interface and the formation of multilayered films, ensuring the stability of the alveoli against physical forces leading to their collapse. To elucidate the molecular organization of SP-B-promoted multilamellar membrane structures, time-resolved Förster Resonance Energy Transfer (FRET) experiments between BODIPY-PC or BODIPY-derivatized SP-B (BODIPY/SP-B), as donor probes, and octadecylrhodamine B, as acceptor probe, were performed in liposomes containing SP-B or BODIPY/SP-B. Our results show that both SP-B and fluorescently labeled SP-B oligomers mediate the connection of adjacent bilayers. Furthermore, by applying rational models to the FRET data, we have been able to provide quantitative details of the structure of SP-B-induced multilayered membrane arrays at the nanometer scale, defining interactions between SP-B rings as key elements for connecting surfactant membranes. The data sustain the structural model and the mechanism of action of SP-B assemblies to sustain the crucial surfactant function.

Förster Resonance Energy Transfer as a Tool for Quantification of Protein-Lipid Selectivity.

This chapter addresses the determination of protein-lipid selectivity, here described as the preference of a protein for having a specific type of lipid in its vicinity, from Förster resonance energy transfer methodologies. These allow a quantification of the effect, that is, the determination of the biasing in distribution of the lipid under study around the protein, as compared to its bulk membrane distribution, with advantages over established approaches that have been used for the same purpose, such as electron spin resonance spectroscopy. The experiment can be carried out with steady-state instrumentation, the formalisms are described in detail, and the model can be applied to a membrane protein of any size.

Interaction of Bile Salts With Lipid Bilayers: An Atomistic Molecular Dynamics Study.

Bile salts (BS) are biosurfactants crucial for emulsification and intestinal absorption of cholesterol and other hydrophobic compounds such as vitamins and fatty acids. Interaction of BS with lipid bilayers is important for understanding their effects on membranes properties. The latter have relevance in passive diffusion processes through intestinal epithelium such as reabsorption of BS, as well as their degree of toxicity to intestinal flora and their potential applications in drug delivery. In this work, we used molecular dynamics simulations to address at the atomic scale the interactions of cholate, deoxycholate, and chenodeoxycholate, as well as their glycine conjugates with POPC bilayers. In this set of BS, variation of three structural aspects was addressed, namely conjugation with glycine, number and position of hydroxyl substituents, and ionization state. From atomistic simulations, the location and orientation of BS inside the bilayer, and their specific interactions with water and host lipid, such as hydrogen bonding and ion-pair formation, were studied in detail. Membrane properties were also investigated to obtain information on the degree of perturbation induced by the different BS. The results are described and related to a recent experimental study (Coreta-Gomes et al., 2015). Differences in macroscopic membrane partition thermodynamics and translocation kinetics are rationalized in terms of the distinct structures and atomic-scale behavior of the bile salt species. In particular, the faster translocation of cholate is explained by its higher degree of local membrane perturbation. On the other hand, the relatively high partition of the polar glycine conjugates is related to the longer and more flexible side chain, which allows simultaneous efficient solvation of the ionized carboxylate and deep insertion of the ring system.

Orientation of nitro-group governs the fluorescence lifetime of nitrobenzoxadiazole (NBD)-labeled lipids in lipid bilayers.

Nitrobenzoxadiazole (NBD) labeled lipids are popular fluorescent probes of membrane structure and dynamics, and have been widely used in both model systems and living cells. Irrespective of attachment to the lipid head group or hydrocarbon chains, the NBD fluorophore generally adopts a transverse bilayer location near the host lipid carbonyl/glycerol moieties. Still, considerable variability is observed in the measured fluorescence lifetimes, indicating that overall fluorophore location is not the determinant of NBD fluorescence properties. Combining fluorescence experiments and molecular dynamics simulations, we show that for two almost identical NBD probes, significant differences in fluorophore orientation and fluorescence lifetime are observed. Integrating these findings with literature data, we demonstrate a correlation between NBD orientation and fluorescence lifetime. The latter is longer when the NBD nitro group is predominantly oriented towards the bilayer interior, compared to probes for which it points to the water medium.

Quantitative Assessment of Methods Used To Obtain Rate Constants from Molecular Dynamics Simulations-Translocation of Cholesterol across Lipid Bilayers.

Accurately calculating rate constants of macroscopic chemical processes from molecular dynamics simulations is a long-sought but elusive goal. The problem is particularly relevant for processes occurring in biological systems, as is the case for ligand-protein and ligand-membrane interactions. Several formalisms to determine rate constants from easily accessible free-energy profiles [Δ Go( z)] of a molecule along a coordinate of interest have been proposed. However, their applicability for molecular interactions in condensed media has not been critically evaluated or validated. This work presents such evaluation and validation and introduces improved methodology. As a case study, we have characterized quantitatively the rate of translocation of cholesterol across 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine bilayers. Translocation across lipid bilayers is the rate-limiting step in the permeation of most drugs through biomembranes. We use coarse-grained molecular dynamics simulations and different kinetic formalisms to calculate this rate constant. A self-consistent test of the applicability of various available formalisms is provided by comparing their predictions with the translocation rates obtained from actual events observed in long unrestrained simulations. To this effect, a novel procedure was used to obtain the effective rate constant, based on an analysis of time intervals between transitions among different states along the reaction coordinate. While most tested formalisms lead to results in reasonable agreement (within a factor of 5) with this effective rate constant, the most adequate one is based on the explicit relaxation frequencies from the transition state in the forward and backward directions along the reaction coordinate.

Homo- and hetero-oligomerization of hydrophobic pulmonary surfactant proteins SP-B and SP-C in surfactant phospholipid membranes.

Pulmonary surfactant is a lipid/protein mixture that reduces surface tension at the respiratory air-water interface in lungs. Among its nonlipidic components are pulmonary surfactant-associated proteins B and C (SP-B and SP-C, respectively). These highly hydrophobic proteins are required for normal pulmonary surfactant function, and whereas past literature works have suggested possible SP-B/SP-C interactions and a reciprocal modulation effect, no direct evidence has been yet identified. In this work, we report an extensive fluorescence spectroscopy study of both intramolecular and intermolecular SP-B and SP-C interactions, using a combination of quenching and FRET steady-state and time-resolved methodologies. These proteins are compartmentalized in full surfactant membranes but not in pure 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) vesicles, in accordance with their previously described preference for liquid disordered phases. From the observed static self-quenching and homo-FRET of BODIPY-FL labeled SP-B, we conclude that this protein forms homoaggregates at low concentration (lipid:protein ratio, 1:1000). Increases in polarization of BODIPY-FL SP-B and steady-state intensity of WT SP-B were observed upon incorporation of under-stoichiometric amounts of WT SP-C. Conversely, Marina Blue-labeled SP-C is quenched by over-stoichiometric amounts of WT SP-B, whereas under-stoichiometric concentrations of the latter actually increase SP-C emission. Time-resolved hetero-FRET from Marina Blue SP-C to BODIPY-FL SP-B confirm distinct protein aggregation behaviors with varying SP-B concentration. Based on these multiple observations, we propose a model for SP-B/SP-C interactions, where SP-C might induce conformational changes on SP-B complexes, affecting its aggregation state. The conclusions inferred from the present work shed light on the synergic functionality of both proteins in the pulmonary surfactant system.

Diphenylhexatriene membrane probes DPH and TMA-DPH: A comparative molecular dynamics simulation study.

Fluorescence spectroscopy and microscopy have been utilized as tools in membrane biophysics for decades now. Because phospholipids are non-fluorescent, the use of extrinsic membrane probes in this context is commonplace. Among the latter, 1,6-diphenylhexatriene (DPH) and its trimethylammonium derivative (TMA-DPH) have been extensively used. It is widely believed that, owing to its additional charged group, TMA-DPH is anchored at the lipid/water interface and reports on a bilayer region that is distinct from that of the hydrophobic DPH. In this study, we employ atomistic MD simulations to characterize the behavior of DPH and TMA-DPH in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and POPC/cholesterol (4:1) bilayers. We show that although the dynamics of TMA-DPH in these membranes is noticeably more hindered than that of DPH, the location of the average fluorophore of TMA-DPH is only ~3-4Å more shallow than that of DPH. The hindrance observed in the translational and rotational motions of TMA-DPH compared to DPH is mainly not due to significant differences in depth, but to the favorable electrostatic interactions of the former with electronegative lipid atoms instead. By revealing detailed insights on the behavior of these two probes, our results are useful both in the interpretation of past work and in the planning of future experiments using them as membrane reporters.

Fluorescence of nitrobenzoxadiazole (NBD)-labeled lipids in model membranes is connected not to lipid mobility but to probe location.

Nitrobenzoxadiazole (NBD)-labeled lipids are popular fluorescent membrane probes. However, the understanding of important aspects of the photophysics of NBD remains incomplete, including the observed shift in the emission spectrum of NBD-lipids to longer wavelengths following excitation at the red edge of the absorption spectrum (red-edge excitation shift or REES). REES of NBD-lipids in membrane environments has been previously interpreted as reflecting restricted mobility of solvent surrounding the fluorophore. However, this requires a large change in the dipole moment (Δµ) of NBD upon excitation. Previous calculations of the value of Δµ of NBD in the literature have been carried out using outdated semi-empirical methods, leading to conflicting values. Using up-to-date density functional theory methods, we recalculated the value of Δµ and verified that it is rather small (∼2 D). Fluorescence measurements confirmed that the value of REES is ∼16 nm for 1,2-dioleoyl-sn-glycero-3-phospho-l-serine-N-(NBD) (NBD-PS) in dioleoylphosphatidylcholine vesicles. However, the observed shift is independent of both the temperature and the presence of cholesterol and is therefore insensitive to the mobility and hydration of the membrane. Moreover, red-edge excitation leads to an increased contribution of the decay component with a shorter lifetime, whereas time-resolved emission spectra of NBD-PS displayed an atypical blue shift following excitation. This excludes restrictions to solvent relaxation as the cause of the measured REES and TRES of NBD, pointing instead to the heterogeneous transverse location of probes as the origin of these effects. The latter hypothesis was confirmed by molecular dynamics simulations, from which the calculated heterogeneity of the hydration and location of NBD correlated with the measured fluorescence lifetimes/REES. Globally, our combination of theoretical and experiment-based techniques has led to a considerably improved understanding of the photophysics of NBD and a reinterpretation of its REES in particular.