The interaction of small molecules, such as drugs or metabolites, with proteins and biomembranes is of fundamental importance for their bioavailability. The systematic characterization of the binding affinity for structurally related ligands may provide rules that allow its prediction for any other relevant molecule. In this work we have studied a homologous series of fluorescent fatty amines with the fluorescent moiety 7-nitrobenz-2-oxa-1,3-diazol-4-yl covalently bound to the amine group (NBD-C(n); n = 4, 6, 8, 10, 12, 14, and 16) in aqueous solution and associated with BSA or lipid bilayers. We have found a linear dependence with the length of the alkyl chain, up to NBD-C(10), for the Gibb's free energy of partition between the aqueous solution and 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers equal to ΔΔG = -2.5 ± 0.3 kJ/mol per methylene group. Additionally, the amphiphiles interacted efficiently with bovine serum albumin, and it was inhibited by fatty acids indicating that binding occurs to the fatty acids highest affinity binding site. The association of the amphiphiles with BSA and POPC bilayers was performed at different temperatures (15-35 °C) allowing for the calculation of the enthalpic and entropic contributions. A value of ΔH = -15 ± 4 kJ/mol was obtained for all amphiphiles and binding agents. The entropy contribution was always positive and increased with the length of the alkyl chain. The location of the ligand in the biological membrane is also of high relevance, namely because this will determine its effect on biomembrane properties at high ligand concentrations. With this goal, we have measured some photophysical properties of the amphiphiles inserted in POPC bilayers, and we found no significant variation along the series, indicating that the NBD group is located in a region with similar properties regardless of the length of the nonpolar group. An exception was noted for the case of NBD-C(14) whose parameters were somewhat different from the trend observed.

One of the great challenges in membrane biophysics is to find a means to foster the transport of drugs across complex membrane structures. In this spirit, we elucidate methodological challenges associated with free energy computations of complex chainlike molecules across lipid membranes. As an appropriate standard molecule to this end, we consider 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled fatty amine, NBD-Cn, which is here dealt with as a homologous series with varying chain lengths. We found the membrane-water interface region to be highly sensitive to details in free energy computations. Despite considerable simulation times, we observed substantial hysteresis, the cause being the small frequency of insertion/desorption events of the amphiphile's alkyl chain in the membrane interface. The hysteresis was most pronounced when the amphiphile was pulled from water to the membrane and compromised the data that were not in line with experiments. The subtleties in umbrella sampling for computing distance along the transition path were also observed to be potential causes of artifacts. With the PGD (pull geometry distance) scheme, in which the distance from the molecule was computed to a reference plane determined by an average over all lipids in the membrane, we found marked deformations in membrane structure when the amphiphile was close to the membrane. The deformations were weaker with the PGC (pull geometry cylinder) method, where the reference plane is chosen based on lipids that are within a cylinder of radius 1.7 nm from the amphiphile. Importantly, the free energy results given by PGC were found to be qualitatively consistent with experimental data, while the PGD results were not. We conclude that with long amphiphiles there is reason for concern with regard to computations of their free energy profiles. The membrane-water interface is the region where the greatest care is warranted.

A complete homologous series of fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled fatty amines of varying alkyl chain length, NBD-C(n), inserted in 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers, was studied using atomistic molecular dynamics (MD) simulations. For all amphiphiles, the NBD fluorophore locates near the glycerol backbone/carbonyl region of POPC and establishes stable hydrogen bonding with POPC ester oxygen atoms. Small differences observed in the transverse location of the fluorophore correlate with other calculated parameters and with small discrepancies recently measured in the photophysical properties of the molecules. The longer-chained NBD-C(n) amphiphiles show significant mass density near the bilayer midplane, and the chains of these derivatives interdigitate to some extent the opposite bilayer leaflet. This phenomenon leads to a slower lateral diffusion for the longer-chained derivatives (n > 12). Effects of these amphiphiles on the structure and dynamics of the host lipid were found to be relatively mild, in comparison with acyl-chain-labeled NBD probes. The molecular details obtained by this work allow the rationalization of the nonmonotonic behavior, recently obtained experimentally, for the photophysical parameters of the amphiphiles and the kinetic and thermodynamic parameters for their interaction with the POPC membranes.

the Czech Republic) and computational chemists Hugo Filipe, João P. Prates Ramalho and Luís Loura (Universities of Coimbra and Évora, Portugal) Title: Fluorescence of nitrobenzoxadiazole (NBD)-labeled lipids in model membranes is connected not to lipid mobility but to probe location By combining computational and experimental approaches, we explain the photophysics of NBD, one of the most frequently used fl uorescent membrane labels. We resolve previously published confl icting results and the long-standing misinterpretation of fl uorescence data of NBD. 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 (Dm) of NBD upon excitation. Previous calculations of the value of Dm 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 Dm and verified that it is rather small (B2 D).Fluorescence measurements confirmed that the value of REES is B16 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.

A complete homologous series of fluorescent phosphatidylethanolamines (diC n PE), labelled at the head group with a 7-nitrobenz-2-oxa-1,3-diazo-4-yl(NBD) fluorophore and inserted in 1-palmitoyl, 2-oleoyl-snglycero-3-phosphocholine (POPC) bilayers, was studied using atomistic molecular dynamics simulations. The longer-chained derivatives of NBD-diC n PE, with n = 14, 16, and 18, are commercially available, and widely used as fluorescent membrane probes. Properties such as location of atomic groups and acyl chain order parameters of both POPC and NBD-diC n PE, fluorophore orientation and hydrogen bonding, membrane electrostatic potential and lateral diffusion were calculated for all derivatives in the series. Most of these probes induce local disordering of POPC acyl chains, which is on the whole counterbalanced by ordering resulting from binding of sodium ions to lipid carbonyl/glycerol oxygen atoms. An exception is found for NBD-diC 16 PE, which displays optimal matching with POPC acyl chain length and induces a slight local ordering of phospholipid acyl chains. Compared to previously studied fatty amines, acyl chain-labelled phosphatidylcholines, and sterols bearing the same fluorescent tag, the chromophore in NBD-diC n PE locates in a similar region of the membrane (near the glycerol backbone/carbonyl region) but adopts a different orientation (with the NO 2 group facing the interior of the bilayer). This modification leads to an inverted orientation of the P-N axis in the labelled lipid, which affects the interface properties, such as the membrane electrostatic potential and hydrogen bonding to lipid head group atoms. The implications of this study for the interpretation of the photophysical properties of NBD-diC n PE (complex fluorescence emission kinetics, differences with other NBD lipid probes) are discussed.

One of the great challenges in pharmacokinetics is to find a means to optimize the transport across cell barriers. In this work, permeation across a cell monolayer, such as the tight endothelia in the blood-brain barrier, was modeled using a homologous series of amphipatic molecules, 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled alkyl chain amphiphiles (NBD-Cn, n = 2 to 16), to obtain rules that relate permeant structure to permeability. The amphiphile enters the system from the serum, equilibrated with serum albumin and lipoproteins, and its sequestration by serum components, interaction with the endothelium, and accumulation in the tissue is followed over time. The dependence of the permeability coefficient on the number of carbons of the amphiphile's alkyl chain has a parabolic-like shape. After a threshold value, an increase in the hydrophobicity of the amphiphile, along the homologous series, results in a decrease in the characteristic rate of permeation to the tissue. A sensitivity analysis was performed, and the rate limiting steps for permeation of each amphiphile were identified. Sequestration in the serum and rate of interaction with the endothelium, particularly the rate of desorption, were found to be the determinant processes for some amphiphiles, while for others translocation was the rate limiting step. Additionally, for some amphiphiles a single rate limiting step could not be identified, with several steps contributing significantly to the overall permeation. Finally, we derived analytical equations that adequately describe the rate of amphiphile accumulation in the tissue for the cases where permeation is controlled by a single rate limiting step.

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 G( 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.

The (de)regulatory effect of quercetin in cholesterol-enriched membrane domains, beyond its antioxidant activity, emerges as an important mode of action.