Region Of Bilayer Research Articles

Membrane effects of N-terminal fragment of apolipoprotein A-I: a fluorescent probe study.

The binding of monomeric and aggregated variants of 1-83N-terminal fragment of apolipoprotein A-I with substitution mutations G26R, G26R/W@8, G26R/W@50 and G26R/W@72 to the model lipid membranes composed of phosphatidylcholine and its mixture with cholesterol has been investigated using fluorescent probes pyrene and Laurdan. Examination of pyrene spectral behavior did not reveal any marked influence of apoA-I mutants on the hydrocarbon region of lipid bilayer. In contrast, probing the membrane effects by Laurdan revealed decrease in the probe generalized polarization in the presence of aggregated proteins. suggesting that oligomeric and fibrillar apoA-I species induce increase in hydration degree and reduction of lipid packing density in the membrane interfacial region. These findings may shed light on molecular details of amyloid cytotoxicity.

Reconstitution and Topological Analysis of Caveolin-1 in Bicelles

Caveolae are 50-100 nm invaginations in the plasma membrane of many cell types that play a critical role in signal transduction. Caveolin-1 is a 21 kD integral membrane protein that is required for the formation of caveolae. Caveolin-1 is thought to adopt an unusual horseshoe topology in the membrane where the N- and C- termini are cytoplasmic. Caveolin-1 has 4 native tryptophan residues in this region (W85, W98, W115, and W128) that can be used as reporters of the micro-environment of the polypeptide chain. Caveolin-1 single tryptophan mutants were successfully reconstituted into CHAPSO/DMPC bicelles. Fluorescence emission measurements were performed on each mutant and analysis of these spectra indicated that tryptophan residues 85 and 128 are in the head group region of the lipid bilayer with λmax values of 344.4 ± 2.4 nm and 338.2 ± 0.6 nm respectively, and tryptophan residues 98 and 115 are inserted into the hydrophobic core with λmax values of 334.4 ± 0.2 nm and 330.2 ± 1.0 nm, respectively. The information gleaned from these studies was supported by MD simulations performed on caveolin-1 in a DMPC bilayer. These data together support the postulation that caveolin-1 contains a membrane embedded turn.

Coarse-grained simulation of dynamin-mediated fission.

Fission is a process in which a region of a lipid bilayer is deformed and separated from its host membrane, so that an additional, topologically independent compartment surrounded by a continuous lipid bilayer is formed. It is a fundamental process in the organization of the compartmentalization of living organisms and carefully regulated by a number of membrane-shaping proteins. An important group within these is the dynamin family of proteins that are involved in the final severance of the hourglass-shaped neck, via which the growing compartment remains connected to the main volume until the completion of fission. We present computer simulations testing different hypotheses of how dynamin proteins facilitate fission by constriction and curvature. Our results on constraint-induced fission of cylindrical membrane tubes emphasize the importance of the local creation of positive curvature and reveal a complex picture of fission, in which the topological transformation can become arrested in an intermediate stage if the proteins constituting the fission machinery are not adaptive.

The Effect of Highly Hydroxylated Fullerenol C60(OH)36on Human Erythrocyte Membrane Organization

The mechanism of the interaction of highly hydroxylated fullerenol C60(OH)36with erythrocyte membranes was studied by electron spin resonance spectroscopy (ESR) of stearic acid derivatives labeled with a nitroxyl radical at C-12 or C-16 and with a nitroxyl derivative of maleimide covalently attached to sulfhydryl groups of membrane proteins. A significant increase in membrane fluidity in the hydrophobic region of the lipid bilayer was observed for 12-doxylstearic acid at fullerenol concentrations of 100 mg/L or 150 mg/L, while for 16-doxylstearic acid significant increase in fluidity was only observed at 150 mg/L. Fullerenol at 100 mg/L or 150 mg/L caused conformational changes in membrane proteins, expressed as an increase in thehw/hsparameter, when fullerenol was added before the maleimide spin label (MSL) to the membrane suspension. The increase of thehw/hsparameter may be caused by changes in lipid-protein or protein-protein interactions which increase the mobility of the MSL label and as a result increase the membrane fluidity. Incubation of the membranes with the MSL before the addition of fullerenol blocked the available membrane protein –SH groups and minimized the interaction of fullerenol with them. This confirms that fullerenol interacts with erythrocyte membrane proteins via available protein –SH groups.

Aromatic Residues Display an Energetic Depth-Dependence in Lipid Bilayers that can be Modulated by Nearest-Neighbor Interactions

The interfacial region of the bilayer is a complex environment composed of lipid head groups, phosphates, diglycerides and a gradient of water molecule density. Due to the statistical preference of the aromatic residues for this area of the bilayer, it is generally believed that these residues are important for both folding and positioning of proteins within the membrane. To interrogate if this preference has a thermodynamic basis, we measured the water-to-lipid free energy changes for the aromatic side chains as a function of depth in the lipid bilayer. We used outer membrane phospholipase A (OmpLA) as a host membrane protein to introduce lipid-facing amino acid substitutions. At twelve sites, we mutated host side chains to alanine, tryptophan, tyrosine and phenylalanine. Chemical denaturation and thermodynamic linkage were employed to measure side chain water-to-lipid free energy changes. These quantities were calculated by taking the difference between stabilities (ΔΔGow,l) of OmpLA aromatic variants from that of alanine, which was used as a reference state. The experimental data can be interpreted as a sum of water-to-lipid transfer free energies and specific nearest neighbor interactions between certain side chains. We used molecular dynamics simulations and double variant cycles to model and quantify these nearest neighbor interactions. Altogether, our findings indicate that the tryptophan and tyrosine aromatic side chains exhibit a depth-dependence in ΔΔGow,l values whereas phenylalanine shows no such trend. These data demonstrate that the enrichment of tryptophan and tyrosine in interfacial regions of bilayers is due to their favorable contributions to the thermodynamic stabilities of membrane proteins.

Electrostatic force microscopy and electrical isolation of etched few-layer graphene nano-domains

Nanostructured bi-layer graphene samples formed through catalytic etching are investigated with electrostatic force microscopy. The measurements and supporting computations show a variation in the microscopy signal for different nano-domains that are indicative of changes in capacitive coupling related to their small sizes. Abrupt capacitance variations detected across etch tracks indicates that the nano-domains have strong electrical isolation between them. Comparison of the measurements to a resistor-capacitor model indicates that the resistance between two bi-layer graphene regions separated by an approximately 10 nm wide etch track is greater than about 1×1012 Ω with a corresponding gap resistivity greater than about 3×1014 Ω⋅nm. This extremely large gap resistivity suggests that catalytic etch tracks within few-layer graphene samples are sufficient for providing electrical isolation between separate nano-domains that could permit their use in constructing atomically thin nanogap electrodes, interconnects, and nanoribbons.

Nano-confined squaraine dye assemblies: new photoacoustic and near-infrared fluorescence dual-modular imaging probes in vivo.

For the purpose of near-infrared (NIR) fluorescence and photoacoustic (PA) tomography dual-modular imaging, self-assembly of squaraine (SQ) dyes is constructed in the hydrophobic phospholipid bilayers of liposomes (SQ⊂L) with variable mixing ratios of SQ and phospholipids from 1:500 to 1:10 (w/w). When doping minimal amounts of SQ, molecularly dispersed SQ in bilayers shows remarkable fluorescence. Interesting, the PA signal is enhanced with increase of SQ in the nanoconfined bilayer region, which is attributed to the formation of SQ-based H-aggregates and enhanced thermal conversion efficiency (η). SQ⊂L shows satisfactory chemical and thermal stabilities and photobleaching resistance. SQ⊂L is well-distributed in the cytoplasm of MCF-7 cells and its fluorescence signal remains for 7 days without dramatic quenching owing to the good stability of SQ⊂L. Furthermore, SQ⊂L is subjected to in vivo NIR fluorescence imaging to evaluate the whole-body biodistribution in organ level. Particularly, PA imaging with deeper tissue penetration capability is utilized to investigate the heterogeneous distribution SQ⊂L inside solid tumor. The majority of SQ⊂L are enriched in the area where the blood vessels are generated, implying that the liposomal nanocarriers exhibit lower tumor tissue penetration capability after the vascular leakage. This result is validated by histological examination of tumor tissue in parallel.

New Insights into Lipid Monolayers from Coarse-Grained Simulation Techniques

New Insights into Lipid Monolayers from Coarse-Grained Simulation Techniques

Effect of dilute solutions of biologically active substances on cell membranes

The article presents data on changes in physicochemical properties of different biological membranes (plasmatic, microsomal, synaptosomes) under the action of biologically active substances, which are different in their chemical structure and the mechanism of action (natural and synthetic antioxidants, thyrotropin--releasing hormone, phorbol esters), in the wide range of concentrations (10(-22)-10(-3)M). Dose dependences of the effect of biologically active substances on the activity of membrane-bound enzymes, lipid peroxidation, the structural state of the various regions of the lipid bilayer of membranes have been obtained and analyzed in terms of their formal generality of polymodality, number and position of the maxima, a sign change of the effect. An attempt to explain the mechanism of each of the observed peaks in these curves has been made. The maximum in the range of relatively high "physiological" concentrations (10(-3)-10(-7)M) is associated with introduction of biologically active substances into biomembranes. In this study maxima in the range of ultra-low doses (10(-11)-10(-16)M) and "apparent" concentrations -(10(-18)M), where the presence of biologically active substance molecule in a reaction volume is probabilistic in nature, are explained by physicochemical properties of diluted biologically active substances solutions. This conclusion is based on our data on the changes in IR spectra of aqueous solutions of biologically active substances and the results obtained by academician A.I. Konovalov et al. concerning the physicochemical properties of dilute solutions of biologically active substances (conductivity, surface tension, charge), due to the formation of so-called "nanoassociates" from biologically active substance molecule and numerous number of water molecules. The nanoassociates formation and biological effect disappear if the low concentration solutions are kept in a special shielded permalloy container protecting its contents from external electromagnetic field. Thus, nanoassociates are the material carriers of the unique ability of the ultra-low doses of biologically active substances to exhibit biological effects.

Water Penetration Profile at the Protein-Lipid Interface in Na,K-ATPase Membranes

The affinity of ionized fatty acids for the Na,K-ATPase is used to determine the transmembrane profile of water penetration at the protein-lipid interface. The standardized intensity of the electron spin echo envelope modulation (ESEEM) from 2H-hyperfine interaction with D2O is determined for stearic acid, n-SASL, spin-labeled systematically at the C-n atoms throughout the chain. In both native Na,K-ATPase membranes from shark salt gland and bilayers of the extracted membrane lipids, the D2O-ESEEM intensities of fully charged n-SASL decrease progressively with position down the fatty acid chain toward the terminal methyl group. Whereas the D2O intensities decrease sharply at the n = 9 position in the lipid bilayers, a much broader transition region in the range n = 6 to 10 is found with Na,K-ATPase membranes. Correction for the bilayer population in the membranes yields the intrinsic D2O-intensity profile at the protein-lipid interface. For positions at either end of the chains, the D2O concentrations at the protein interface are greater than in the lipid bilayer, and the positional profile is much broader. This reveals the higher polarity, and consequently higher intramembrane water concentration, at the protein-lipid interface. In particular, there is a significant water concentration adjacent to the protein at the membrane midplane, unlike the situation in the bilayer regions of this cholesterol-rich membrane. Experiments with protonated fatty acid and phosphatidylcholine spin labels, both of which have a considerably lower affinity for the Na,K-ATPase, confirm these results.

Interaction of neurotransmitters with a phospholipid bilayer: A molecular dynamics study

We have performed a series of molecular dynamics simulations to study the interactions between the neurotransmitters (NTs) γ-aminobutyrate (GABA), glycine (GLY), acetylcholine (ACH) and glutamate (GLU) as well as the amidated/acetylated γ-aminobutyrate (GABAneu) and the osmolyte molecule glycerol (GOL) with a dipalmitoylphosphatidylcholine (DPPC) bilayer. In agreement with previously published experimental data, we found the lowest membrane affinity for the charged molecules and a moderate affinity for zwitterionic and polar molecules. The affinity can be ranked as follows: ACH–GLU<<GABA<GLY<<GABAneu<<GOL. The latter three penetrated the bilayer at most with the deepest location being close to the glycerol backbone of the phospholipids. Even at that position, these solutes were noticeably hydrated and carried ∼30–80% of the bulk water along. The mobility of hydration water at the solute is also affected by the penetration into the bilayer. Two time scales of exchanging water molecules could be determined. In the bulk phase, the hydration layer contains ∼20% slow exchanging water molecules which increases 2–3 times as the solutes entered the bilayer. Our results indicate that there is no intermediate exchange of slow moving water molecules from the solutes to the lipid atoms and vice versa. Instead, the exchange relies on the reservoir of unbounded (“free”) water molecules in the interfacial bilayer region. Results from the equilibrium simulations are in good agreement with the results from umbrella sampling simulations, which were conducted for the four naturally occurring NTs. Free energy profiles for ACH and GLU show a minimum of ∼2–3kJ/mol close to the bilayer interface, while for GABA and GLY, a minimum of respectively ∼2kJ/mol and ∼5kJ/mol is observed when these NTs are located in the vicinity of the lipid glycerol backbone. The most important interaction of NTs with the bilayer is the charged amino group of NTs with the lipid phosphate group.

Synthesis of Atomically Thin &lt;inline-formula&gt;&lt;tex-math&gt;${\bf MoS}_{\bf 2}$&lt;/tex-math&gt;&lt;/inline-formula&gt; Triangles and Hexagrams and Their Electrical Transport Properties

Atomically thin molybdenum disulfide <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math>$({\rm MoS}_{2})$</tex-math></inline-formula> triangles and hexagrams were prepared by a two-step growth ambient pressure chemical vapor deposition (APCVD) process. Molybdenum Trioxide <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math>$({\rm MoO}_{3})$</tex-math></inline-formula> nanobelts, a few microns in length and width, were prepared using a hydrothermal technique and utilized as the starting material. High temperature treatment of the <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math>${\rm MoO}_{3}$</tex-math></inline-formula> nanobelts followed by a rigorous sulfurization via APCVD processing provided different morphologies of <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math>${\rm MoS}_{2}$</tex-math></inline-formula> monolayers and bilayer (BL) sheets. Triangle and hexagram morphologies were characterized using Raman spectroscopy, photoluminescence (PL) measurements, scanning electron microscopy and atomic force microscopy (AFM). The regrowth step in the CVD process was proven to be ideal in enlarging the grain size. PL and Raman spectroscopy and AFM results confirmed the presence of monolayer and BL regions in the regrowth growth process. Triangle and hexagram domains are observed to be cooperatively nucleating and coalescing together to form large-area layers. Furthermore, the electrical transport properties of the synthesized <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math>${\rm MoS}_{2}$</tex-math></inline-formula> layers were studied. Electron mobility based on back gated field effect transistors was measured to be approximately 0.02 <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math>${\rm cm}^{2}$</tex-math></inline-formula> /V. S.

Liposomes physically coated with peptides: preparation and characterization.

Physically coating liposomes with peptides of desirable functions is an economic, versatile, and less time-consuming approach to prepare drug delivery vehicles. In this work, we designed three peptides-Ac-WWKKKGGNNN-NH2 (W2K3), Ac-WWRRRGGNNN-NH2(W2R3), Ac-WWGGGGGNNN-NH2(W2G3)-and studied their coating ability on negatively charged liposomes. It was found that the coating was mainly driven by the electrostatic interaction between the peptides' cationic side groups and the acidic lipids, which also mediated the "anchoring " of Trp residuals in the interfacial region of lipid bilayers. At the same conditions, the amount of the coated W2R3 was more than that of W2K3, but the stability of the liposome coated with W2R3 was deteriorated. This was caused by the delocalized charge of the guanidinium group of arginine. The coating of the peptide rendered the liposome pH-responsive behavior but did not prominently change the phase transition temperature. The liposome coated with peptides displayed appropriate pH/temperature dual responsive characteristics and was able to release the content in a controlled manner.

Role of structural changes induced in biological membranes by hydrolysable tannins from sumac leaves (Rhus typhina L.) in their antihemolytic and antibacterial effects.

In this study, we found that the sumac tannins (Rhus typhina L.) exert to a various extent antihemolytic effects and antibacterial activity against Bacillus cereus and Pseudomonas aeruginosa depending on structural specificity of bacteria and different mechanisms of their toxic action. The sumac tannins exert the most expressed activity against B. cereus. The antihemolytic effect of the sumac tannins seems to be connected to a greater extent with their modifying action on the erythrocyte membrane structure. It was found that the sumac tannins are incorporated into the erythrocyte membrane, causing transformation of discocytes into echinocytes and enhancing the rigidity of the hydrophilic region of the lipid bilayer. We suggest that the embedding of sumac tannins into the membrane of erythrocytes alters their physical properties and, as a consequence, can limit their interaction with bacterial toxins.

Epitaxial Growth of Asymmetrically‐Doped Bilayer Graphene for Photocurrent Generation

An asymmetrically doped bilayer graphene is grown by modulation-doped chemical vapor deposition, which consists of one intrinsic layer and one nitrogen-doped layer according to AB stacking. The asymmetrically doped bilayer crystalline profile is found to extend the identical registry as adjacent pristine bilayer region, thus forming single-crystalline bilayer graphene p-n junctions. Efficient photocurrent with responsivity as high as 0.2 mA/W is generated at the bilayer p-n junctions via a hot carrier-assisted mechanism.

Structure of the membrane anchor of pestivirus glycoprotein E(rns), a long tilted amphipathic helix.

Erns is an essential virion glycoprotein with RNase activity that suppresses host cellular innate immune responses upon being partially secreted from the infected cells. Its unusual C-terminus plays multiple roles, as the amphiphilic helix acts as a membrane anchor, as a signal peptidase cleavage site, and as a retention/secretion signal. We analyzed the structure and membrane binding properties of this sequence to gain a better understanding of the underlying mechanisms. CD spectroscopy in different setups, as well as Monte Carlo and molecular dynamics simulations confirmed the helical folding and showed that the helix is accommodated in the amphiphilic region of the lipid bilayer with a slight tilt rather than lying parallel to the surface. This model was confirmed by NMR analyses that also identified a central stretch of 15 residues within the helix that is fully shielded from the aqueous layer, which is C-terminally followed by a putative hairpin structure. These findings explain the strong membrane binding of the protein and provide clues to establishing the Erns membrane contact, processing and secretion.

Antimicrobial properties and interaction of two Trp-substituted cationic antimicrobial peptides with a lipid bilayer

Tryptophan (Trp) residues reportedly exhibit a strong membrane-disruptive activity, and this property endows Trp-containing antimicrobial peptides (AMPs) with a unique ability to interact with the surface of bacterial cell membranes, possibly improving antimicrobial properties. In this study, we investigated the influence of Trp residues engineered to have a distinct preference for the interface region of lipid bilayers on antimicrobial activity. We designed two Trp-substituted AMPs (I1WL5W and I4WL5W) by replacing Ile or Leu residues with two Trp residues at different positions in the L-K6 peptide, and determined their antimicrobial activity and mechanism of membrane action. Both I1WL5W and I4WL5W exhibited significantly higher antimicrobial activity and lower cytotoxicity against Gram-negative and Gram-positive bacteria compared with L-K6. The Trp-substituted peptides had a disordered structure in aqueous solution and adopted an α-helical structure in solutions of 50% trifluoroethanol/water and 30 mM SDS. I1WL5W and I4WL5W caused a significant leakage of calcein from liposomes containing membranes that mimicked those of Escherichia coli and Staphylococcus aureus. Scanning electron microscopy analysis suggested that I1WL5W and I4WL5W killed bacteria by disrupting bacterial cell membranes. Furthermore, fluorescence and quenching data from a variety of liposomes, which mimic different cell membranes, indicated that the Trp-substituted peptides could insert into the lipid bilayers and induce blue shifts in the emission spectra of the Trp residues. I1WL5W and I4WL5W were also less susceptible to acrylamide or KI quenchers. The current work may be important for designing novel Trp-containing peptides exhibiting strong antimicrobial abilities by penetrating bacterial membranes.

Effect of cholesterol on behavior of 5-fluorouracil (5-FU) in a DMPC lipid bilayer, a molecular dynamics study

In this work, molecular dynamics (MD) simulations were performed to investigate the effects of cholesterol on the interaction between the hydrophilic anticancer drug, 5-FU, and fully hydrated 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayer. Several structural and dynamical parameters of DMPC bilayers with varying amounts of cholesterol (0, 25, and 50mol%) in the presence and absence of drug molecules were calculated. Moreover, the free energy barriers for translocation of one 5-FU molecule from water to the lipid bilayer were determined by using the potential of mean force (PMF). PMF studies indicated that the location of the maximum free energy barrier was in the hydrophobic middle region of bilayer, while the minimums of the barrier were located at the hydrophilic part of bilayer at the interface with water. The minimum and maximum of the free energy profiles were independent of cholesterol concentration and suggested that the drug molecules 5-FU were accumulated in the vicinity of the polar head group of lipid bilayers. Moreover, the results showed that with increasing cholesterol concentration in the bilayer, the free energy barrier for translocation of 5-FU across the bilayer also increases which can be attributed to the condensing effect of the cholesterol on the bilayer.

Lipid Selectivity in Lipid Efflux Induced by Proteins and Peptides

Some proteins and peptides have the ability to extract lipids from membranes. In some systems, such as some toxins, it leads to cell death. In other systems, this process is vital. For example, bovine seminal plasma contains phosphocholine-binding proteins that associate with sperm membranes upon ejaculation and cause lipid efflux; this effect modifies the membrane lipid composition, an essential step in the maturation process of fertile sperm. In general, the affinity of these peptides/proteins for membranes and their impact on membrane permeability are well characterized. However the induced lipid efflux and particularly its lipid specificity are much less investigated. Using model membranes with different compositions, we identified the lipid selectivity of the membrane lipid efflux of two systems. First, we characterized the lipid efflux induced by BSP1, the most abundant Binder-of-Sperm protein in bull seminal plasma. The protein binding displays a distinct affinity for phosphatidylcholine. However, even though the protein anchoring on membranes is PC-specific, we demonstrated that the lipid extraction is practically without any lipid specificity; BSP1 molecules solubilise a lipid patch to form small complexes with a stoichiometry of 10-15 lipids per protein. Second, we characterized the lipid specificity of lipid efflux induced by melittin, a helical peptide with a secondary amphipathic character. Phosphatidylethanolamine, and cholesterol, two molecular species ordering bilayers, reduce the membrane affinity of melittin. We determined that, in parallel, the lipid fraction extracted by melittin is depleted of these lipids. It is proposed that the peptide accumulates in regions of bilayers where the bilayers are less ordered and, when its concentration is sufficient, detaches this patch from the membrane. These findings illustrate the need to investigate globally lipid efflux processes, from the peptide/protein binding to the mixed lipid-peptide/protein self-assembly formation.

Theoretical Investigation of TrHbN Association to Biological Membranes

TrHbN is a group I truncated hemoglobin from Mycobacterium tuberculosis (Mtb). TrHbN protects Mtb aerobic respiration from •NO inhibition and is thought to play pivotal roles in the persistence of mycobacterial infection. Its 3D structure is characterized by the presence of hydrophobic tunnels. Previous MD simulations revealed that three of theses tunnels are used by •NO to reach the active site. Despite the fact that trHbN is soluble and highly active in aqueous solvent, there is compelling evidence suggesting that trHbN is associated with biological membranes. We have investigated the association of trHbN with membranes differing in lipid compositions (DOPC, CL and CL:DOPE) using all-atom MD simulations.We found that trHbN penetrates the peripheral regions of the lipid bilayer for all the lipid compositions. Two portions of trHbN make a dominant contribution to the membrane binding: the N-terminal pre-A helix, and the G and H helices from the core of the protein. The pre-A helix has four charged residues that interact with the lipid phosphates, and several hydrophobic residues that insert under the lipid headgroups. The G and H helices have several hydrophobic residues in contact with the aliphatic chains. These residues were identified to form a large hydrophobic funnel-shape entrance of the Short tunnel (ST), previously shown to be preferentially used by •NO to reach the distal heme pocket. As a consequence, the ST has its entrance pointing towards the lipid bilayer, while the Long tunnel (LT) entrance is pointing towards the aqueous solvent. The orientation of trHbN differs slightly in presence of cardiolipins due to additional lipid phosphate-protein core interactions. These results suggest that the substrates reach the active site from the membrane interior using the ST and the products escape to the solvent using the LT.