Hydrophobic Region Of Membrane Research Articles

Location, Partitioning Behavior, and Interaction of Capsaicin with Lipid Bilayer Membrane: Study Using Its Intrinsic Fluorescence.

Capsaicin is an ingredient of a wide variety of red peppers, and it has various pharmacological and biological applications. The present study explores the interaction of capsaicin with dimyristoylphosphatidylcholine (DMPC) lipid bilayer membrane by monitoring various photophysical parameters using its intrinsic fluorescence. In order to have a clearer understanding of the photophysical responses of capsaicin, studies involving (i) its solvation behavior in different solvents, (ii) the partition coefficient of capsaicin in different thermotropic phase states of lipid bilayer membrane, and (iii) its location inside lipid bilayer membrane have been carried out. Capsaicin has a reasonably high partition coefficient for DMPC liposome membrane, in both solid gel (2.8 ± 0.1 × 10(5)) and liquid crystalline (2.6 ± 0.1 × 10(5)) phases. Fluorescence quenching study using cetylpyridinium chloride (CPC) as quencher suggests that the phenolic group of capsaicin molecule is generally present near the headgroup region and hydrophobic tail present inside hydrophobic core region of the lipid bilayer membrane. The intrinsic fluorescence intensity and lifetime of capsaicin sensitively respond to the temperature dependent phase changes of liposome membrane. Above 15 mol %, capsaicin in the aqueous liposome suspension medium lowers the thermotropic phase transition temperature by about 3 °C, and above 30 mol %, the integrity of the membrane is significantly lost.

Exploring O2 diffusion in A-type cytochrome c oxidases: molecular dynamics simulations uncover two alternative channels towards the binuclear site.

Cytochrome c oxidases (Ccoxs) are the terminal enzymes of the respiratory chain in mitochondria and most bacteria. These enzymes couple dioxygen (O2) reduction to the generation of a transmembrane electrochemical proton gradient. Despite decades of research and the availability of a large amount of structural and biochemical data available for the A-type Ccox family, little is known about the channel(s) used by O2 to travel from the solvent/membrane to the heme a3-CuB binuclear center (BNC). Moreover, the identification of all possible O2 channels as well as the atomic details of O2 diffusion is essential for the understanding of the working mechanisms of the A-type Ccox. In this work, we determined the O2 distribution within Ccox from Rhodobacter sphaeroides, in the fully reduced state, in order to identify and characterize all the putative O2 channels leading towards the BNC. For that, we use an integrated strategy combining atomistic molecular dynamics (MD) simulations (with and without explicit O2 molecules) and implicit ligand sampling (ILS) calculations. Based on the 3D free energy map for O2 inside Ccox, three channels were identified, all starting in the membrane hydrophobic region and connecting the surface of the protein to the BNC. One of these channels corresponds to the pathway inferred from the X-ray data available, whereas the other two are alternative routes for O2 to reach the BNC. Both alternative O2 channels start in the membrane spanning region and terminate close to Y288I. These channels are a combination of multiple transiently interconnected hydrophobic cavities, whose opening and closure is regulated by the thermal fluctuations of the lining residues. Furthermore, our results show that, in this Ccox, the most likely (energetically preferred) routes for O2 to reach the BNC are the alternative channels, rather than the X-ray inferred pathway.

Interactions of amelogenin with phospholipids.

Amelogenin protein has the potential to interact with other enamel matrix proteins, mineral, and cell surfaces. We investigated the interactions of recombinant amelogenin rP172 with small unilamellar vesicles as model membranes, toward the goal of understanding the mechanisms of amelogenin-cell interactions during amelogenesis. Dynamic light scattering (DLS), fluorescence spectroscopy, circular dichroism (CD), and nuclear magnetic resonance (NMR) were used. In the presence of phospholipid vesicles, a blue shift in the Trp fluorescence emission maxima of rP172 was observed (∼334 nm) and the Trp residues of rP172 were inaccessible to the aqueous quencher acrylamide. DLS studies indicated complexation of rP172 and phospholipids, although the possibility of fusion of phospholipids following amelogenin addition cannot be ruled out. NMR and CD studies revealed a disorder-order transition of rP172 in a model membrane environment. Strong fluorescence resonance energy transfer from Trp in rP172 to DNS-bound-phospholipid was observed, and fluorescence polarization studies indicated that rP172 interacted with the hydrophobic core region of model membranes. Our data suggest that amelogenin has ability to interact with phospholipids and that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities.

Vesicles protect activated acetic acid.

Abstract Methyl thioacetate, or activated acetic acid, has been proposed to be central to the origin of life and an important energy currency molecule in early cellular evolution. We have investigated the hydrolysis of methyl thioacetate under various conditions. Its uncatalyzed rate of hydrolysis is about 3 orders of magnitude faster (K=0.00663 s(-1); 100°C, pH 7.5, concentration=0.33 mM) than published rates for its catalyzed production, making it unlikely to accumulate under prebiotic conditions. However, our experiments showed that methyl thioacetate was protected from hydrolysis when inside its own hydrophobic droplets. Further, we found that methyl thioacetate protection from hydrolysis was also possible in droplets of hexane and in the membranes of nonanoic acid vesicles. Thus, the hydrophobic regions of prebiotic vesicles and early cell membranes could have offered a refuge for this energetic molecule, increasing its lifetime in close proximity to the reactions for which it would be needed. This model of early energy storage evokes an additional critical function for the earliest cell membranes.

Effect of chlorogenic acid on the phase transition in phospholipid and phospholipid/cholesterol membranes

Chlorogenic acid (CGA) is present in many plants, especially in green coffee, dry plums, and bilberries. It is an important bioactive polyphenol. Studies showed that CGA has an antioxidative, bacteriostatic, anticancer, antiviral, and anti-inflammatory activity. Despite great interest in this compound, its interaction with the lipid model membrane has not yet been investigated. To better understand the relationship between the biological activity of CGA and its interaction with biological membranes, the thermotropic behavior of model lipid membranes was investigated. The effect of CGA on the model lipid membrane, specifically on the lipid bilayer phase transitions, was examined by the combined methods: differential scanning calorimetry and fluorescence spectroscopy. In particular, the degree of packing order of the hydrophilic phase of the lipid bilayer was determined using the fluorimetric method with Laurdan and Prodan probes, while the fluorescence anisotropy of the hydrophobic phase with the DPH and TMA-DPH probes. The results of the study show that CGA incorporates mainly into the hydrophilic part of membrane, changing the packing order of the polar heads of lipids. No significant changes were recorded in membrane fluidity of the hydrophobic membrane region, for the fluorescence anisotropy practically did not change. One can thus infer that CGA does not penetrate deep into the hydrophobic area of the membrane.

Impact of histidine residues on the transmembrane helices of viroporins

Abstract The role of histidine in channel-forming transmembrane (TM) helices was investigated by comparing the TM helices from Virus protein 'u' (Vpu) and the M2 proton channel. Both proteins are members of the viroporin family of small membrane proteins that exhibit ion channel activity, and have a single TM helix that is capable of forming oligomers. The TM helices from both proteins have a conserved tryptophan towards the C-terminus. Previously, alanine 18 of Vpu was mutated to histidine in order to artificially introduce the same HXXXW motif that is central to the proton channel activity of M2. Interestingly, the mutated Vpu TM resulted in an increase in helix tilt angle of 11° in lipid bilayers compared to the wild-type Vpu TM. Here, we find the reverse, when histidine 37 of the HXXXW motif in M2 was mutated to alanine, it decreased the helix tilt by 10° from that of wild-type M2. The tilt change is independent of both the helix length and the presence of tryptophan. In addition, compared to wild-type M2, the H37A mutant displayed lowered sensitivity to proton concentration. We also found that the solvent accessibility of histidine-containing M2 is greater than without histidine. This suggests that the TM helix may increase the solvent exposure by changing its tilt angle in order to accommodate a polar/charged residue within the hydrophobic membrane region. The comparative results of M2, Vpu and their mutants demonstrated the significance of histidine in a transmembrane helix and the remarkable plasticity of the function and structure of ion channels stemming from changes at a single amino acid site.

Imbalance in redox system of rat liver following permethrin treatment in adolescence and neonatal age

1. The effect of different permethrin treatments on the redox system of rat liver, is presented. Two types of oral administration were chosen: (i) sub-chronic treatment (1/10 of LD50 for 60 days) during adolescence (5 weeks old) and (ii) sub-acute treatment (1/44 of LD50 for 15 days) during early life (from postnatal days 6–21).2. The results show that adolescent permethrin treatment induces damage to the liver redox system, increasing lipid and protein peroxidation and reducing membrane fluidity in the hydrophilic--hydrophobic region of the bilayer. In addition, glutathione peroxidase (GPx) and GSH levels resulted decreased, while glutathione transferase (GST) and catalase (CAT) levels increased.3. The rats treated in early life with permethrin and sacrificed in adult age, showed less signs of damage compared to those exposed during adolescence in which lipid peroxidation was increased by 32%, whereas for the first group the raise was only 11%. Moreover, fluidity improved in the deeper hydrophobic membrane region of the treated group, while the level of CAT was significantly lower compared to the control one.4. Although sub-chronic treatment increased CAT and GST and decreased GPx and GSH levels, the present data suggest that a shorter exposure to permethrin during neonatal age decreased CAT level and it could represent an important risk factor for the onset of long-term liver damage.

Interaction of free fatty acids with mitochondria during uncoupling of oxidative phosphorylation

The activity of free saturated fatty acids (caprylic, capric, lauric, myristic, palmitic and stearic) as inducers and regulators of uncoupling of oxidative phosphorylation with participation of ADP/ATP antiporter, aspartate/glutamate antiporter and cyclosporin A-sensitive structure was investigated in experiments on rat liver mitochondria. It is established that at equal uncoupling activity of fatty acids the regulatory effect is minimal for caprylic acid and raised with increasing the hydrophobicity of fatty acids reaching the maximum value for stearic acid. There exists the linear dependence of the regulatory effect value of fatty acids on fatty acids content in the hydrophobic region of the inner membrane. The model that describes the interaction of fatty acids with the hydrophobic region of the mitochondrial inner membrane preserving functional activity of organelles is developed. It is established that if molecules of various fatty acids being in the hydrophobic region of the membrane are equally effective as uncoupling regulators, their specific uncoupling activity is different. Caprylic acid, a short-chain fatty acid, possesses the highest uncoupling activity. As the acyl chain length increases, the specific uncoupling activity of fatty acids reduces exponentially. Under these conditions components of the uncoupling activity sensitive to glutamate and carboxyatractylate and glutamate and insensitive to these reagents (but sensitive to cyclosporin A) change approximately equally.

Molecular Interactions of Alzheimer's Aβ Peptide Fibrils and Oligomers with Lipid Membranes

The toxicity of amyloid oligomers and larger fibrillar aggregates formed by of Alzheimer's disease amyloid beta (Aβ) peptides is related to perturbing the normal biological function of neighboring cellular membranes. We probed the first steps in the mechanism of Aβ-membrane interactions using atomistic molecular dynamics simulations of Aβ9-40 fibrillar oligomers modeled as protofilament segments, including lipid bilayers and explicit water molecules. Our study identified as principal driving force the electrostatic interaction between charged peptide residues and the lipid headgroups, which can modulate the further penetration of the C-termini of amyloid fibrils or fibrillar oligomers into the hydrophobic region of lipid membranes. These findings unveil detailed molecular mechanisms of Aβ-membrane interactions, and suggest a polymorphic structural character of amyloid ion channels embedded in lipid bilayers.

Photophysical properties and photodynamic activity of a novel menthol–zinc phthalocyanine conjugate incorporated in micelles

Abstract The synthesis, photophysical properties, and photodynamic activity of the lipophilic tetra-menthyl-substituted zinc phthalocyanine (ZnMintPc, main regioisomer {2,9,16,23-tetrakis{[(1 S ,2 R ,5 S )-5-methyl-2-(1-methylethyl)cyclohexyl]oxy}-phthalocyaninato}zinc(II)) are described. This compound was synthesized under cyclotetramerization conditions by reaction of substituted phthalonitrile prepared from 4-nitrophthalonitrile with natural l -menthol. This new phthalocyanine presented remarkable structural and photophysical features with potential application in photodynamic therapy (PDT) involving hydrophobic membrane regions. This photosensitizer was incorporated in micelles of 12 different surfactants, namely ionic detergents SDS, CTAB, and HPS, the non-ionic detergents Tween ® 20, Tween ® 80, C 12 E 9 , Brij ® 30, Brij ® 35, Brij ® 97, and Brij ® 98, and the triblock copolymers Pluronic F-68 and Pluronic F-127. Solubilization or aggregation in the lipophilic compartment of the micelles was assessed by UV–vis absorption and photoluminescence spectroscopies. Singlet oxygen was produced by irradiation of the micelles with light from red LEDs, peaked at 635 nm, and the quantum yields were obtained using 1,3-diphenylisobenzofuran (DPBF) as probe. Two micellar systems, more specifically Brij ® 30 and Pluronic ® F-127, displayed excellent ability to generate singlet oxygen. Photobleaching rates of the ZnMintPc incorporated into the micelles were also obtained. The results demonstrated the lipophilic ZnMintPc is suitable for PDT applications and suggest the use of Pluronic ® F-127 in the formulation of adequate drug delivery systems for this photosensitizer.

Vitex Agnus-Castus L. Essential Oil Increases Human Erythrocyte Membrane Fluidity

Vitex Agnus-CastusL. Essential Oil Increases Human Erythrocyte Membrane FluidityErythrocyte membrane fluidity is related to their rheologic behavior, the dynamic quality of erythrocytes, which is tempted in hypertension and atherosclerosis. An increased risk of these and other cardiovascular diseases occurs in ageing women. Menopause-related conditions are often treated with hormone replacement therapy that may increase the risk of malignancies.Vitex agnus-castusL. essential oil contains various organic compounds (monoterpenes, sesquiterpenes and terpenoids), and is increasingly used as an alternative therapy for menopausal symptoms. These components of the oil may be incorporated into cell membranes, thereby changing the membrane fluidity. The aim of this study was to determine the effects ofVitex agnus-castusessential oil on human erythrocyte membrane fluidity at graded depths. We used Electron Paramagnetic Resonance spectroscopy and fatty acid spin probes (5-doxyl stearic acid and 12-doxyl stearic acid), whose spectra depend on membrane fluidity. After treatment withVitex agnus-castusessential oil the erythrocytes had a significant (p=0.029) and reversible increase in membrane fluidity in the deeper hydrophobic membrane regions, with no change (p>0.05) in fluidity near the membrane's hydrophilic surface. These results document increased fluidity of the human erythrocyte membrane byVitex agnus-castusessential oil, and this action may be useful in patients with menopause-related hypertension and other cardiovascular conditions.

Cholesterol Effect on Water Permeability through DPPC and PSM Lipid Bilayers: A Molecular Dynamics Study

Water permeability of two different lipid bilayers of dipalmitoylphosphatidylcholine (DPPC) and palmitoylsphingomyelin (PSM) in the absence and presence of cholesterol (0-50 mol %) have been studied by molecular dynamics simulations to elucidate the molecular mechanism of the reduction in water leakage across the membranes by the addition of cholesterol. An enhanced free energy barrier was observed in these membranes with increased cholesterol concentration, and this was explained by the reduced cavity density around the cholesterol in the hydrophobic membrane core. There was an increase of trans conformers in the hydrophobic lipid chains adjacent to the cholesterol, which reduced the cavity density. The enhanced free energy barrier was found to be the main reason to reduce the water permeability with increased cholesterol concentration. At low cholesterol concentrations the PSM bilayer exhibited a higher free energy barrier than the DPPC bilayer for water permeation, while at greater than 30 mol % of cholesterol the difference became minor. This tendency for the PSM and DPPC bilayers to resemble each other at higher cholesterol concentrations was similar to commonly observed trends in several structural properties, such as order parameters, cross-sectional area per molecule, and cavity density profiles in the hydrophobic regions of bilayer membranes. These results demonstrate that DPPC and PSM bilayers with high cholesterol contents possess similar physical properties, which suggests that the solubility of cholesterol in these lipid bilayers has importance for an understanding of multicomponent lipid membranes with cholesterol.

Site-specific solvent exposure analysis of a membrane protein using unnatural amino acids and 19F nuclear magnetic resonance

Membrane proteins play an essential role in cellular metabolism, transportation and signal transduction across cell membranes. The scarcity of membrane protein structures has thus far prevented a full understanding of their molecular mechanisms. Preliminary topology studies and residue solvent exposure analysis have the potential to provide valuable information on membrane proteins of unknown structure. Here, a 19F-containing unnatural amino acid (trimethylfluoro-phenylalanine, tfmF) was applied to accomplish site-specific 19F spin incorporation at different sites in diacylglycerol kinase (DAGK, an Escherichia coli membrane protein) for site-specific solvent exposure analysis. Due to isotope effect on 19F spins, a standard curve for 19F-tfmF chemical shifts was drawn for varying solvent H2O/D2O ratios. Further site-specific 19F solvent isotope shift analysis was conducted for DAGK to distinguish residues in water-soluble loops, interfacial areas or hydrophobic membrane regions. This site-specific solvent exposure analysis method could be applied for further topological analysis of other membrane proteins.

Interactions of biocidal guanidine hydrochloride polymer analogs with model membranes: a comparative biophysical study

Four synthesized biocidal guanidine hydrochloride polymers with different alkyl chain length, including polyhexamethylene guanidine hydrochloride and its three new analogs, were used to investigate their interactions with phospholipids vesicles mimicking bacterial membrane. Characterization was conducted by using fluorescence dye leakage, isothermal titration calorimetry, and differential scanning calorimetry. The results showed that the gradually lengthened alkyl chain of the polymer increased the biocidal activity, accompanied with the increased dye leakage rate and the increased binding constant and energy change value of polymer-membrane interaction. The polymer-membrane interaction induced the change of pretransition and main phase transition (decreased temperature and increased width) of phospholipids vesicles, suggesting the conformational change in the phospholipids headgroups and disordering in the hydrophobic regions of lipid membranes. The above information revealed that the membrane disruption actions of guanidine hydrochloride polymers are the results of the polymer's strong binding to the phospholipids membrane and the subsequent perturbations of the polar headgroups and hydrophobic core region of the phospholipids membrane. The alkyl chain structure significantly affects the binding constant and energy change value of the polymer-membrane interactions and the perturbation extent of the phospholipids membrane, which lead to the different biocidal activity of the polymer analogs. This work provides important information about the membrane disruption action mechanism of biocidal guanidine hydrochloride polymers.

Proton transfer via a transient linear water-molecule chain in a membrane protein

High-resolution protein ground-state structures of proton pumps and channels have revealed internal protein-bound water molecules. Their possible active involvement in protein function has recently come into focus. An illustration of the formation of a protonated protein-bound water cluster that is actively involved in proton transfer was described for the membrane protein bacteriorhodopsin (bR) [Garczarek F, Gerwert K (2006) Nature 439:109-112]. Here we show through a combination of time-resolved FTIR spectroscopy and molecular dynamics simulations that three protein-bound water molecules are rearranged by a protein conformational change that resulted in a transient Grotthuss-type proton-transfer chain extending through a hydrophobic protein region of bR. This transient linear water chain facilitates proton transfer at an intermediate conformation only, thereby directing proton transfer within the protein. The rearrangement of protein-bound water molecules that we describe, from inactive positions in the ground state to an active chain in an intermediate state, appears to be energetically favored relative to transient incorporation of water molecules from the bulk. Our discovery provides insight into proton-transfer mechanisms through hydrophobic core regions of ubiquitous membrane spanning proteins such as G-protein coupled receptors or cytochrome C oxidases.

Cholesterol Transport in Model Lipid Membranes

Proper intracellular cholesterol transport is required for a healthy functioning of the cell and any disorder can lead to diseases, ranging from neurodegenerative Niemann Pick TYPE-C and Alzheimer, to cholelithiasis and atherosclerosis. However, the progress in the area of intra-cellular cholesterol transport is hampered by the huge inconsistencies in the reported value of cholesterol transport rates probably linked to the requirement of cholesterol tag or extraneous compounds such as cyclodextrine for such measurements. We recently reported a novel approach which employs Small Angle Neutron Scattering as a tool for in situ measurement of the cholesterol intra and inter-membrane exchange rates without recourse of cholesterol tags or extraneous compounds. Interestingly we found that cholesterol inter-membrane flipping is rather slow with half-life of a few hours. These results are in odds with the widely accepted belief that the presence of small hydrophilic group on cholesterol head should allow cholesterol to flip quickly through the hydrophobic acyl chain region of lipid membrane. In addition, we show that replacing cholesterol with the relatively benign dehydroergosterol or simply adding 2mM of cyclodextrine significantly affects and accelerates cholesterol flipping rates. To further develop the biophysical mechanism for cholesterol trans-membrane flipping, transfer rates were measured for few cholesterol analogues with a slightly different molecular structure and thereby the geometrical and chemical compatibility with lipids. In addition, MD simulations were performed to measure the energetic and get the better understanding at molecular level for cholesterol transport. Overall, this work provides new insight in to cholesterol transport behavior in model lipid membrane.

Effect of Cholesterol on Distribution of Stable, Hydrophobic Perchlorotriphenylmethyl Triethylester Radical Incorporated in Lecithin Liposomal Membranes

Perchlorotriphenylmethyl triethylester radical (PTM-TE) is a hydrophobic, stable radical giving a narrow singlet ESR signal with a small satellite signal for (13)C in organic solvents. In order to use PTM-TE as a label of liposomal membranes, its manner of incorporation into liposomal membranes was studied. Two components, broad and narrow signals, were observed on the ESR spectrum of PTM-TE incorporated into liposomal membranes composed of egg yolk phosphatidylcholine (egg-PC). The broad signal was increased by the presence of cholesterol in the membranes. The spectral anisotropy of the broad signal was very small as analyzed with oriented planar multilamellar membranes. The narrow signal increased with an increase in temperature in the absence of cholesterol, whereas only a small increase in the signal was observed in the presence of cholesterol. The g-value and line width of the narrow signal were very close to those of PTM-TE in mineral oil, whose viscosity is close to the microviscosity in the hydrophobic region of egg-PC membranes. On the other hand, the g-value and line width of the broad signal were close to those of solid PTM-TE. These observations indicate that the broad signal observed in liposomes originates from PTM-TE clusters in the membranes. The clusters were dissolved in egg-PC membranes at a PTM-TE/egg-PC molar ratio of less than 0.017. However, the clusters were hardly dissolved in the presence of cholesterol.

The Negative Effect of Soy Extract on Erythrocyte Membrane Fluidity: An Electron Paramagnetic Resonance Study

A decrease of erythrocyte membrane fluidity can contribute to the pathophysiology of hypertension. Soy products, which are used as alternative therapeutics in some cardiovascular conditions, contain various isoflavones (genistein, daidzein, and their glucosides, genistin and daidzin), which can incorporate cellular membrane and change its fluidity. The aim of this study was to examine the effects of soy extract (which generally corresponds to the soy products of isoflavone composition) on erythrocyte membrane fluidity at graded depths. We used electron paramagnetic resonance spectroscopy and fatty acid spin probes (5-DS and 12-DS), the spectra of which are dependent on membrane fluidity. After being treated with soy extract, erythrocytes showed a significant (P = 0.016) decrease of membrane fluidity near the hydrophilic surface, while there were no significant changes of fluidity in deeper hydrophobic membrane regions. These results suggest that soy products containing high levels of genistein and isoflavone glucosides may not be suitable for use in hypertension because they decrease erythrocyte membrane fluidity.

Interaction of 1,3,4-thiadiazolium mesoionic derivatives with mitochondrial membrane and scavenging activity: Involvement of their effects on mitochondrial energy-linked functions

The aim of this work was to assess the significance of the interaction of the 1,3,4-thiadiazolium derivatives MI-J, MI-4F and MI-2,4diF with mitochondrial membrane and their effects on energy-linked functions. Mitochondrial swelling in the absence of substrate was inhibited by all derivatives; however, the fluorine derivatives were most effective. MI-4F decreased swelling by ∼32% even at the lowest concentration (65nmolmg−1 protein), reaching ∼67% at the concentration of 130nmolmg−1 protein. Swelling of mitochondria in the presence of oxidizable substrates was also strongly decreased by all derivatives. This effect was more pronounced when using glutamate plus malate, and also fluorine derivatives, which promoted complete inhibition at all concentrations (6.5–130nmolmg−1 protein). Swelling occurred when succinate was the substrate in the presence of MI-J (6.5–65nmolmg−1 protein); however, the shrinkage rate was strongly decreased. MI-4F and MI-2,4diF also inhibited swelling, with total inhibition occurring at a concentration of 65nmolmg−1 protein. Lipid peroxidation induced by Fe3+–ADP/2-oxoglutarate in isolated mitochondria was inhibited time- and dose-dependently by the derivatives, reaching complete inhibition at the highest concentration (80nmolmg−1 protein). However, when lipid peroxidation was initiated by peroxyl radicals generated from AAPH, the inhibition was less intense, reaching ∼50%, ∼40% and ∼58% with MI-J, MI-4F and MI-2,4diF (80nmolmg−1 protein), respectively. The mesoionic compounds also showed superoxide radical scavenging ability of ∼22%, ∼32% and ∼40% (80nmolmg−1 protein), respectively. Fluorescence polarization experiments showed that the derivatives are able to enter the bilayer, decreasing its fluidity in the hydrophobic DMPC membrane region and ordering the fluid phase. Our results suggest that MI-J, MI-4F and MI-2,4diF interact significantly, albeit in different modes, with mitochondrial membrane, and that fluorine derivatives seem to alter the membrane's properties more markedly.

Characterization of the Effects of Aryl‐azido Compounds and UVA Irradiation on the Viral Proteins and Infectivity of Human Immunodeficiency Virus Type 1

Hydrophobic UV-activatable compounds have been shown to partition into the hydrophobic region of biological membranes to selectively label transmembrane proteins, and to inactivate enveloped viruses. Here, we analyze various UV-activatable azido- and iodo-based hydrophobic compounds for their ability to inactivate a model-enveloped virus, human immunodeficiency virus (HIV-1 MN). Treatment of HIV-1 with 1,5-diazidonapthalene (DAN), 1-iodo, 5-azidonaphthalene (INA), 1-azidonaphthalene (AzNAP) or 4,4'-diazidobiphenyl (DABIPH) followed by UVA irradiation for 2 min resulted in complete viral inactivation, whereas treatment using analogous non-azido-containing controls had no effect. Incorporation of an azido moiety within these hydrophobic compounds to promote photoinduced covalent reactions with proteins was found to be the primary mechanism of viral inactivation for this class of compounds. Prolonged UVA irradiation of the virus in the presence of these azido compounds resulted in further modifications of viral proteins, due to the generation of reactive oxygen species, leading to aggregation as visualized via Western blot analysis, providing additional viral modifications that may inhibit viral infectivity. Furthermore, inactivation using these compounds resulted in the preservation of surface antigenic structures (recognized by neutralizing antibodies b12, 2g12 and 4e10), which is favorable for the creation of vaccines from these inactivated virus preparations.