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.

A new tool for quantification of membrane protein partitioning between different cell membranes.

The aqueous two-phase partition system (ATPS) is a method widely used to separate and purify plant and animal membranes carrying bound proteins. However, a common problem of this separation is a mutual contamination of obtained phases. Such contamination adversely affects the accuracy of values of the protein of interest partition between particular membranes when determined by direct measurement. In order to overcome this problem, we have developed a fairly simple mathematical algorithm and found formulas designed to quantify correctly the distribution of the protein of interest between two different membranes. This new tool makes it possible to determine the bias-adjusted ratio of protein distribution between the membranes, regardless of the efficiency of membrane separation in a two-phase system. By means of this algorithm, not only current, but also a number of previously published ATPS-based experiments were (re)analyzed and quantified. The quantitative results of this large-scale analysis of the subcellular localization of various membrane proteins from Arabidopsis, potato, melon, and corn including cytokinin and ethylene receptors, ABCG14 cytokinin transporters, LRR receptor-like protein kinases, and BAK1 co-receptors are presented and discussed here.

A computational study of effects on membrane recruitment of the polar linkers in Vitamin E derivatives.

BACKGROUND: Previous studies found that Vitamin E (VE) could recruit protein kinase B (Akt1) to the membrane by targeting its unconventional lipid-binding site, which led to the dephosphorylation of Akt1 at Ser473, eventually deactivating the enzyme. METHODS: A series of VE-like compounds with varying types and lengths of the linker groups are designed to study the VE-driven membrane recruitment of Akt1 using a combined molecular docking and molecular dynamics (MD) simulation approach. RESULTS: We find that the linker groups with only one methylene linker and multiple hydrogen bond donors are optimal for achieving a balance between binding to the protein and partitioning into the membrane to form a stable protein-ligand-membrane ternary complex. These polar linkers are found to form stable hydrogen bonds with the lipid head groups during the MD simulations, which turns out critical for ensuring that the chromanol ring of the VE-like compounds resides above the membrane surface to fully engage in the protein. CONCLUSIONS: Our results reveal the molecular determinants of the linker groups for VE derivatives' ability to anchor Akt1 to the membrane. GENERAL SIGNIFICANCE: These findings will facilitate the design of membrane interfacial compounds to recruit specific proteins to the membrane to modulate the protein function.

The influence of myristoylation, liposome surface charge and nucleic acid interaction in the partition properties of HIV-1 Gag-N-terminal peptides to membranes.

The group-specific antigen (GAG) polyprotein of HIV-1 is the main coordinator of the virus assembly process at the plasma membrane (PM) and is directed by its N-terminal matrix domain (MA). MA is myristoylated and possess a highly basic region (HBR) responsible for the interaction with the negative lipids of the PM, especially with PIP2. In addition, MA binds RNA molecules proposed as a regulatory step of the assembly process. Here we study the interaction of a synthetic peptide (N-terminal 21 amino acids of MA) and liposomes of different compositions using a variety of biophysical techniques. Particularly, we use the fluorescence properties of the single tryptophan of the peptide to analyze its partition to membranes, where we harness for first time the analytical ability of spectral phasors method to study this interaction. We found that electrostatic interactions play an important role for peptide partition to membranes and myristoylation reduces the free energy of the process. Interestingly, we observe that while the presence of PIP2 does not cause measurable changes on the peptide-membrane interaction, the interaction is favored by cholesterol. Additionally, we found that the partition process goes through a transition state involving peptide disaggregation and changes in the peptide secondary structure. On the other hand, we found that the presence of oligonucleotides competes with the interaction with lipids by increasing peptide solubility. In summary, we think that our results, in context of the current knowledge of the role of HIV-1 MA, contribute to a better molecular understanding of the membrane association process.

The Unusual Transmembrane Partition of the Hexameric Channel of the Hepatitis C Virus.

The p7 protein of the hepatitis C virus (HCV) can oligomerize in membrane to form cation channels. Previous studies showed that the channel assembly in detergent micelles adopts a unique flower-shaped oligomer, but the unusual architecture also presented problems for understanding how this viroporin resides in the membrane. Moreover, the oligomeric state of p7 remains controversial, as both hexamer and heptamer have been proposed. Here we address the above issues using p7 reconstituted in bicelles that mimic a lipid bilayer. We found, using a recently developed oligomer-labeling method, that p7 forms hexamers in the bicelles. Solvent paramagnetic relaxation enhancement analyses showed that the bilayer thickness around the HCV ion channel is substantially smaller than expected, and thus a significant portion of the previously assigned membrane-embedded region is solvent exposed. Our study provides an effective approach for characterizing the transmembrane partition of small ion channels in near lipid bilayer environment.

Propofol affinity to mitochondrial membranes does not alter mitochondrial function.

The molecular mechanisms of hepatotoxicity after propofol anaesthesia have not been fully elucidated, although there is a relation with mitochondrial dysfunction. The action of propofol on mitochondrial hepatic functions in a rat model was evaluated by infusion for 4h with 25 and 62.5mg/kg/h propofol or 3.125ml/kg/h (vehicle). Liver mitochondrial respiratory rates were evaluated as well as mitochondrial transmembrane potential (ΔΨ), calcium fluxes, mitochondrial enzymatic activities (Complex I-V) and oxidative stress biomarkers (superoxide dismutase, catalase, glutathione reductase, glutathione S-transferase, lipid peroxidation and the oxidised/reduced glutathione ratio). Biophysical interactions with membrane models were also performed. The mitochondrial transmembrane potential was decreased and the opening time of the mitochondrial permeability transition pore was slightly reduced for the highest dose. The activity of complex II was stimulated by propofol, which also causes fluctuations on some respiratory parameters, whereas the antioxidant system was affected in a nonspecific manner. Fluorescence quenching studies suggested that propofol is preferably located in deeper regions of the bilayer and has a high affinity to mitochondrial membranes. It is suggested that propofol interacts with liver mitochondrial membranes with mild modification in mitochondrial function.

Quantitative Measurement of Cationic Polymer Vector and Polymer-pDNA Polyplex Intercalation into the Cell Plasma Membrane.

Cationic gene delivery agents (vectors) are important for delivering nucleotides, but are also responsible for cytotoxicity. Cationic polymers (L-PEI, jetPEI, and G5 PAMAM) at 1× to 100× the concentrations required for translational activity (protein expression) induced the same increase in plasma membrane current of HEK 293A cells (30-50 nA) as measured by whole cell patch-clamp. This indicates saturation of the cell membrane by the cationic polymers. The increased currents induced by the polymers are not reversible for over 15 min. Irreversibility on this time scale is consistent with a polymer-supported pore or carpet model and indicates that the cell is unable to clear the polymer from the membrane. For polyplexes, although the charge concentration was the same (at N/P ratio of 10:1), G5 PAMAM and jetPEI polyplexes induced a much larger current increase (40-50 nA) than L-PEI polyplexes (<20 nA). Both free cationic lipid and lipid polyplexes induced a lower increase in current than cationic polymers (<20 nA). To quantify the membrane bound material, partition constants were measured for both free vectors and polyplexes into the HEK 293A cell membrane using a dye influx assay. The partition constants of free vectors increased with charge density of the vectors. Polyplex partition constants did not show such a trend. The long lasting cell plasma permeability induced by exposure to the polymer vectors or the polyplexes provides a plausible mechanism for the toxicity and inflammatory response induced by exposure to these materials.

Interaction of fullerene nanoparticles with biomembranes: from the partition in lipid membranes to effects on mitochondrial bioenergetics.

Partition and localization of C60 and its derivative C60(OH)18-22 in lipid membranes and their impact on mitochondrial activity were studied, attempting to correlate those events with fullerene characteristics (size, surface chemistry, and surface charge). Fluorescence quenching studies suggested that C60(OH)18-22 preferentially populated the outer regions of the bilayer, whereas C60 preferred to localize in deeper regions of the bilayer. Partition coefficient values indicated that C60 exhibited higher affinity for dipalmitoylphosphatidylcholine and mitochondrial membranes than C60(OH)18-22. Both fullerenes affected the mitochondrial function, but the inhibitory effects promoted by C60 were more pronounced than those induced by C60(OH)18-22 (up to 20 nmol/mg of mitochondrial protein). State 3 and p-trifluoromethoxyphenylhydrazone-uncoupled respirations are inhibited by both fullerenes when glutamate/malate or succinate was used as substrate. Phosphorylation system and electron transport chain of mitochondria are affected by both fullerenes, but only C60 increased the inner mitochondrial membrane permeability to protons, suggesting perturbations in the structure and dynamics of that membrane. At concentrations of C60(OH)18-22 above 20 nmol/mg of mitochondrial protein, the activity of FoF1-ATP synthase was also decreased. The evaluation of transmembrane potential showed that the mitochondria phosphorylation cycle decreased upon adenosine diphosphate addition with increasing fullerenes concentration and the time of the repolarization phase increased as a function of C60(OH)18-22 concentration. Our results suggest that the balance between hydrophilicity and hydrophobicity resulting from the surface chemistry of fullerene nanoparticles, rather than the cluster size or the surface charge acquired by fullerenes in water, influences their membrane interactions and consequently their effects on mitochondrial bioenergetics.