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.

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.

Molecular crowding effects on the distribution of amphiphiles in biological media.

Biological systems are the result of the interactions established among their many distinct molecules and molecular assemblies. The high concentration of small molecules dissolved in the aqueous media alter the water properties with important consequences in the interactions established. In this work, the effects of high concentrations of the disaccharide trehalose on the solubility of a homologous series of fluorescent amphiphiles (NBD-Cn, n=4-16) and on their interaction with a lipid bilayer and a serum protein are quantitatively characterized. Both kinetic and equilibrium aspects are reported for a better understanding of the effects observed. The aqueous solubility of the most hydrophobic amphiphiles (n ≥ 8) is strongly increased by 1 M trehalose, while no signifcant effect is observed for the most polar amphiphile (n = 4). This results from a decrease in the magnitude of the hydrophobic effect at molecular crowding conditions. A small decrease is observed on the equilibrium association with serum albumin. This is most significant for amphiphiles with longer alkyl chains, in agreement with their increased solubility in the aqueous media containing trehalose. The effects on the association of the amphiphiles with lipid bilayers are influenced by both equilibrium and kinetic aspects. On the one hand, the decreased magnitude of the hydrophobic effect leads to a decrease in the affinity of the amphiphiles towards the membrane. However, this tendency may be overbalanced by the effects on the kinetics of the interaction (insertion/desorption) due to the increase in the viscosity of the aqueous media. It is shown that the distribution of amphiphilic drugs in the crowded biological media is significantly different from that predicted from studies in dilute solutions and that the effects are dependent on the solute's hydrophobicity.

Partition of Amphiphilic Molecules to Lipid Bilayers by ITC: Low-Affinity Solutes.

A protocol is developed to allow the accurate characterization of partition to lipid bilayers for solutes with low affinity, using isothermal titration calorimetry. The methodology proposed is suitable for studies using complex membranes, such as intact biomembranes or whole cells. In the method developed, the association is characterized at increasing solute concentrations. This allows the characterization of solute partition into unperturbed membranes, as well as effects induced by high solute concentrations. Most druglike molecules are expected to interact with low-to-moderate affinity with relevant cell membranes. This is due to both the need for a relatively high aqueous solubility of the drug and the poor binding properties of the cell membranes. The methodology is applied to characterize the interaction of antibiotic Rifampicin with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and with lipid bilayers representative of bacterial membranes.