Partitioning of 2,6-Bis(1H-Benzimidazol-2-yl)pyridine fluorophore into a phospholipid bilayer: complementary use of fluorescence quenching studies and molecular dynamics simulations

Abstract

Successful use of fluorescence sensing in elucidating the biophysical properties of lipid membranes requires knowledge of the distribution and location of an emitting molecule in the bilayer. We report here that 2,6-bis(1H-benzimidazol-2-yl)pyridine (BBP), which is almost non-fluorescent in aqueous solutions, reveals a strong emission enhancement in a hydrophobic environment of a phospholipid bilayer, making it interesting for fluorescence probing of water content in a lipid membrane. Comparing the fluorescence behavior of BBP in a wide variety of solvents with those in phospholipid vesicles, we suggest that the hydrogen bonding interactions between a BBP fluorophore and water molecules play a crucial role in the observed "light switch effect". Therefore, the loss of water-induced fluorescence quenching inside a membrane are thought to be due to deep penetration of BBP into the hydrophobic, water-free region of a bilayer. Characterized by strong quenching by transition metal ions in solution, BBP also demonstrated significant shielding from the action of the quencher in the presence of phospholipid vesicles. We used the increase in fluorescence intensity, measured upon titration of probe molecules with lipid vesicles, to estimate the partition constant and the Gibbs free energy (ΔG) of transfer of BBP from aqueous buffer into a membrane. Partitioning BBP revealed strongly favorable ΔG, which depends only slightly on the lipid composition of a bilayer, varying in a range from -6.5 to -7.0kcal/mol. To elucidate the binding interactions of the probe with a membrane on the molecular level, a distribution and favorable location of BBP in a POPC bilayer were modeled via atomistic molecular dynamics (MD) simulations using two different approaches: (i) free, diffusion-driven partitioning of the probe molecules into a bilayer and (ii) constrained umbrella sampling of a penetration profile of the dye molecule across a bilayer. Both of these MD approaches agreed with regard to the preferred location of a BBP fluorophore within the interfacial region of a bilayer, located between the hydrocarbon acyl tails and the initial portion of the lipid headgroups. MD simulations also revealed restricted permeability of water molecules into this region of a POPC bilayer, determining the strong fluorescence enhancement observed experimentally for the membrane-partitioned form of BBP.

FIGURE 1

Example of fluorescence titration of BBP upon adding POPC vesicles, measured in 50 mM sodium phosphate buffer at pH 8. An increase in the LUV concentration results in the strong fluorescence enhancement of BBP, which was also accompanied by a blue shift of its emission maximum. The arrows indicate the direction of changes at increasing LUV concentrations, varied in a range from 0 to 1.8 mM.

FIGURE 2

Plots of the fluorescence enhancement of BBP observed upon LUV titration; POPC (green), 75POPC/25POPC (red), 90POPC/10Chol (cyan), and 25POPC/75POPG (blue). The titrations were performed in 50 mM sodium phosphate buffer at pH 8 at T=298 K. The relative fluorescence intensity at 379 nm was fitted to Eq. 1. Fitting results are shown with the colour-coded solid curves.

FIGURE 3

Stern-Vollmer plots for BBP fluorescence quenching by Co²⁺ ions in sodium phosphate buffer at pH 8 in the absence and in the presence of lipid vesicles. The extent of fluorescence quenching of unbound BBP (○) changes strongly upon partitioning into LUVs (POPC (green □), 90POPC/10Chol (cyan ), and 25POPC/75POPG (blue △)). The changes in quenching suggest that Co²⁺ ions do not readily penetrate the hydrophobic region of the lipid vesicle; so that the deeply buried BBP molecules become significantly inaccessible to the quencher. Fluorescence intensities were excited at 315 nm and measured at either 397 nm (free dyes) or 379 nm (in LUV). The quenching experiments in the presence of LUV were carried out at lipid saturation of 2 mM (see Figure 2 for more details).

FIGURE 4

Typical snapshots of MD simulations show the partitioning kinetics of BBP into a POPC bilayer at different simulation times. The MD simulations were based on free, passive distribution of probe molecules between bulk water and a POPC bilayer. Four probe molecules were sampled to ensure better MD statistics. The lipid tails are shown as sticks in green, the phosphorus and nitrogen atoms of the lipid headgroups are shown by red and cyan balls, respectively. For clarity, water molecules are not shown.

FIGURE 5

MD trajectories of the partitioning kinetics of BBP into a POPC bilayer monitored by movements of the center-of-mass (COM) of each probe molecules with respect to the POPC bilayer normal z. The COM movements of the sampled molecules were traced to insure the complete equilibration and convergence of the MD system. The COM trajectories are schematically superimposed on a POPC snapshot.

FIGURE 6

A schematic presentation of the umbrella sampling scheme applied to evaluate a free energy profile for partitioning of BBP to a POPC bilayer. To gather additional MD sampling statistics, the two probe molecules were sampled simultaneously, so that when the first molecule was in the center of the bilayer, the second one was in bulk water. The harmonic restraint potential was applied to the distance d_c between the center of mass of the pyridine moiety (colored yellow) and the center of mass of the bilayer in direction Z normal to the bilayer. During the MD umbrella sampling, the two probe molecules were kept to be at the distance d_1-2 = 25 Å apart each other.

FIGURE 7

MD simulations of a free energy profile of BBP penetration across a POPC bilayer calculated using the potential of mean constraint force. The PMF obtained according to the umbrella sampling scheme shown in Figure 6. The PMF was calculated from the biased distributions using the weighted histogram analysis method. The two BBP molecules were sampled simultaneously allowing for estimating sampling errors shown with corresponding error bars. The black solid line represents the average profile of the partitioning free energy. The partitioning profile is set to zero in bulk water. For the purpose of visualization, the PMF is schematically superimposed on a MD snapshot of a POPC bilayer.

FIGURE 8

(A) Geometric criteria for the formation of hydrogen bond between the benzimidazole N-H groups of BBP and water molecules. (B) The probability profile for hydrogen-bonding between BBP and water molecules estimated during MD umbrella sampling of a penetration depth of BBP across a POPC bilayer (see Figure 7 for more details). The probability of BBP–water H-bonding is schematically superimposed on a snapshot of a POPC bilayer. (C) The BBP–water H-bonding probability is now superimposed on mass density profiles of a POPC bilayer (filled green) and water (filled cyan) showing that the profile of the probe−water H-bonding correlates with a profile of water permeability into a bilayer.

FIGURE 9

Example of MD umbrella sampling of BBP in a POPC bilayer showing that excess water molecules can enter a bilayer in hydrogen-bonded associates with BBP. (A) The snapshot was obtained after 2 ns of MD umbrella sampling (see Figure 6 for more details), during which the pyridine ring of the fluorophore was constrained near the center of the bilayer. The lipid tails are green and the phosphorus and nitrogen atoms of the head groups of POPC are shown as cyan and yellow balls. (B) The insert demonstrates that the formation of a stable hydrogen-bonding complex between BBP and water molecules has led to the trapping of the water molecules into the hydrophobic core of the bilayer.

SCHEME 1

Chemical structure of 2,6-bis(1H-benzimidazol-2-yl)pyridine (BBP).

SCHEME 2

Possible hydrogen-bonded forms of BBP: Form I shows a bifunctional hydrogen-bonding character of BBP, acting both as a donor and as an acceptor of a hydrogen bond. Forms II and III are capable of either direct intramolecular or solvent-assisted phototautomerization.