Fluorescent Probes cis- and trans-Parinaric Acids in Fluid and Gel Lipid Bilayers: A Molecular Dynamics Study

Abstract

Fluorescence probes are indispensable tools in biochemical and biophysical membrane studies. Most of them possess extrinsic fluorophores, which often constitute a source of uncertainty and potential perturbation to the host system. In this regard, the few available intrinsically fluorescent membrane probes acquire increased importance. Among them, cis- and trans-parinaric acids (c-PnA and t-PnA, respectively) stand out as probes of membrane order and dynamics. These two compounds are long-chained fatty acids, differing solely in the configurations of two double bonds of their conjugated tetraene fluorophore. In this work, we employed all-atom and coarse-grained molecular dynamics simulations to study the behavior of c-PnA and t-PnA in lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), representative of the liquid disordered and solid ordered lipid phases, respectively. All-atom simulations indicate that the two probes show similar location and orientation in the simulated systems, with the carboxylate facing the water/lipid interface and the tail spanning the membrane leaflet. The two probes establish interactions with the solvent and lipids to a similar degree in POPC. However, the almost linear t-PnA molecules have tighter lipid packing around them, especially in DPPC, where they also interact more with positively charged lipid choline groups. Probably for these reasons, while both probes show similar partition (assessed from computed free energy profiles across bilayers) to POPC, t-PnA clearly partitions more extensively than c-PnA to the gel phase. t-PnA also displays more hindered fluorophore rotation, especially in DPPC. Our results agree very well with experimental fluorescence data from the literature and allow deeper understanding of the behavior of these two reporters of membrane organization.

Keywords: fluorescence spectroscopy; lipid membranes; membrane probe; molecular dynamics simulations.

Figure 1

Structures of ionized cis-parinaric acid (c-PnA, a) and trans-parinaric acid (t-PnA, b), and phospholipids POPC (c) and DPPC (d), with indication of relevant atoms or groups of atoms.

Figure 2

Final snapshots of the two runs of c-PnA in POPC (a,b), t-PnA in POPC (c,d), c-PnA in DPPC (e,f) and t-PnA in DPPC (g,h). For each lipid system, the four images are drawn on the same scale. However, for the sake of representation, the DPPC snapshots are drawn on a slightly reduced scale compared with the POPC ones.

Figure 3

Average area per lipid molecule a (a) and bilayer thickness (evaluated as the distance between the average planes of the lipid P atoms in opposite leaflets, d_PP; (b)) for the studied systems.

Figure 4

Average transverse distances to the bilayer center of mass for three probe groups (in columns) and, as reference, for different lipid atoms in probe-free systems, calculated for the POPC (a) and DPPC (b) simulations. Error bars reflect standard errors for the 95% confidence level.

Figure 5

Mass density profiles of the different species in the simulations with c-PnA (a,c) or t-PnA (b,d), in POPC (a,b) or DPPC (c,d).

Figure 6

Long axis (defined as the vector from the terminal to the first C atom) tilt angle distributions of the parinaric acid and lipid acyl chains in POPC (a) and DPPC (b). Lipid distributions refer to probe-free simulations.

Figure 7

sn-1 acyl chain long axis (defined as the vector from the terminal to the first C atom) distributions of POPC (a,b) and DPPC (c,d), in the presence of c-PnA (a,c) or t-PnA (b,d). Each curve concerns a different range of distances R between the centers of mass of the lipid and the nearest probe.

Figure 8

Atom–atom radial distribution density functions (RDFs, left plots) and their cumulative counterparts (cumulative RDFs, right plots) of lipid around PnA (a,b) and water (c,d), lipid choline (e,f) and sodium ion (g,h) around PnA carboxylate, calculated for the POPC simulations. Red and green curves refer to simulations with c-PnA and t-PnA, respectively.

Figure 9

Atom–atom radial distribution density functions (RDFs, left plots) and their cumulative counterparts (cumulative RDFs, right plots) of lipid around PnA (a,b) and water (c,d), lipid choline (e,f) and sodium ion (g,h) around PnA carboxylate, calculated for the DPPC simulations. Red and green curves refer to simulations with c-PnA and t-PnA, respectively.

Figure 10

Rotational ACFs C(t) of c-PnA (a,c) and t-PnA (b,d) in POPC (a,b) and DPPC (c,d). Each thin blue line represents the C(t) of an individual fluorophore. The red and green lines (for c-PnA and t-PnA, respectively) represent the average over the eight simulated molecules of each probe in a given system. The black lines are fits to multiexponential functions (Equation (2); see Table 1 for best fit parameters).

Figure 11

Potential of mean force (PMF) profiles across the lipid bilayers of c-PnA in POPC (a), c-PnA in DPPC (b), t-PnA in POPC (c) and t-PnA in DPPC (d). Data are presented with the following color code: ionized c-PnA (red), neutral c-PnA (black/gray), ionized t-PnA (green) and neutral t-PnA (blue). Curves with different shades of these colors correspond to profiles obtained from replicate sets of umbrella sampling simulations.

Figure 12

Energy barriers for the insertion (blue), desorption (orange) and translocation (gray) processes of ionized and neutral t-PnA and c-PnA in POPC and DPPC lipid bilayers.