Probing Liquid-Ordered and Disordered Phases in Lipid Model Membranes: A Combined Theoretical and Spectroscopic Study of a Fluorescent Molecular Rotor

Abstract

An integrated theoretical/experimental strategy has been applied to the study of environmental effects on the spectroscopic parameters of 4-(diphenylamino)phtalonitrile (DPAP), a fluorescent molecular rotor. The computational part starts from the development of an effective force field for the first excited electronic state of DPAP and proceeds through molecular dynamics simulations in solvents of different polarities toward the evaluation of Stokes shifts by quantum mechanics/molecular mechanics (QM/MM) approaches. The trends of the computed results closely parallel the available experimental results thus giving confidence to the interpretation of new experimental studies of the photophysics of DPAP in lipid bilayers. In this context, results show unambiguously that both flexible dihedral angles and global rotations are significantly retarded in a cholesterol/DPPC lipid matrix with respect to the DOPC matrix, thus confirming the sensitivity of DPAP to probe different environments and, therefore, its applicability as a probe for detecting different structures and levels of plasma membrane organization.

Figure 1

(a) 4-(Diphenylamino)phthalonitrile (DPAP) 2D molecular structure; (b) a configuration of the DPAP rotor (in green) embedded within the DPPC/CHOL matrix. In orange, the cholesterol molecules (with hydroxyl groups in purple); in red and white, DPPC lipid polar heads and nonpolar tails, respectively; in yellow and blue, chloride and sodium ions, respectively. For clarity, water molecules and hydrogen atoms of DPAP and lipids are omitted.

Figure 2

DPAP propeller-like conformation as optimized in butanoic acid (solvent effect modeled by the C-PCM). Flexible dihedral angles are indicated with green arrows. The ipso (C1, C1′, and C″), the ortho (C2, C2′, and C2″) carbon atoms are labeled in black together with the nitrogen (N′ and N″) and the carbon (C3 and C3′) atoms of the two cyano groups.

Figure 3

Frontier molecular orbitals of DPAP: HOMO (a) and LUMO (b). An isosurface value of 0.02 au has been used.

Figure 4

Potential energy profiles of dihedral angle 1 (a) and 2/3 (b). QM data, open circles; MM data, solid lines.

Figure 5

Distribution of the emission wavelengths computed for 200 DPAP geometries extracted from the MD trajectories in cyclohexane (a), o-xylene (b), and acetonitrile (c). The solid lines represent the fitting to a Gaussian curve. For sake of clarity a different number of bins have been used in the three panels.

Figure 6

Conformational dependence of the fluorescence wavelength computed in acetonitrile. (a) Energy profile of dihedral angle 1. (b) Relation between DPAP emission energy and dihedral angle 1 amplitude. (c) Dihedral 1 distribution for the selected conformations taken from the MD trajectory and used for fluorescence wavelength calculations.

Figure 7

ph-FLIM of multilamellar vesicles characterized by homogeneous lipid phases. (a) Fluorescence image of a multilamellar vesicle characterized by the homogeneous L_o phase (i.e., DPPC/cholesterol). (b) Fluorescence image of a multilamellar vesicle characterized by the homogeneous L_d phase (i.e., DOPC). (c) Phasor plot relevant to vesicles (a and b), as superimposed on the same diagram: the segment connecting the averages of the two reference phasor clouds is depicted in red.

Figure 8

Computed deuterium order parameters: (a) DPPC and (b) DOPC. Solid line, average over all DPPC/DOPC lipids; dotted line, average over DPPC/DOPC lipids within 5 Å from DPAP. In black sn-1 chain, in red sn-2 chain.