Effects of lateral diffusion on the fluorescence anisotropy in hexagonal lipid phases. II. An experimental study

Abstract

The polymorphic phase behavior of unsaturated phosphatidylethanolamine (PE)/diacylglycerol (DG) binary lipid mixtures was investigated by the use of time-resolved fluorescence anisotropy. Using a fluorescent lipid, 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenylethyl] carbonyl]3-sn-phosphatidyl-choline (DPH-PC), the orientational order and rotational dynamics of the above lipid mixtures in the liquid crystalline lamellar (L alpha) and inverted hexagonal (HII) phases were studied. By employing a one-exponential model (Cheng, K.H. 1989: Biophys. J. 55:1025-1031) to fit the anisotropy decay data, abrupt decreases in the normalized initial anisotropy decay slope and the residual anisotropy of DPH-PC were observed at approximately 6-8% DG, signifying a L alpha/HII phase transition. Using our new theoretical WOBHOP and P2P4HOP models as described in a preceding paper (Van Der Meer, B.W., K.H. Cheng, and S.Y. Chen. 1990. Biophys. J. 58:000-000), two or more rotational correlation times were required to describe the anisotropy decay behavior of DPH-PC in the HII phase. These rotation correlation times were further related to the second and fourth rank order parameters, and the wobbling and hopping diffusion constants of the fluorescent probe in the highly curved lipid cylindrical tubes of the HII phase. The hopping diffusion constant (DH) equals the lateral diffusion constant (DL) divided by R2 (R = radius of the lipid cylindrical tubes). The value of DL was estimated by measuring the excimer formation rate of 1-palmitoyl-2-[10-(1-pyrenl)decanoyl] phosphatidyl choline (py-PC) in the same PE/DG mixtures. Upon comparing the values of DH and DL, the value of R was determined to be approximately 10-15 A, and agreed with that derived from x-ray diffraction (Tate, M.W., and S.M. Gruner, 1989, Biochemistry. 28:4245-4253; Rand, R.P., N.L. Fuller, S.M. Gruner, and V.A. Parsegian. 1990. Biochemistry. 29:76-87).

Detection of membrane packing defects by time-resolved fluorescence depolarization

“…We assume that the lateral diffusion of the rod-shaped (or cylindrical) DPH-PC around a curved layer, the wobbling diffusion of the whole DPH-PC molecule with respect to the normal of the membranes (local director), and the orientational distribution of the long DPH-PC axis with respect to the local director are the only major factors contributing to the fluorescence depolarization of DPH-PC. The justifications of the above assumptions have been discussed in detail elsewhere (Cheng, 1989;van der Meer et al, 1990). The anisotropy decay r(t) of DPH-PC in a lipid layer can be written as…”

“…where r. anisotropy is the anisotropy of the fluorophore at time 0, and Qw and Qc are the time-dependent depolarization factors due to wobbling and curvature-associated rotational diffusion of the fluorophore. It has been shown (van der Meer et al, 1990) that Qw can be expressed as QW= [(P2)2 + (1-(P2)2)exp[ -6Dwt/(l-(p2)2)] (4)…”

“…It is believed that the appearance of localized curvature in the lipid layers is an important signature of the D state (Boni and Hui, 1983, and references therein). Our previous fluorescence depolarization studies (Cheng, 1989;Chen et al, 1990) have established that the rotational dynamics behavior of rod-shaped fluorophore is very sensitive to changes in the curvature of the membranes due to the first-order transition of lamellar-to-inverted hexagonal phases. In this study, a time-resolved fluorescence depolarization technique (Gratton et al, 1984;Cheng, 1989;Chen et al, 1992) was employed to test an important hypothesis.…”

“…A fluorescent lipid probe, diphenylhexatriene (DPH) chain-labeled PC (DPH-PC) (Cheng, 1989), was used to investigate the rotational diffusion and conformational order of the host PE/PC mixtures, which exhibit the mesoscopic D state, as well as one-dimensional lamellar liquid crystalline (La,) and twodimensional inverted hexagonal (HI,) ordered phases. The rotational diffusion model for rod-shaped fluorophore in curved matrix has been introduced and discussed in great detail in our previous studies of ordered phases (van der Meer et al, 1990;Chen et al, 1990Chen et al, , 1992. A very brief outline of this model is presented here.…”

Frequency-resolved intramolecular excimer fluorescence study of lipid bilayer and nonbilayer phases

“…In this HI, phase, the lipids form hexagonally packed long watercored cylindrical tubes in which the lipid polar headgroups are facing the long symmetrical axes ofthe cylinders. Although the structure and morphologies of this HI, phase have been investigated extensively by the x-ray (7)(8)(9) and electron microscopic (1) techniques, the detailed molecular dynamics of the lipids in the HI, phase, particularly within the nanosecond (or fluorescence) time regime, has become a subject of interest only recently (2)(3)(4)(5)(6)(10)(11)(12). It is believed that knowledge of both the structure and molecular dynamics of the lipids in the HI, phase is required in order to better understand the role of nonbilayer-preferring lipids in the cell membrane function at the molecular level (2).…”