Intramembrane polarity by electron spin echo spectroscopy of labeled lipids

ABSTRACT The association of water (D^sub 2^O) with phospholipid membranes was studied by using pulsed-electron spin resonance techniques. We measured the deuterium electron spin echo modulation of spin-labeled phospholipids by D^sub 2^O in membranes of dipalmitoyl phosphatidylcholine with and without 50 mol% of cholesterol. The Fourier transform of the relaxation-- corrected two-pulse echo decay curve reveals peaks, at one and two times the deuterium NMR frequency, that arise from the dipolar hyperfine interaction of the deuterium nucleus with the unpaired electron spin of the nitroxide-labeled lipid. For phosphatidylcholine spin-labeled at different positions down the sn-2 chain, the amplitude of the deuterium signal decreases toward the center of the membrane, and is reduced to zero from the C-12 atom position onward. At chain positions C-5 and C-7 closer to the phospholipid headgroups, the amplitude of the deuterium signal is greater in the presence of cholesterol than in its absence. These results are in good agreement with more indirect measurements of the transmembrane polarity profile that are based on the ^sup 14^N-hyperfine splittings in the conventional continuous-wave electron spin resonance spectrum.

INTRODUCTION

The penetration of water into lipid membranes and the resultant transmembrane polarity profile have important consequences not only for membrane permeability but also for the energetics of protein insertion into the membrane and the stability of transmembrane helices in integral proteins. Recently, the polarity profile of lipid membranes was mapped by using the isotropic ^sup 14^N-hyperfine splittings in the continuous-wave (CW) electron spin resonance (ESR) spectra of phospholipids that were spin-labeled in the sn-2 chain (Marsh, 2001). A sigmoidal, trough-like profile was obtained in which a transition from the high polarity region at chain positions closer to the phospholipid polar headgroups occurs, in the range C-8 to C-9, to a low polarity region in the center of the membrane. Addition of cholesterol to the membranes was found to increase the polarity in the outer regions, and to decrease it almost to zero in the inner regions of the membrane. This increases the gradient of the polarity transition and shifts it by 1-2 C-atom positions at 50 mol% cholesterol content.

From the known dependence of nitroxide isotropic hyperfine splittings on hydrogen bonding (Gagua et al., 1978; Marsh, 2002a), the polarity profiles obtained in the above study (Marsh, 2001) were related to penetration of water into the membrane. Nevertheless, a more direct approach to the detection of membrane-associated water is desirable, not least to substantiate the interpretation given to the polarity dependence of the spin-label hyperfine splittings in membranes. This is done here by using pulse-Fourier transform ESR techniques with deuterium-labeled water.

Kevan and co-workers (Szajdzinska-Pietek et al., 1984) have used modulation of the electron spin echo decays of spin-labeled fatty acids by the deuterium hyperfine interactions with D^sub 2^O to investigate the association of water with detergent micelles. Here we use this approach, specifically the Fourier transform of the relaxation-corrected echo decay, to probe the direct interactions of water with spin-labeled lipid chains in phospholipid bilayer membranes. Both the transmembrane profile and the effect of cholesterol on water association with the phospholipids is found to parallel the rather more indirect results based on nitroxide hyperfine splittings in CW-ESR spectra (Griffith et al., 1974; Marsh, 2001; Subczynski et al., 1994).

MATERIALS AND METHODS

Materials

Dipalmitoyl phosphatidylcholine (DPPC), cholesterol, and heavy water (D^sub 2^O) were from Sigma/Aldrich (St. Louis, MO). Spin-labeled phosphatidylcholines (1-acyl-2(n-doxyl-stearoyl)-sn-glycero-phosphocholine, n-PCSL) were from Avanti Polar lipids (Birmingham, AL) or synthesized according to Marsh and Watts (1982).

Sample preparation

DPPC with 1 mol% of n-PCSL, with and without 50 mol% cholesterol, were codissolved in chloroform. Solvent was evaporated with a nitrogen gas stream and residual traces removed by drying under vacuum overnight. The lipid (15 mg) was dispersed either in H^sub 2^O (phosphate-buffered saline, pH 7.5) or in D^sub 2^O at a concentration of -100 mg/ml by vortex mixing with heating to 60 deg C, i.e., above the chain-melting phase transition. The sample was then transferred to a standard 4 mm-diameter, quartz ESR tube, concentrated by pelleting in a bench-top centrifuge (the sample in D^sub 2^O floats) and the excess water removed.

We thank Dr. D. Erilov for recording 3-pulse echo spectra and Brigitta Angerstein for spin-label synthesis.

This work was performed in the framework of the project CIPE-MIA26WP3.

[Reference]

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[Author Affiliation]

Rosa Bartucci,* Rita Guzzi,* Derek Marsha^ and Luigi Sportelli*

[Author Affiliation]

*Dipartimento di Fisica and Unita INFM, Universita della Calabria, 1-87036 Arcavacata di Rende (CS), Italy; and ^Max-Planck-Institut fur biophysikalische Chemie, Abteilung Spektroskopie, 37077 Gottingen, Germany

[Author Affiliation]

Submitted July 8, 2002, and accepted for publication October 11, 2002.

[Author Affiliation]

Address reprint requests to Dr. R. Bartucci, Dipartimento di Fisica ed Unita INFM, Universita della Calabria, 1-87036 Arcavacata di Rende (CS), Italia. Tel.: + 39-0984-496074; Fax: + 39-0984-494401; E-mail: bartucci@fis. unical.it.