Effects of cholesterol or gramicidin on slow and fast motions of phospholipids in oriented bilayers ("F NMR/nuclear spin-lattice relaxation/rotating-frame relaxation/molecular motions/difluorodeuterated phospholipid)

Nuclear spin-lattice relaxation both in the rotating frame and in the laboratory frame is used to investigate the slow and fast molecular motions of phospholipids in oriented bilayers in the liquid crystalline phase. The bilayers are prepared from a perdeuterated phospholipid labeled with a pair of 'IF atoms at the 7 position of the 2-sn acyl chain. Phospholipid-cholesterol or phospholipid-gramicidin interactions are characterized by measuring the relaxation rates as a function of the bilayer orientation, the locking field, and the temperature. Our studies show that cholesterol or gramicidin can specifically enhance the relaxation due to slow motions in phospholipid bilayers with correlation times Ts longer than 10-8 sec. The perturbations of the geometry ofthe slow motions induced by cholesterol are qualitatively different from those induced by gramicidin. In contrast, the presence of cholesterol or gramicidin slightly suppresses the fast motions with correlation times if = 10-9 to 10-10 sec without significantly affecting their geometry. Weak locking-field and temperature dependences are observed for both pure lipid bilayers and bilayers containing either cholesterol or gramicidin, suggesting that the motions of phospholipid acyl chains may have dispersed correlation times. To establish a relationship among the structure, dynamics, and function of biological membranes requires knowledge of (i) the packing and motion of phospholipid molecules in a membrane and (ii) the mechanisms for lipid-sterol and lipidprotein interactions. During the last three decades, considerable efforts have been devoted to the biophysical studies of phospholipid bilayers, which are often viewed as a simplified model of natural membranes (1). Although the static properties of a lipid bilayer, such as the conformation and order ofphospholipids, have been carefully characterized (2, 3), the dynamic behavior of phospholipids in a bilayer environment is less well understood (4). The physical state of a phospholipid bilayer under physiological conditions is that of a lyotropic liquid crystal surrounded by water molecules. Due to the amorphous packing and prevalence of molecular motions, high-resolution x-ray diffraction cannot be applied to lipid bilayers in a liquid crystalline phase. Hence, magnetic resonance, which can give both static and dynamic information, is of vital importance in the field of membrane biophysics. In particular, when combined with isotopic labeling, nuclear relaxation becomes a unique technique which can monitor the microscopic dynamics of a specific site within a macromolecule or a supermolecular assembly. Normally, the spin-lattice relaxation in the laboratory frame (T1) is sensitive to molecular motions on the time scale of 10-1o to 10-1 sec, while the spin-lattice relaxation in the rotating frame (T1p) is sensitive to motions on the time scale of 10-6 to 10-4 sec (5). A number of nuclear magnetic resonance (NMR) investigations have focused on the molecular motions of phospholipids in a pure lipid bilayer, and various theoretical models have been proposed (6-22). Aside from those studies emphasizing lateral diffusion (9, 19), most existing models of the dynamics of phospholipids belong to one of two classes: (i) one or more noncollective anisotropic rotations of a lipid molecule (or a segment of the molecule) with well-defined correlation times (6-8, 10, 11, 16-18, 22); or (ii) collective bilayer disturbances with small angular modulations and a broad distribution of correlation times (13-15, 20). Multiple motions coexist in a bilayer, even though different motions may have quite different geometries and time scales (20, 21). Since most biochemical and biophysical processes occur on a microsecond or slower time scale, we believe that the slow motions of phospholipids (i.e., correlation time, r 2 10-6 sec) are more relevant to the function of biological membranes than their fast motions. Several studies have suggested that slow motions play an important role in lipidprotein interactions or lipid-mediated protein-protein interactions (4, 7, 14, 23). Cholesterol and gramicidin are commonly chosen as prototypes for investigating interactions with phospholipids (24, 25). -Cholesterol can be found in many biological membranes in ratios that range from 10 to 40 mol % (26). In the liquid crystalline phase, cholesterol increases the order of the phospholipid acyl chains and decreases the membrane permeability; at low temperatures, it prevents the acyl chains from forming a highly ordered gel phase (26-28). Gramicidin is a pentadecapeptide which can be considered as a model of membrane proteins; it folds into a helical structure and forms channels in a lipid bilayer (29, 30). In general, membrane peptides and proteins have little or no effect on the chain ordering of phospholipids (24, 31-34). For recent reviews on using NMR to study lipid bilayers containing cholesterol or gramicidin, see refs. 4 and 35. To investigate the effects of membrane-intercalating molecules on the dynamics of phospholipids, Cornell and coworkers (23, 36) have determined the ratios of averaged TU1 to Tj' for methylene protons in dispersions of pure phospholipids and phospholipid mixed with cholesterol or gramicidin, as well as for erythrocyte membranes. They have found that both additives cause the Tlp to shorten significantly, which indicates that the intensity of the slow motions of the phospholipid has increased. On the other hand, only small effects on the fast motion of the acyl chain have been observed. Since the ratio of the relaxation rates observed for Abbreviations: T1, spin-lattice relaxation in the laboratory frame; T1P, spin-lattice relaxation in the rotating frame; 2-[7,7-19F2]DMPCd52, 1-(myristoyl-d27)-2-(7,7-difluoromyristoyl-d25)-sn-glycero3-phosphocholine; Ho, static magnetic field; H1, locking field; CSA, chemical shift anisotropy. *Present address: Department of Chemistry, University of California, Berkeley, CA 94720. tTo whom reprint requests should be addressed. 8758 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 86 (1989) 8759 phospholipid dispersions with cholesterol or gramicidin is closer to that found for natural membranes, they conclude that biological membranes are rich in slow motions, and such motions can be induced or enhanced by adding foreign molecules to pure phospholipid bilayers. Using macroscopically oriented bilayers prepared from a phospholipid with perdeuterated acyl chains in which a single CF2 group occurs at the 7 position of the sn-2 acyl chain, 1-(myristoyl-d27)-2-(7,7-difluoromyristoyl-d25)-sn-glycero3-phosphocholine (2-[7,7-19F2]DMPC-d52), we have investigated both slow and fast motions by measuring 19F Tjp and 771 as a function of the bilayer orientation, the locking field, and the temperature. The advantages of using a '9F label include the following: the NMR sensitivity is high and there is no natural background (37); the relaxation of '9F is dominated by intramolecular interactions (21); and the crosspolarization between '9F and 2H is negligible in the range of locking fields employed in this study. We have found that adding cholesterol to bilayers of the perdeuterated fluorolipid can dramatically alter the orientation dependence of Tj7l, indicating that, besides increasing the intensity of the slow motions, a large perturbation of the geometry of these motions is induced compared to the pure phospholipid bilayers. The addition of gramicidin produces a smaller change in the geometry of the slow motions, although there is still a significant increase in the relaxation rate. For all cases, the geometry of the fast motions remains basically the same. Several motional models have been tested against our experimental results. The systematic differences observed between the effects produced by cholesterol and gramicidin can lead to new insight into the nature of the structure-function relationship of various membrane components. MATERIALS AND METHODS The synthesis of 2-[7,7-19F2]DMPC-d52 will be described elsewhere. Cholesterol and gramicidin (Dubos) were purchased from Sigma and used without further purification. Oriented bilayers of phospholipid were prepared by dissolving about 30 mg of 2-[7,7-'9F2]DMPC-d52 with or without 12 wt % cholesterol (22 mol %) or 14 wt % gramicidin (6.2 mol %) in 0.6 ml of 3:1 (vol/vol) chloroform/methanol. Aliquots (20,l) were deposited on microscope coverslips (7.8 x 20 mm) and dried under reduced pressure. The slips were then stacked and hydrated in a closed chamber in the presence of 1 M MgCl2 at 32°C (relative humidity 90%) for 5-15 days, during which the lipids gradually oriented. The relaxation measurements were performed on a modified WH-300 spectrometer operating at 282.4 MHz for 19F. A home-built probe with a loop-gap resonator as the NMR detector was employed. The relaxation time T, was determined by inversion recovery and Tlp was determined by spin locking, with the lengths of the 900 pulses being 3.7 and 3.2 ,usec, respectively. For the Tlp measurements, the lockingfield (Hl) intensity was controlled by an electronic attenuator and calibrated by the corresponding 3600 pulse duration. As the sample orientation was changed, the carrier frequency of the spin-locking pulse was adjusted such that the offresonance effect caused by the chemical shift anisotropy (CSA) could be compensated. The temperature was held at 32°C for Tlp measurements and varied from 28°C to 48°C for T, measurements. At all temperatures used for the NMR studies, the bilayer was in the liquid crystalline phase.