Fluorine-19 nuclear magnetic resonance studies of ... - Europe PMC

Proc. Nati. Acad. Sci. USA Vol. 75, No. 2, pp. 630-634, February 1978

Biochemistry

Fluorine-19 nuclear magnetic resonance studies of Escherichia coli membranes* (fluorinated myristic acid/fluorinated membranes/E. coli membrane vesicle/membrane fluidity)

MARTIN P. N. GENT, PATRICIA F. COTTAM, AND CHIEN Hot Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

Communicated by Britton Chance, October 17, 1977

ABSTRACT Several fluorinated fatty acids of the general structure CH13(CH2)I3.mCFg(CH2)m_2COOH are incorporated biosynthetically as unsaturated fatty acid analogues into the phospholipids of Escherichia coli. Under optimum conditions an unsaturated fatty acid autotroph, K1060B5, can be grown so that 50% of the total phospholipid fatty acids are 8,8-difluoromyristate. Conditions are found for which more than 20% of the fatty acids are fluorinated before a decrease in growth rate is observed. We have used 19F nuclear magnetic resonance to examine membranes isolated from E. coli grown under the latter conditions. A comparison is made with spectra of aqueous dispersions of extracted E. coli phospholipids and model multilayer phospholipid membranes. An explanation of the '9F resonance line shape in these membrane systems and the relationship to a molecular order parameter is given. It is apparent that I9F nuclear magnetic resonance is more sensitive to the degree of ordering or fluidity of phospholipids than spin labels or fluorescent probes. For instance, a dramatic effect of membrane protein on lipid fluidity can be seen. Finally, this method can be used to measure the proportion of frozen and fluid lipid in biological membranes at temperatures within the span of the gel-to-lipid phase transition.

studies using nitroxide spin labels suffer two disadvantages. First, the probe molecule may not be incorporated biosynthetically into the biological membrane. Second, the EPR signals may be lost due to reduction of the nitroxide spin labels by the reducing systems of the host, such as the electron transport chain in Escherichia coli membrane vesicles (10). Also, the nitroxide moiety may have different chemical properties from the methylene group that it replaces. The last effect could result in the spectroscopic probe being in a different environment from that of the bulk lipid in the intact biological membrane (11). Although NMR probes, such as 1H, 13C, and 31P nuclei, occur naturally in cell membrane lipids, the examination of their nuclear resonances suffers other drawbacks. The 31P resonance can only report on the head group region of the membrane. 1H and 13C lipid resonances overlap each other and resonances due to proteins and other membrane constituents (12, 13). This problem can be surmounted, in 13C and 2H nuclei, by specific isotropic enrichment so that interference with other resonances is negligible (14, 15). However, these nuclei have very low sensitivity, which, coupled with the broad line width and low molar concentration of lipids in biological samples, makes the NMR experiments difficult. The sensitivity problem is worse for observation of the relaxation effects that report on lipid molecular motions. These problems are reflected in the fact that few NMR studies of intact cell membranes have been published. 19F NMR spectroscopy of specifically incorporated fluorine circumvents all of the problems described above except the inherently low sensitivity of the NMR technique coupled to the lower molar concentration. However, 19F is superior to most nuclei in this regard since it has 83% of the sensitivity of the protons or higher if advantage is taken of the nuclear Overhauser effect. The 19F nuclei can be incorporated biosynthetically as a difluoromethylene-labeled fatty acid whose chemistry is similar to its hydrocarbon analogue. The CF2 group is similar to the CH2 group in its size, geometry, and physical characteristics so it will not greatly perturb the membrane. Since fluorine is not present normally in biological systems, there are no complications due to overlapping resonances. Thus, 19F NMR has the advantage of a single resonance of high sensitivity due to a nucleus incorporated into a specific location in

The large number of biochemical processes that occur within the plasma membrane of cells have prompted many studies of structure-function relationships in such membranes. In particular, the composition and physical state of the phospholipid have been shown to be important factors in such diverse phenomena as transport, oxidative phosphorylation, metabolism, and mitosis (1). These effects must be due to lipid-lipid and lipid-protein interactions. Based on this consideration, model systems consisting of pure phospholipids in the presence of selected purified proteins have been extensively studied by physical and spectroscopic techniques (2). Magnetic resonance studies have been very useful in describing the structure and dynamics of the phospholipids of model membrane systems. Such studies can give information on the rate (3, 4) and extent (5, 6) of the restricted phospholipid motions and the rate of lateral diffusion (7-9) within the bilayer. Moreover, nuclear magnetic resonance (NMR) spectroscopy and electron paramagnetic resonance (EPR) spectroscopy using spin labels can report on the environment of specific chemical groups within the phospholipids. Since the NMR signal results from the bulk phospholipid, it is more direct than many other spectroscopic techniques that require the addition of small amounts of esoteric chemical structures as reporter groups. For the above stated reasons, NMR and EPR should also be powerful tools for studying the lipid component of intact biological membranes. In practice, however, the full potential of the magnetic resonance techniques is hard to realize. EPR

Abbreviations: NMR, nuclear magnetic resonance; EPR, electron paramagnetic resonance; R1, spin-lattice relaxation rate; M2, second moment; Sm. molecular order parameter; CSA, chemical shift anisotropy. * This paper was presented in part at the VII International Conference on Magnetic Resonance in Biological Systems, St. Jovite, Quebec, Canada, September 19-24, 1976. t From whom reprints should be requestedat: Department of Biological Sciences, 378 Crawford Hall, University of Pittsburgh, Pittsburgh, PA 15260.

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Gent et al.

the membrane. Finally, the '9F resonance shows a complex relaxation behavior that could be very informative with regard to the dynamics of lipid fatty acid chains in cell membranes (16). The experimental benefits of 19F NMR motivated our attempt to incorporate fluorinated fatty acids into bacterial membranes. Several difluoromethylene-labeled analogues of myristic acid were synthesized by standard procedures. At least two such derivatives were incorporated into the membrane phospholipids of a wild-type strain of E. coli and, to a larger extent, into the phospholipids of a strain that requires unsaturated fatty acids. Membrane preparations were examined by I9F NMR to determine the line shape and spin-lattice relaxation rates (R1). These were compared to similar results from aqueous dispersions of the extracted lipids and from liposomes consisting of the fluorinated fatty acid dispersed in egg lecithin. A preliminary interpretation is given in terms of the nature of the lipid molecular motions in the several membrane preparations studied. MATERIALS AND METHODS The difluoromethylene fatty acids were synthesized from the corresponding keto acid methyl esters by a mild and specific technique involving MoF6 in CH2Cl2 solution (17) (Fluoreze M., PCR, Gainsville, FL) that resulted in a 20% yield in our hands. The keto acids were synthesized by standard methods (5). w-l-Keto acids were produced by oxidation of 1-methylcyclotridecanol in CrO3/acetic acid solution (18). All chemicals used were reagent grade. Escherichia coli K12 (X) ATCC10798, a wild-type strain, was supplied by L. A. Jacobson. Strain K1060B5, FabB FadE ThiStrr, was kindly supplied by D. F. Silbert. It is an unsaturated fatty acid auxotroph, and its fatty acid incorporation behavior has been described in detail (19). Organisms were grown at the required temperature in a New Brunswick shaking-water bath in a medium composed of 60 mM potassium phosphate buffer at pH 7.0, 0.01% MgSO47H20, 0.1% (NH4)2SO4, 1.0% Casamino acids (Difco), and 0.2% glycerol. Brij-58 (Sigma) was added to a final concentration of 1 mg/ml. Oleate and thiamine were used at a final concentration of 100 ,tg/ml. Overnight cultures were harvested at 40 by centrifugation at 3000 X g and washed in 0.15 M NaCI. The pellet was resuspended in medium containing 100 ,ug of fatty acid supplement per ml that had been warmed to the corresponding overnight growth temperature. Growth was followed by measurement of optical density (OD) on a Bausch and Lomb "Spectronic 20" at 425 nm. Lipid composition was examined as a function of growth time and temperature. The fatty acid composition was determined from gas chromatographic analysis of the methyl esters of phosphatidylethanolamine by the procedure of Silbert et al. (20). The amount of free fatty acid in the membrane vesicles was undetectable and must be less than 5% of the amount of fatty acid acylated to phospholipids. All membrane samples were dissolved in D20 buffer containing 50 mM potassium phosphate, 10 mM MgSO4, and 50 ,ug of chloramphenicol per ml at pH 6.6. The model bilayer membranes consisted of 20 mg of fluorinated fatty acids and 400 mg of egg lecithin [purified from fresh eggs by the method of Singleton et al. (21)] that were dissolved in CHC13, dried under reduced pressure, and then mixed in a Vortex with 1.6 ml of D20 buffer at a temperature greater than 300. Dispersions were made of the total extracted lipids of the E. coli membrane vesicles. The lipids were extracted by the

Proc. Natl. Acad. Sci. USA 75 (1978)

631

method of Bligh and Dyer (22). The CHCl3 solution was washed once with water and dried under reduced pressure for 12 hr. The lipids were hydrated at approximately 20 mg/ml in D20 buffer by sonication at a temperature greater than 300, then frozen and thawed to aggregate any small particles. Membrane vesicles were prepared from whole cells by the Kaback procedure (23) with minor modifications. The vesicles were pelleted from -20 ml of D20 buffer and resuspended in the same buffer at 25 mg of membrane protein per ml, as determined by a modified Lowry assay. 19F NMR spectra at 84.7 MHz were taken on a Bruker HFX-90 spectrometer modified for pulse Fourier transform and equipped with a deuterium lock in 10-mm sample tubes. After a 40-,usec acquisition delay, data points were collected at a rate equivalent to a 60 KHz sweep width. Baseline artifacts were removed by substracting the spectrum of a sample containing no fluorine (detailed methods to be published elsewhere). Spin-lattice relaxation rates (R1) were measured by the 90--r-90 saturation recovery method and calculated for a straight line logarithmic fit to the data points. 19F NMR spectra at 235.2 MHz were taken on the MPG-HF 250 MHz NMR correlation spectrometer (24). A modulation frequency of 50,000 Hz was used to sweep a 30,000 Hz sweep width at 0.5 sec/scan. For weak signals, a baseline correction had to be applied. No frequency-dependent phase correction was necessary. RESULTS AND DISCUSSION Incorporation of fluorinated fatty acids into E. coli We have found that an unsaturated fatty acid auxotroph (K1060B5) with a penchant for branched chain fatty acids shows a significant incorporation of fluorinated myristic acid into the cell membrane. The three positional isomers of difluoromyristate are incorporated to very different extents. In the most favorable circumstances, cells can be grown so that 50% of the fatty acids are 8,8-difluoromyristate. Difluoromyristic acid appears to act as an unsaturated fatty acid analogue (Table 1). It competes poorly with oleic acid for incorporation and is taken up by K1060B5 only when no source of unsaturates is available. One has to weigh the benefits of such high incorporation against the decreased viability and abnormal behavior of cells grown under these conditions. The effects of 8,8-difluoromyristate on the growth curves of K1060B5 are presented in Fig. 1. In all cases growth persisted longer with the 8,8-difluoromyristate than with either myristic acid or no fatty acid supplement. At 370 the difluoromyristate supported normal growth rates for two generations, while myristate was not effective in maintaining growth for more than one generation. After two generations a significant difference in the growth of oleate- and difluoromyristate-supplemented cells was seen, however. This occurred sooner at lower temperatures where a greater unsaturated fatty acid content is necessary to keep the membrane lipid fluid. The percent incorporation of the fluorinated fatty acids depends on both the temperature and the number of generations of growth. Table 1 shows the fatty acid composition of the phosphatidylethanolamine of E. coli grown at 440, 370, and 300 until the optical density of the culture stopped increasing. The greatest incorporation of difluoromyristate occurred at 370. At this most favorable temperature there was a significant variation in the uptake of the three positional isomers synthesized. 4,4-Difluoromyristate was not taken up to a measurable extent. However, 8,8-difluoromyristate was incorporated to the same extent as oleate. 13,13-Difluoromyristate showed an interme-

Biochemistry:

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Gent et al.

Table 1. E. coli phosphatidylethanolamine fatty acid composition at the maximum incorporation of fluorinated myristic acids

Strain Fatty Fatty acid composition, mol % and acid Difluoro temp. supplement 14:0 16:0 16:1 18:1 14:0 K1060B5 18:1 300 8,8-fluoro

10.4 14.5

37.1 33.1

K1060B5* 18:1

7.8 22.0 24.5 35.1

15.9 66.0 19.9 24.6

8.6 32.4

40.6 37.7

2.0 1.2

35.1 32.7

370

4,4-fluoro 8,8-fluoro

13,13-fluoro K1060B5 18:1 440 8,8-fluoro

K12(X)

18:1

300

8,8-fluoro

8.3

9.0

29.3 31.9

41.0 7.0

44.2

64.2 6.0 2.1 0 41.4 7.2

0 50.9 36.6 21.8

31.3 24.9

8.0

K12(X) 18:1 3.9 42.0 11.7 14.9 440 3.3 41.2 13:7 7.7 4.2 8,8-fluoro * Data at 370 include unpublished observations of J. J. Baldassare and D. F. Silbert.

diate behavior, and was generally incorporated to two-thirds the extent of the 8,8-isomer. For the purpose of the NMR experiment, incorporation of difluoromyristate into the K1060B5 strain for one generation at 30 gave good results. The membranes were fluid enough to see I9F NMR spectra above 30', and 30% of the phospholipid fatty acids were 8,8-difluoromyristate. Under the same conditions, 20% incorporation of 13,13-difluoromyristate occurred. In both cases the cells were harvested during the period of exponential growth that is the same rate as oleate-grown cells. Although we have not investigated all possibilities, Table 1

shows that the wild-type E. coli will incorporate at least 8% of 8,8-difluoromyristate. No metabolism of the fluorinated fatty acid was detected by gas chromatography. Thus, difluoromethylene fatty acids can also be incorporated into wild-type cells. '9F NMR studies There are several aspects of the 19F NMR spectroscopy in lipid bilayer membranes that could give information about intraand intermolecular interactions of the phospholipid hydrocarbon chains. The most informative aspect in biological membranes is the resonance relaxation behavior. More direct measurements of structural perturbations, such as sterically induced chemical shifts and altered spin-spin couplings, are of value in studying sonicated model bilayer membranes (25). Unfortunately they cannot be utilized in biological membranes because of the broadness of the resonance. For this reason we will restrict our comments to the relaxation behavior of the difluoromyristate spectroscopic probe. An understanding of the basis of the relaxation effects can be found in the comparison of the 19F resonance in egg lecithin multilayers to the resonances of other nuclei incorporated into the phospholipid of this well-studied model membrane. The 19F resonances for 4,4-, 8,8-, and 13,13-difluoromyristate incorporated to 5% by weight in egg lecithin are shown at two different magnetic field strengths in Fig. 2. It is apparent that the line shape and width of the resonance is field dependent. There is also a large difference in line width for the three positional isomers. The 19F NMR resonance at 84.7 MHz appears as a sharp central peak superimposed on a much broader resonance that is asymmetric (Fig. 2A). The approximately "super-lorentzian" shape of the resonance is the same for the three difluoromethylene positions studied. However, the width, as indicated by the decreased intensity of the central spike and the increased intensity in the wings of the resonance, substantially decreases for the difluoromethylene group adjacent to the methyl terminus of the hydrocarbon chain. The resonance line shape at 235.2 MHz is very asymmetric and does not show a sharp central peak (Fig. 2B). It is reminiscent of "powder-type" spectra due to a resonance affected by chemical shift anisotropy (CSA). Again, the width of the resonance decreases for groups farther away from the carboxyl moiety. Previous magnetic resonance studies of other nuclei in egg

5 kHz

Growth period, hr FIG.

1.

Growth

curves

for E. coli K1060B5 grown

on

different

fatty acid supplements: *, oleate; 0, 8,8-difluoromyristate; *, myristate; and Vj none. The extent of growth was assumed to be proportional to the optical density at 450 nm.

10 kHz

Ho --e Ho FIG. 2. 19F NMR spectra of various isomers of difluoromyristate in egg lecithin multilayers. (A) 84.7 MHz spectra, 370, resulting from 1000 scans and 100 Hz line broadening: a, 13,13-difluoromyristate; b, 8,8-difluoromyristate; c, 4,4-difluoromyristate. (B) 235.2 MHz spectra, 350, resulting from 1000 scans and 100 Hz line broadening: a, 13,13-difluoromyristate; b, 8,8-difluoromyristate; c, 1% trifluoroacetic acid at pH 6.7 in D20. -s

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lecithin membranes have described line shapes similar to those seen above. 'H and 13C resonances of methylene groups in the lipid fatty acid chain show "super-lorentzian" shapes whose widths decrease for groups toward the methyl terminus (26, 27). However, the line widths do not depend on the applied magnetic field (26). These 1H and '3C NMR results are explained by dipole-dipole interactions that are quickly spatially averaged around the hydrocarbon chain axis while spatial averaging in other directions is restricted (28). The motional gradient down the fatty acid chain is a reflection of the decreased ordering effect, of adjacent molecules on the hydrocarbon chain- methylenes moving away from the aqueous interface of the bilayer membrane. The 19F NMR results at 84.7 MHz could be explained in a similar way. The 19F line shapes at 235.2 MHz are more similar to 31P NMR spectra of phospholipids. At high magnetic fields the CSA of the phosphate bonds, coupled to the very restricted motion of the phosphate group in bilayer membranes, leads to a powder-type spectrum similar to those in Fig. 2 (29, 30). The asymmetric line shape occurs because the most probable bond orientations with respect to the applied magnetic field lead to a resonance displaced from the average value. If Teflon is taken as a model compound, a chemical shift difference of 117 ppm is expected for parallel compared to perpendicular orientations of the C-F bond in a difluoromethylene group (31). This value is more than large enough to account for the line width of the 19F resonance at 235.2 MHz. Both CSA and dipole-dipole interactions must be taken into account to relate the observed line shape to an order parameter. The Notionally averaged values of these two magnetic interactions can be obtained by a computer-assisted line fitting procedure (see ref. 29, for instance). Because averaging by fast rotational motion about the hydrocarbon chain axis, z, generates an axially symmetric interaction Hamiltonian, both the average dipole-dipole and CSA are proportional to Sz. Therefore, their ratio once determined at a given Lamor frequency, Po, is independent of molecular motion. The order parameter can be obtained from the observed spectrum by the simple relation

S=V=DD (observed) = PCSA (observed) VDD(O

VCSA(O)

in which (0) is the line width calculated if the CF2 group undergoes rotation about the z axis only. The molecular order parameter, Sm. is 0.5 Sz in all cases, due to complete rotational averaging about the hydrocarbon chain axis on the NMR time scale. We find that values of VDD(O) = 15 kHz and PCSA(O) = 110 ppm best fit the experimental spectra at 84.7 and 235.2 MHz. A complete theoretical analysis of the 19F NMR line shape in this partially ordered system will be given elsewhere. To check the assumptions made in the line shape analysis, the order parameter, Sm, obtained from 19F NMR can be compared to that found by other magnetic resonance techniques. The most unambiguous results come from 2H NMR studies, where there should be minimal perturbation of the bilayer structure due to the resonance probe. Sm can be measured directly from the splitting due to nuclear quadrupole interactions. From 2H NMR, a value of Sm of about 0.2 is found for positions 2-10 and a lower value of -