Proc. Nati. Acad. Sci. USA Vol. 74, No. 12, pp. 5435-5439, December 1977 Biochemistry
Deuterium magnetic resonance studies of the interaction of lipids with membrane proteins (membranes/cytochrome c oxidase/quadrupolar splittings/order parameters)
F. W. DAHLQUIST*, D. C. MUCHMORE*, J. H. DAVISt , AND M.
BLOOMf
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403; and f Department of Physics, University of British Columbia, Vancouver, British Columbia, Canada V6T IW5 *
Communicated by V. Boekeiheide, September 26, 1977
The deuterium magnetic resonance spectra of ABSTRACT lipid-protein particles containing cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) isolated from beef heart mitochondria and the specifically deuterated lipid 1416,16,16-trideuteropalmitoyl-2-palmitoleoyl phosphatidylcholine are presented. These reconstituted particles are of uniform lipid and protein content; however, the spectra clearly show two environments characterized by distinctly different residual quadrupolar splittings or order parameters. The lessordered environment shows a splitting similar to but slightly less than that of the pure lipid alone at a given temperature. The more restricted environment appears to be induced by the presence of the protein. The amount of the restricted lipid is clearly temperature dependent with a 2- to 3fold decrease in relative amount from 2 to 220. The rate of exchange of lipid between the free and restricted environments is slower than 103/sec. The significance of these phenomena is discussed.
There is now convincing evidence that some membrane proteins extend into and, in some cases, through the lipid bilayer (1, 2). For such proteins, the interactions of the lipid with the protein and with other lipids have an important role in establishing the architecture of the membrane and the function of the proteins included in that membrane. Recently, it has been possible to apply various forms of spectroscopy to the problem of lipid-protein interactions. In a pioneering work, Jost et al. (3) used electron spin resonance techniques to study the interaction of beef heart mitochondrial cytochrome c oxidase with the natural mixtures of mitochondrial lipids. This work demonstrated a highly restricted or immobilized component of the lipid corresponding to about 0.2 mg of lipid per mg of protein, independent of the ratio of lipid to protein. Presumably, this immobilized component is due to the interaction between the protein and the lipid. The authors named this restricted lipid "boundary lipid" and suggested that 0.2 mg/mg corresponds to a single solvation layer of lipid about the circumference of protein where it is in contact with lipid bilayer. A number of further questions concerning the nature of the boundary lipid are raised by this important study. Some examples are: Does the protein preferentially sequester some lipids as opposed to others? What is the energy difference between boundary lipid and free lipid under a given set of conditions? What is the time scale of the exchange of the boundary and free lipid? Deuterium nuclear magnetic resonance (DMR) offers a powerful technique for probing these questions (4-6). When rapid and complete reorientation of the deuterated molecule with respect to the applied magnetic fields takes place in an The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
isotropic medium such as a typical fluid, the spectrum consists of a single absorption line. In systems such as oriented lipid bilayers, however, not all reorientations of the molecules are equally probable. Then, a splitting of the DMR line is observed due to the time-averaged deuterium quadrupolar interactions, even though the molecule reorients extremely rapidly. In our systems, the magnitude of the quadrupolar splitting is a measure of the orientational order of the molecule due to the combined influence of the bilayer and the lipid protein interface. In a sample consisting of a superposition of randomly oriented bilayers, these effects appear as a powder pattern spectrum characterized by two maxima. The separation of the maxima, the residual quadrupolar splitting, is a measure of orientational order of the molecule at the position of the deuterium nucleus. This splitting ranges from a maximum of about 130 kHz for complete order to 0 in the isotropic limit in which all orientations of the molecule are equally probable and molecular reorientation is fast. If the deuterated molecules exist in several sites or environments, each characterized by a different degree of restriction of motion, and the rate at which a given molecule can exchange from one environment to the others is small compared to the difference in residual splitting characterizing those environments, the observed DMR spectrum would be the superposition of several powder pattern spectra. The contribution of each site to the spectrum is characterized by a residual quadrupolar splitting that reflects the motional properties of that environment. Thus, dynamic information about the exchange process can be deduced from the observed spectra as well as information concerning the relative amounts of free and restricted lipid present in the sample. This communication describes an investigation of the interaction of beef heart mitochondrial cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) with the specifically deuterated lipid 1-(16,16,16-trideuteropalmitoyl)-2-palmitoleoyl phosphatidylcholine in reconstituted, large, single-layer lipid-protein particles. A quadrupolar spin echo technique ("solid echo") was used to obtain undistorted spectra (7). This is essential to the proper analysis of the spectra because one must be able to analyze the line shape precisely. The results clearly demonstrate that any exchange of lipid between a relatively free and a restricted environment must be slow (i.e., longer than 0-3 sec). The restricted environment is due to the presence of cytochrome c oxidase and the amount of lipid in this restricted environment is strongly temperature dependent at temperatures well above the phase transition temperature of the pure lipid. Abbreviation: DMR, deuterium nuclear magnetic resonance.
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5436
Biochemistry: Dahlquist et al.
Proc. Natl. Acad. Sci. USA .74 (1977)
MATERIALS AND METHODS The synthetic route used to prepare 16,16,16-trideuterohexadanoic acid is outlined below. Details of the procedure are available from F.W.D. and D.C.M. upon request.
CH30H
(CH2)-C'
HOC H2(C
HCI
H21 -C-OCH3
0-I CH2 (CH2)|C-0CH 19
p-CH3C6H4 SO3H
1. LiAI D4
Qo-CH2(CH2) 4C D20-SO2CH3
2. CH3SO2CI,
1. Li BEt3D, THF 2.
"'H2 C r04' CH3CO2H,
C
D3(CH2)4CO?2H
(CH3j2Ca
Diacyl Lecithin. In a typical preparation, 960 mg (3.7 mmol) of deuterated hexadecanoic acid and 600 mg (3.7 mmol) of carbonyldiimidazole were mixed in 10 ml of dry chloroform. After cessation of CO2 evolution, this solution was added to 200 mg (8.1 mmol) of glycerophosphorylcholine that had been dried on the bottom of a pear-shaped flask. Stirring by means of a small magnetic bar was initiated, and the bath temperature was raised to 650. A stream of nitrogen was blown across the surface until the solvent was gone (-3 hr). After 4 additional hr, 10 ml of chloroform was added. The cycle was repeated twice, and then 1 ml of water was added. The chloroform/water was blown off slowly under a stream of nitrogen, and the residue was applied to the top of a 135 X 2.3 cm column of Sephadex LH-20 packed in 95% ethanol. The material (513 mg) in the clearly resolved lecithin peak was judged to be homogeneous by thin-layer chromatographic analysis. 1(16,16,16-Trideuteropalmitoyl) Lysolecithin. The satu*rated lecithin was hydrolyzed to the corresponding 1-acyl lysolecithin by the procedure of Wells and Hanahan (8). In a typical preparation, 100 mg of dry lipid was suspended in 10 ml of diethyl ether/methanol, 95:5 (vol/vol). To this mixture was added 100 ,ug of phospholipase A from Crotalus durissus terrificus (Sigma) in a 0.2 ml of 0.22 M NaCI/0.02 M CaCl2/1 mM EDTA, pH 6. After the reaction, the solvents were removed and the lysolecithin was separated from the fatty acid by chromatography on Sephadex LH-20 (250, 95% ethanol solvent). Metal cations were removed by washing the lysolecithin-containing column fractions through another column containing 5 g of Amberlite MB-1 ion-exchange resin in 95% ethanol solvent.
1416,16,16-Trideuteropalmitoyl)2-palmitoleoyl
Phos-
phatidyicholine. A 260-mg portion of lysolecithin dissolved in S ml of CHC13 was treated with the reagent formed from 430 mg (1.7 mmol) of palmitoleic acid and 272 mg (1.7 mmol) of carbonyldiimidazole in 5 ml of chloroform. The bath temperature was raised to 400, and a stream of nitrogen was blown across the surface. After 5 hr, the mixture was diluted with chloroform, and the cycle was repeated. After an additional 4 hr, the reaction was quenched with methanol. The components
were separated on a silica gel column with a chloroform/ methanol gradient as eluant. The lecithin fraction was applied to the top of a 135 X 2.3 cm Sephadex LH-20 column and eluted with 95% ethanol. The diacyl phosphatidylcholine thus obtained was judged to be homogeneous by thin-layer chromatographic analysis. Isolation of Cytochrome c Oxidase. Cytochrome c oxidase was purified from beef heart mitochondria by the method described by Capaldi and Hayashi (9). Enzyme activity was measured by following the rate of oxidation of ferrocytochrome c at 550 nm in a solution containing 0.5% Tween-80 detergent (10, 11). Protein concentration was determined by the method of Lowry et al. (12) with bovine serum albumin as a standard. Heme concentration was measured by the difference spectrum at 603-630 nm of the reduced heme in 1% Triton X-100 (13). Preparation of Lipid-Protein Particles. In a typical preparation, 100 mg (6 imol) of lipid in ethanol solution was dried onto a pear-shaped flask, dried under reduced pressure, and then suspended in 10 ml of sodium cholate (twice recrystallized from 70% ethanol), 20 mg/ml/0.090 M NaCl/0.020 M TrisHC1, pH 7.4. Cytochrome c oxidase (200 mg) was suspended in 10 ml of the same buffer. The lipid and protein solutions were then combined and mixed by vortexing. The lipid/protein mixture was dialyzed at 40 against four 0.5-liter washes of 0.090 M NaCl/0.020 M Tris-HCl, pH 7.4, over a 24-h period. The solution was then layered onto a 10-ml 10-60% (wt/vol) sucrose gradient buffered with 0.090 M NaCl/0.020 M Tris1HCl, pH 7.4. The sample was centrifuged at 75,000 X g for 2 hr at 4°. A tight band of lipid-protein complex formed at a density corresponding to 35% sucrose. Lipid-protein determinations were performed after the sucrose was removed by dialysis against the buffer described above. To obtain the lipid/protein ratio (mg/mg), lipid phosphorus was assayed by the method of Ames and Dubin (13), and the protein was determined by the method of Lowry et al. (12). DMR spectra were obtained at 13.8 MHz with a Bruker SXP 4-100 spectrometer. All spectra were acquired by using a quadrupolar echo pulse technique in order to overcome the line shape distortions encountered when observing deuterium spectra (7). RESULTS Fig. 1 shows the DMR spectra of an aqueous suspension of the lipid at various temperatures. These temperatures are well above the fluid-gel phase transition temperature of this lipid (-15°), and the powder pattern spectra were sharp and characterized by a single residual quadrupolar coupling. This value ranged from 3.3 kHz at 00 to 2.4 kHz at 200. These values should be compared to the splitting expected, if no motional averaging were taking place, of approximately 130 kHz. Thus, nearly complete averaging was observed because the labeled methyl group can spin freely and the chain motions are relatively extensive. As the temperature increased, the motional averaging became more complete, and the observed splitting decreased slightly. Fig. 2 shows the DMR spectra of reconstituted cytochrome c oxidase-lipid particles over the same range of temperatures used for the data in Fig. 1. These particles were of uniform density (equal to 35% sucrose) and therefore of uniform ratio of lipid to protein, corresponding to 0.5 mg of lipid per mg of protein. It is clear that the protein had a dramatic effect on the spectra. A single residual quadrupolar splitting is no longer sufficient to describe the observed spectra. In addition, the spectra show considerable variation with temperature when
Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Dah1quist et al.
5437
t
A
B
D
V-V0, kHz FIG. 1. DMR spectra at various temperatures of 1-(16,16,16trideuteropalmitoyl)-2-palmitoleoyl phosphatidylcholine suspended in 15% sucrose 0.01 M Tris-HCl, pH 7.6/0.09 M NaCl. The lipid concentration was approximately 50 mg/ml. The spectra are the result of 4000 echo accumulations acquired at a rate of 5/sec.
cytochrome c oxidase was present. The spectrum obtained at 20 clearly shows the superposition of at least two powder pattern spectra. One is characterized by a residual splitting of about 2.6 kHz and the other by 4.6 kHz. The lower value is similar to that obtained for the free lipid at this temperature. The larger value I21.5 0
~~~90
I
~~~2.5
I
-10
-5
0
5
10
V-VO, kHz FIG. 2. DMR spectra at various temperatures of cytochrome c
oxidase-lipid particles. The buffer was the same as that used in Fig. 1. The lipid concentration was approximately 50 mg/ml. The particles were of uniform lipid/protein ratio, equal to 0.5 mg of lipid per mg of protein. These spectra are the result of 20,000 echo accumulations at
21.50, 51,000 at 90, and 80,000 at 2.50, all at a rate of 2/sec.
r
4 kHz
-n
r
4 kHz-.
FIG. 3. Comparisons of simulated DMR spectra (Right) and observed spectra (Left) with free lipid and lipid-protein particles. The parameters used to calculate the simulated spectra are given in Table 1. Spectra: A, free lipid at 90, 4000 scans; B, lipid-protein particles at 2.50, 80,000 scans; C, lipid-protein particles at 90, 31,000 scans; D, lipid-protein particles at 180, 20,000 scans. For the purposes of simulation, the center line due to natural abundance of deuterium in the water was neglected.
represents a population of lipid that is substantially more restricted in its motion as a result of the presence of the protein. As the temperature increased, the amount of this restricted lipid clearly decreased. A quantitative analysis of Fig. 2 can be made by computer simulation of the observed spectra. These simulations require a knowledge of the amount of lipid in a particular environment and of the residual quadrupolar splitting and linewidth associated with that environment. The linewidth could be due to line broadening effects associated with exchange of lipid between different environments or to local variations of quadrupolar splittings attributable to influences such as different mechanical strains or protein concentrations in the particles. For the purpose of the calculation, we assumed a Lorentzian line shape; however, because the linewidths are small relative to the powder pattern widths, the exact form of the line shape seems to be unimportant. Fig. 3 shows the comparison of the observed spectra and the simulated ones. We assumed that only two environments are present. This assumption leads to an adequate fit of the simulated and observed spectra, although small, systematic inconsistencies appear in the outer portions of the spectra. The parameters used to calculate the spectra shown in Fig. 3 are summarized in Table 1. The spectrum of the lipid alone at 9° (A in Fig. 3) was simulated by using a single residual quadrupolar splitting of 2.80 kHz. This gives excellent agreement with the observed spectrum, including the shapes of the maxima and the presence of the "shoulders" in the powder pattern in the outer portion of the spectrum. The powder pattern was calculated as the superposition of 1000 doublets with Lorentzian line shapes and linewidths of 120 Hz. The splitting of the doublets was allowed to vary according to (3 cos2 0- 1) in 1000 increments uniformly spaced in cos 0.
Biochemistry: Dahlquist et al.
5438
Proc. Natl. Acad. Sci.. USA 74 .1977)
Table 1. Parameters used for spectral simulation
Spectrum* A B
0C
C
9
D
21.5
92.5
Residual splitting, kHz
Linewidth, Hz
Fraction of total
2.8 2.6 4.6 2.3 3.6 2.1 4.4
120 350 350 300 300 250 250
1.0 0.37 0.63 0.56 0.44 0.77 0.23
* See Fig. 3.
A similar procedure to that of the free lipid was used to simulate the spectrum of the lipid-protein complex at 2.50. However, two complete powder patterns were calculated by using residual splitting of 2.6 kHz and 4.6 kHz and equal linewidths of 30 Hz. The composite spectrum was then generated by using an intensity ratio of the broader to sharper components of 1.7. Values of this ratio of 1.6 and 1.8 gave visually less satisfactory fits to the observed spectrum. Similar procedures were used to analyze the two other spectra. The best fits were obtained with ratios of restricted to free lipid of 0.8 at 90 and 0.3 at 21.50, with estimated errors of 0.1 in each case. These data clearly demonstrate a dramatic decrease in the proportion of restricted lipid with increasing temperature, ranging from about 0.32 mg at 2° to about 0.11 + 0.03 mg at 21.50 per mg of protein. The residual splittings of the restricted lipid in spectra B, C, and D are compatible with a temperature-independent value of 4.2 ± 0.4 kHz, within experimental error. Because the spectra of the lipid-protein particles always show at least two environments, the exchange rate of lipid between these two environments must be slow relative to the difference in residual quadrupole splitting (approximately 2wr 2 X 103/sec 104/sec) characterizing the free and restricted environments. If we assume that the linewidths are completely due to broadening, the exchange rate is estimated to be about 103 sec1. This represents an upper limit to the exchange rate because there may be other contributions to the linewidth. DISCUSSION The results presented here clearly demonstrate the presence of a population of lipid whose motion is more restricted in the presence of cytochrome c oxidase than in its absence. In addition to this restricted lipid, a component of the lipid is observed that is characterized by a restriction of motion similar to that of the pure lipid but always slightly less restricted than the pure lipid at the same temperature. Thus, it is clear that the protein both restricts some lipid, presumably that which is nearest the protein, and also appears to modify the packing of the lipid further away from its surface, so that slightly more motional averaging is observed in the DMR signal of the lipid in this more distant environment. Because we used the quadrupolar echo technique which, in principle, introduces no intensity or line shape artifacts due to data collection, it is reasonable to attempt to simulate the observed spectra by using theoretical powder pattern spectra. The pure lipid is fit very well by a powder pattern spectrum characterized by one residual quadrupolar splitting or order parameter. The spectra of the lipid-protein particles are more complicated and require at least two residual splittings (or order parameters) to fit the data. Two residual splittings are sufficient to fit the data quite well. However, this does not imply that only -
two environments are actually present. In fact, a range of restricted environments characterized by a distribution of order parameters about the mean is possible. Such a situation cannot be readily distinguished from a single environment whose intrinsic linewidth is large. Significantly, the relative amounts of lipid present in the two motionally defined environments are clearly temperature dependent. Thus, the average free energy difference of the lipidin these two environments is small [on the order of 1 kcal (4.2 kJ)/mol] at temperatures between 00 and 200. It is conceivable that a decrease in the amount of restricted lipid is seen with increasing temperature because the exchange of lipid between the restricted and free environments becomes fast enough to give exchange averaging of a portion of the restricted lipid with free. This possibility is essentially ruled out because the observed residual splitting of the free component of lipid-protein particle spectra decreases with increasing temperature in a way similar to that of the pure lipid. If exchange averaging were occurring, one would expect the average of free lipid and that part of the restricted lipid capable of exchanging to result in an increase in the residual splitting observed for the averaged environment with increasing temperature. This was not observed. We also see little exchange-dependent broadening and conclude, therefore, that the rate of exchange of lipid between the free and restricted environments must be slower than 103/sec. This is similar to the time scale for turnover of cytochrome c oxidase (11) and suggests that bulk lipid exchanges during a catalytic turnover are unlikely. The amount of restricted lipid observed by using DMR is found to range between 0.32 mg/mg of cytochrome c oxidase at 20 to 0.11 mg/mg at 210. These values agree nicely with the value, about 0.2 mg/mg, determined by Jost et al. (3) with electron spin resonance techniques and a nitroxide derivative of stearic acid as the spin label probe. However, no temperature dependence was observed in the amount of restricted lipid seen by these workers (P. C. Jost, R. A. Capaldi, and 0. H. Griffith, personal communication). The exact reasons for this difference are unclear at this time, although a number of explanations are possible. Clearly, the time scale for motional averaging in the electron spin resonance and DMR experiments are vastly different. It is possible that a given environment shows restricted motion on one time scale and not the other. In addition, the electron spin resonance technique used a dilute spin label probe with a natural mixture of mitochondrial lipids whereas the DMR experiment used a chemically homogeneous deuterated phosphatidylcholine both as the bulk lipid and as the probe. It is possible that some compensating effects of the partitioning of the electron spin resonance probe and natural lipid mixture occurs which obscures the temperature-dependent effects for a single lipid. The answer to this question must await further experimentation. The data presented here clearly demonstrate the utility of DMR in the study of lipid-protein interactions. The technique uses a relatively nonperturbing deuterated lipid to probe the motion of the lipid itself. The time scale of the experiment appears to be ideal for most biological systems and offers a dramatic sensitivity to environment as judged by its motional properties. The data obtained may be fairly easily quantitated. We anticipate considerable application of these approaches to the general question of lipid-protein interactions with emphasis on the possible lipid specificity apparently seen for certain membrane proteins. The work at The University of British Columbia was supported by the National Research Council of Canada and a special Killam-Canada Council Interdisciplinary Grant. The work at The University of Oregon
Biochemistry: Dahlquist et al. was supported by National Science Foundation Grant BMS 75-10422. F.W.D. is an A. P. Sloan Foundation Fellow, 1975-1977.
1.
2. 3. 4.
5. 6.
Henderson, R., Capaldi, R. A. & Leigh, J. S. (1977) J. Mol. Biol. 112,631-648. Eytan, G. D;, Carroll, R. C., Schatz, G. & Racker, E. (1975)J. Biol. Chem. 250,8598-8603. Jost, P. C., Griffith, 0. H., Capaldi, R. A. & Vanderkooi, G. (1973) Proc. Natl. Acad. Sci. USA 70,480-484. Stockton, G. W., Johnson, K. G., Butler, K. W., Tulloch, A. P., Boulanger, Y., Smith, I. C. P., Davis, J. H. & Bloom, M. (1977) Nature, in press. Seelig, A. & Seelig, J. (1974) Biochemistry 13, 4839-4845. Stockton, G. W., Polnaszek, C. F., Tulloch, A. P., Hasan, F. &
Proc. Nati. Acad. Sci. USA 74 (1977)
5439
Smith, I. C. P. (1976) Biochemistry 15, 954-966. 7. Davis, J. H., Jeffrey, K. R., Bloom, M., Valic, M. I. & Higgs, T. P. (1976) Chem, Phys. Lett. 42,390-394. 8. Wells, M. A. & Hanahan, D. J. (1969) Biochemistry 8, 414424. 9. Capaldi, R. A. & Hayashi, H. (1972) FEBS Lett. 26,261-263. 10. Smith, L. (1955) in Methods in Enzymology, eds. Colowick, S. & Kaplan, N. 0. (Academic Press, New York), Vol. 2 pp. 732764. 11. Vanneste, W. H., Ysebaert-Vanneste, M. & Mason, H. (1974) J. Blol. Chem. 249,7390-7401. 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 13. Ames, B. N. & Dubin, D. T. (1960) J. Biol. Chem. 235, 769775.
Deuterium magnetic resonance studies of the interaction of lipids with membrane proteins (membranes/cytochrome c oxidase/quadrupolar splittings/order parameters)
F. W. DAHLQUIST*, D. C. MUCHMORE*, J. H. DAVISt , AND M.
BLOOMf
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403; and f Department of Physics, University of British Columbia, Vancouver, British Columbia, Canada V6T IW5 *
Communicated by V. Boekeiheide, September 26, 1977
The deuterium magnetic resonance spectra of ABSTRACT lipid-protein particles containing cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) isolated from beef heart mitochondria and the specifically deuterated lipid 1416,16,16-trideuteropalmitoyl-2-palmitoleoyl phosphatidylcholine are presented. These reconstituted particles are of uniform lipid and protein content; however, the spectra clearly show two environments characterized by distinctly different residual quadrupolar splittings or order parameters. The lessordered environment shows a splitting similar to but slightly less than that of the pure lipid alone at a given temperature. The more restricted environment appears to be induced by the presence of the protein. The amount of the restricted lipid is clearly temperature dependent with a 2- to 3fold decrease in relative amount from 2 to 220. The rate of exchange of lipid between the free and restricted environments is slower than 103/sec. The significance of these phenomena is discussed.
There is now convincing evidence that some membrane proteins extend into and, in some cases, through the lipid bilayer (1, 2). For such proteins, the interactions of the lipid with the protein and with other lipids have an important role in establishing the architecture of the membrane and the function of the proteins included in that membrane. Recently, it has been possible to apply various forms of spectroscopy to the problem of lipid-protein interactions. In a pioneering work, Jost et al. (3) used electron spin resonance techniques to study the interaction of beef heart mitochondrial cytochrome c oxidase with the natural mixtures of mitochondrial lipids. This work demonstrated a highly restricted or immobilized component of the lipid corresponding to about 0.2 mg of lipid per mg of protein, independent of the ratio of lipid to protein. Presumably, this immobilized component is due to the interaction between the protein and the lipid. The authors named this restricted lipid "boundary lipid" and suggested that 0.2 mg/mg corresponds to a single solvation layer of lipid about the circumference of protein where it is in contact with lipid bilayer. A number of further questions concerning the nature of the boundary lipid are raised by this important study. Some examples are: Does the protein preferentially sequester some lipids as opposed to others? What is the energy difference between boundary lipid and free lipid under a given set of conditions? What is the time scale of the exchange of the boundary and free lipid? Deuterium nuclear magnetic resonance (DMR) offers a powerful technique for probing these questions (4-6). When rapid and complete reorientation of the deuterated molecule with respect to the applied magnetic fields takes place in an The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
isotropic medium such as a typical fluid, the spectrum consists of a single absorption line. In systems such as oriented lipid bilayers, however, not all reorientations of the molecules are equally probable. Then, a splitting of the DMR line is observed due to the time-averaged deuterium quadrupolar interactions, even though the molecule reorients extremely rapidly. In our systems, the magnitude of the quadrupolar splitting is a measure of the orientational order of the molecule due to the combined influence of the bilayer and the lipid protein interface. In a sample consisting of a superposition of randomly oriented bilayers, these effects appear as a powder pattern spectrum characterized by two maxima. The separation of the maxima, the residual quadrupolar splitting, is a measure of orientational order of the molecule at the position of the deuterium nucleus. This splitting ranges from a maximum of about 130 kHz for complete order to 0 in the isotropic limit in which all orientations of the molecule are equally probable and molecular reorientation is fast. If the deuterated molecules exist in several sites or environments, each characterized by a different degree of restriction of motion, and the rate at which a given molecule can exchange from one environment to the others is small compared to the difference in residual splitting characterizing those environments, the observed DMR spectrum would be the superposition of several powder pattern spectra. The contribution of each site to the spectrum is characterized by a residual quadrupolar splitting that reflects the motional properties of that environment. Thus, dynamic information about the exchange process can be deduced from the observed spectra as well as information concerning the relative amounts of free and restricted lipid present in the sample. This communication describes an investigation of the interaction of beef heart mitochondrial cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) with the specifically deuterated lipid 1-(16,16,16-trideuteropalmitoyl)-2-palmitoleoyl phosphatidylcholine in reconstituted, large, single-layer lipid-protein particles. A quadrupolar spin echo technique ("solid echo") was used to obtain undistorted spectra (7). This is essential to the proper analysis of the spectra because one must be able to analyze the line shape precisely. The results clearly demonstrate that any exchange of lipid between a relatively free and a restricted environment must be slow (i.e., longer than 0-3 sec). The restricted environment is due to the presence of cytochrome c oxidase and the amount of lipid in this restricted environment is strongly temperature dependent at temperatures well above the phase transition temperature of the pure lipid. Abbreviation: DMR, deuterium nuclear magnetic resonance.
5435
5436
Biochemistry: Dahlquist et al.
Proc. Natl. Acad. Sci. USA .74 (1977)
MATERIALS AND METHODS The synthetic route used to prepare 16,16,16-trideuterohexadanoic acid is outlined below. Details of the procedure are available from F.W.D. and D.C.M. upon request.
CH30H
(CH2)-C'
HOC H2(C
HCI
H21 -C-OCH3
0-I CH2 (CH2)|C-0CH 19
p-CH3C6H4 SO3H
1. LiAI D4
Qo-CH2(CH2) 4C D20-SO2CH3
2. CH3SO2CI,
1. Li BEt3D, THF 2.
"'H2 C r04' CH3CO2H,
C
D3(CH2)4CO?2H
(CH3j2Ca
Diacyl Lecithin. In a typical preparation, 960 mg (3.7 mmol) of deuterated hexadecanoic acid and 600 mg (3.7 mmol) of carbonyldiimidazole were mixed in 10 ml of dry chloroform. After cessation of CO2 evolution, this solution was added to 200 mg (8.1 mmol) of glycerophosphorylcholine that had been dried on the bottom of a pear-shaped flask. Stirring by means of a small magnetic bar was initiated, and the bath temperature was raised to 650. A stream of nitrogen was blown across the surface until the solvent was gone (-3 hr). After 4 additional hr, 10 ml of chloroform was added. The cycle was repeated twice, and then 1 ml of water was added. The chloroform/water was blown off slowly under a stream of nitrogen, and the residue was applied to the top of a 135 X 2.3 cm column of Sephadex LH-20 packed in 95% ethanol. The material (513 mg) in the clearly resolved lecithin peak was judged to be homogeneous by thin-layer chromatographic analysis. 1(16,16,16-Trideuteropalmitoyl) Lysolecithin. The satu*rated lecithin was hydrolyzed to the corresponding 1-acyl lysolecithin by the procedure of Wells and Hanahan (8). In a typical preparation, 100 mg of dry lipid was suspended in 10 ml of diethyl ether/methanol, 95:5 (vol/vol). To this mixture was added 100 ,ug of phospholipase A from Crotalus durissus terrificus (Sigma) in a 0.2 ml of 0.22 M NaCI/0.02 M CaCl2/1 mM EDTA, pH 6. After the reaction, the solvents were removed and the lysolecithin was separated from the fatty acid by chromatography on Sephadex LH-20 (250, 95% ethanol solvent). Metal cations were removed by washing the lysolecithin-containing column fractions through another column containing 5 g of Amberlite MB-1 ion-exchange resin in 95% ethanol solvent.
1416,16,16-Trideuteropalmitoyl)2-palmitoleoyl
Phos-
phatidyicholine. A 260-mg portion of lysolecithin dissolved in S ml of CHC13 was treated with the reagent formed from 430 mg (1.7 mmol) of palmitoleic acid and 272 mg (1.7 mmol) of carbonyldiimidazole in 5 ml of chloroform. The bath temperature was raised to 400, and a stream of nitrogen was blown across the surface. After 5 hr, the mixture was diluted with chloroform, and the cycle was repeated. After an additional 4 hr, the reaction was quenched with methanol. The components
were separated on a silica gel column with a chloroform/ methanol gradient as eluant. The lecithin fraction was applied to the top of a 135 X 2.3 cm Sephadex LH-20 column and eluted with 95% ethanol. The diacyl phosphatidylcholine thus obtained was judged to be homogeneous by thin-layer chromatographic analysis. Isolation of Cytochrome c Oxidase. Cytochrome c oxidase was purified from beef heart mitochondria by the method described by Capaldi and Hayashi (9). Enzyme activity was measured by following the rate of oxidation of ferrocytochrome c at 550 nm in a solution containing 0.5% Tween-80 detergent (10, 11). Protein concentration was determined by the method of Lowry et al. (12) with bovine serum albumin as a standard. Heme concentration was measured by the difference spectrum at 603-630 nm of the reduced heme in 1% Triton X-100 (13). Preparation of Lipid-Protein Particles. In a typical preparation, 100 mg (6 imol) of lipid in ethanol solution was dried onto a pear-shaped flask, dried under reduced pressure, and then suspended in 10 ml of sodium cholate (twice recrystallized from 70% ethanol), 20 mg/ml/0.090 M NaCl/0.020 M TrisHC1, pH 7.4. Cytochrome c oxidase (200 mg) was suspended in 10 ml of the same buffer. The lipid and protein solutions were then combined and mixed by vortexing. The lipid/protein mixture was dialyzed at 40 against four 0.5-liter washes of 0.090 M NaCl/0.020 M Tris-HCl, pH 7.4, over a 24-h period. The solution was then layered onto a 10-ml 10-60% (wt/vol) sucrose gradient buffered with 0.090 M NaCl/0.020 M Tris1HCl, pH 7.4. The sample was centrifuged at 75,000 X g for 2 hr at 4°. A tight band of lipid-protein complex formed at a density corresponding to 35% sucrose. Lipid-protein determinations were performed after the sucrose was removed by dialysis against the buffer described above. To obtain the lipid/protein ratio (mg/mg), lipid phosphorus was assayed by the method of Ames and Dubin (13), and the protein was determined by the method of Lowry et al. (12). DMR spectra were obtained at 13.8 MHz with a Bruker SXP 4-100 spectrometer. All spectra were acquired by using a quadrupolar echo pulse technique in order to overcome the line shape distortions encountered when observing deuterium spectra (7). RESULTS Fig. 1 shows the DMR spectra of an aqueous suspension of the lipid at various temperatures. These temperatures are well above the fluid-gel phase transition temperature of this lipid (-15°), and the powder pattern spectra were sharp and characterized by a single residual quadrupolar coupling. This value ranged from 3.3 kHz at 00 to 2.4 kHz at 200. These values should be compared to the splitting expected, if no motional averaging were taking place, of approximately 130 kHz. Thus, nearly complete averaging was observed because the labeled methyl group can spin freely and the chain motions are relatively extensive. As the temperature increased, the motional averaging became more complete, and the observed splitting decreased slightly. Fig. 2 shows the DMR spectra of reconstituted cytochrome c oxidase-lipid particles over the same range of temperatures used for the data in Fig. 1. These particles were of uniform density (equal to 35% sucrose) and therefore of uniform ratio of lipid to protein, corresponding to 0.5 mg of lipid per mg of protein. It is clear that the protein had a dramatic effect on the spectra. A single residual quadrupolar splitting is no longer sufficient to describe the observed spectra. In addition, the spectra show considerable variation with temperature when
Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Dah1quist et al.
5437
t
A
B
D
V-V0, kHz FIG. 1. DMR spectra at various temperatures of 1-(16,16,16trideuteropalmitoyl)-2-palmitoleoyl phosphatidylcholine suspended in 15% sucrose 0.01 M Tris-HCl, pH 7.6/0.09 M NaCl. The lipid concentration was approximately 50 mg/ml. The spectra are the result of 4000 echo accumulations acquired at a rate of 5/sec.
cytochrome c oxidase was present. The spectrum obtained at 20 clearly shows the superposition of at least two powder pattern spectra. One is characterized by a residual splitting of about 2.6 kHz and the other by 4.6 kHz. The lower value is similar to that obtained for the free lipid at this temperature. The larger value I21.5 0
~~~90
I
~~~2.5
I
-10
-5
0
5
10
V-VO, kHz FIG. 2. DMR spectra at various temperatures of cytochrome c
oxidase-lipid particles. The buffer was the same as that used in Fig. 1. The lipid concentration was approximately 50 mg/ml. The particles were of uniform lipid/protein ratio, equal to 0.5 mg of lipid per mg of protein. These spectra are the result of 20,000 echo accumulations at
21.50, 51,000 at 90, and 80,000 at 2.50, all at a rate of 2/sec.
r
4 kHz
-n
r
4 kHz-.
FIG. 3. Comparisons of simulated DMR spectra (Right) and observed spectra (Left) with free lipid and lipid-protein particles. The parameters used to calculate the simulated spectra are given in Table 1. Spectra: A, free lipid at 90, 4000 scans; B, lipid-protein particles at 2.50, 80,000 scans; C, lipid-protein particles at 90, 31,000 scans; D, lipid-protein particles at 180, 20,000 scans. For the purposes of simulation, the center line due to natural abundance of deuterium in the water was neglected.
represents a population of lipid that is substantially more restricted in its motion as a result of the presence of the protein. As the temperature increased, the amount of this restricted lipid clearly decreased. A quantitative analysis of Fig. 2 can be made by computer simulation of the observed spectra. These simulations require a knowledge of the amount of lipid in a particular environment and of the residual quadrupolar splitting and linewidth associated with that environment. The linewidth could be due to line broadening effects associated with exchange of lipid between different environments or to local variations of quadrupolar splittings attributable to influences such as different mechanical strains or protein concentrations in the particles. For the purpose of the calculation, we assumed a Lorentzian line shape; however, because the linewidths are small relative to the powder pattern widths, the exact form of the line shape seems to be unimportant. Fig. 3 shows the comparison of the observed spectra and the simulated ones. We assumed that only two environments are present. This assumption leads to an adequate fit of the simulated and observed spectra, although small, systematic inconsistencies appear in the outer portions of the spectra. The parameters used to calculate the spectra shown in Fig. 3 are summarized in Table 1. The spectrum of the lipid alone at 9° (A in Fig. 3) was simulated by using a single residual quadrupolar splitting of 2.80 kHz. This gives excellent agreement with the observed spectrum, including the shapes of the maxima and the presence of the "shoulders" in the powder pattern in the outer portion of the spectrum. The powder pattern was calculated as the superposition of 1000 doublets with Lorentzian line shapes and linewidths of 120 Hz. The splitting of the doublets was allowed to vary according to (3 cos2 0- 1) in 1000 increments uniformly spaced in cos 0.
Biochemistry: Dahlquist et al.
5438
Proc. Natl. Acad. Sci.. USA 74 .1977)
Table 1. Parameters used for spectral simulation
Spectrum* A B
0C
C
9
D
21.5
92.5
Residual splitting, kHz
Linewidth, Hz
Fraction of total
2.8 2.6 4.6 2.3 3.6 2.1 4.4
120 350 350 300 300 250 250
1.0 0.37 0.63 0.56 0.44 0.77 0.23
* See Fig. 3.
A similar procedure to that of the free lipid was used to simulate the spectrum of the lipid-protein complex at 2.50. However, two complete powder patterns were calculated by using residual splitting of 2.6 kHz and 4.6 kHz and equal linewidths of 30 Hz. The composite spectrum was then generated by using an intensity ratio of the broader to sharper components of 1.7. Values of this ratio of 1.6 and 1.8 gave visually less satisfactory fits to the observed spectrum. Similar procedures were used to analyze the two other spectra. The best fits were obtained with ratios of restricted to free lipid of 0.8 at 90 and 0.3 at 21.50, with estimated errors of 0.1 in each case. These data clearly demonstrate a dramatic decrease in the proportion of restricted lipid with increasing temperature, ranging from about 0.32 mg at 2° to about 0.11 + 0.03 mg at 21.50 per mg of protein. The residual splittings of the restricted lipid in spectra B, C, and D are compatible with a temperature-independent value of 4.2 ± 0.4 kHz, within experimental error. Because the spectra of the lipid-protein particles always show at least two environments, the exchange rate of lipid between these two environments must be slow relative to the difference in residual quadrupole splitting (approximately 2wr 2 X 103/sec 104/sec) characterizing the free and restricted environments. If we assume that the linewidths are completely due to broadening, the exchange rate is estimated to be about 103 sec1. This represents an upper limit to the exchange rate because there may be other contributions to the linewidth. DISCUSSION The results presented here clearly demonstrate the presence of a population of lipid whose motion is more restricted in the presence of cytochrome c oxidase than in its absence. In addition to this restricted lipid, a component of the lipid is observed that is characterized by a restriction of motion similar to that of the pure lipid but always slightly less restricted than the pure lipid at the same temperature. Thus, it is clear that the protein both restricts some lipid, presumably that which is nearest the protein, and also appears to modify the packing of the lipid further away from its surface, so that slightly more motional averaging is observed in the DMR signal of the lipid in this more distant environment. Because we used the quadrupolar echo technique which, in principle, introduces no intensity or line shape artifacts due to data collection, it is reasonable to attempt to simulate the observed spectra by using theoretical powder pattern spectra. The pure lipid is fit very well by a powder pattern spectrum characterized by one residual quadrupolar splitting or order parameter. The spectra of the lipid-protein particles are more complicated and require at least two residual splittings (or order parameters) to fit the data. Two residual splittings are sufficient to fit the data quite well. However, this does not imply that only -
two environments are actually present. In fact, a range of restricted environments characterized by a distribution of order parameters about the mean is possible. Such a situation cannot be readily distinguished from a single environment whose intrinsic linewidth is large. Significantly, the relative amounts of lipid present in the two motionally defined environments are clearly temperature dependent. Thus, the average free energy difference of the lipidin these two environments is small [on the order of 1 kcal (4.2 kJ)/mol] at temperatures between 00 and 200. It is conceivable that a decrease in the amount of restricted lipid is seen with increasing temperature because the exchange of lipid between the restricted and free environments becomes fast enough to give exchange averaging of a portion of the restricted lipid with free. This possibility is essentially ruled out because the observed residual splitting of the free component of lipid-protein particle spectra decreases with increasing temperature in a way similar to that of the pure lipid. If exchange averaging were occurring, one would expect the average of free lipid and that part of the restricted lipid capable of exchanging to result in an increase in the residual splitting observed for the averaged environment with increasing temperature. This was not observed. We also see little exchange-dependent broadening and conclude, therefore, that the rate of exchange of lipid between the free and restricted environments must be slower than 103/sec. This is similar to the time scale for turnover of cytochrome c oxidase (11) and suggests that bulk lipid exchanges during a catalytic turnover are unlikely. The amount of restricted lipid observed by using DMR is found to range between 0.32 mg/mg of cytochrome c oxidase at 20 to 0.11 mg/mg at 210. These values agree nicely with the value, about 0.2 mg/mg, determined by Jost et al. (3) with electron spin resonance techniques and a nitroxide derivative of stearic acid as the spin label probe. However, no temperature dependence was observed in the amount of restricted lipid seen by these workers (P. C. Jost, R. A. Capaldi, and 0. H. Griffith, personal communication). The exact reasons for this difference are unclear at this time, although a number of explanations are possible. Clearly, the time scale for motional averaging in the electron spin resonance and DMR experiments are vastly different. It is possible that a given environment shows restricted motion on one time scale and not the other. In addition, the electron spin resonance technique used a dilute spin label probe with a natural mixture of mitochondrial lipids whereas the DMR experiment used a chemically homogeneous deuterated phosphatidylcholine both as the bulk lipid and as the probe. It is possible that some compensating effects of the partitioning of the electron spin resonance probe and natural lipid mixture occurs which obscures the temperature-dependent effects for a single lipid. The answer to this question must await further experimentation. The data presented here clearly demonstrate the utility of DMR in the study of lipid-protein interactions. The technique uses a relatively nonperturbing deuterated lipid to probe the motion of the lipid itself. The time scale of the experiment appears to be ideal for most biological systems and offers a dramatic sensitivity to environment as judged by its motional properties. The data obtained may be fairly easily quantitated. We anticipate considerable application of these approaches to the general question of lipid-protein interactions with emphasis on the possible lipid specificity apparently seen for certain membrane proteins. The work at The University of British Columbia was supported by the National Research Council of Canada and a special Killam-Canada Council Interdisciplinary Grant. The work at The University of Oregon
Biochemistry: Dahlquist et al. was supported by National Science Foundation Grant BMS 75-10422. F.W.D. is an A. P. Sloan Foundation Fellow, 1975-1977.
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