APPLICATION OF PROTON-ENHANCED NUCLEAR INDUCTION SPECTROSCOPY TO THE

STUDY OF MEMBRANES

Julio Urbina and J. S. Waugh

Department of Chemistry and Research Laboratory of Electronics Massachusetts Institute of Technology

Cambridge, Massachrtsetts 02139

There is growing interest in the study of the molecular organization of biological membranes in relation to the fundamental functions performed by these structures, such as passive, facilitated, and active transport of ions and nonelectrolytes, electron transport coupled to the production of high energy substances such as ATP, cell recognition and its relationship to malignant transformation and development, the relationship of membranes to protein synthesis, the membrane-mediated effects of hormones, anesthetics and anti- biotics, and so on. Characterization of the molecular architecture of mem- branes has been a difficult task because of the enormous complexity of the macromolecular aggregrate which constitutes a functional membrane and because the size and extreme insolubility of the membrane’s lipoprotein com- plexes render many traditional methods for studying macromolecules in solution useless. At the same time the impossibility of crystallizing membranes di- minishes the utility of otherwise powerful x-ray diffraction methods.

Magnetic resonance relaxation times (TI and T2), alteration and anisot- ropies of chemical shifts, and cross-relaxation effects can in principle be used in selected membrane systems to study molecular mobility, interactions between membrane components, phase transitions in the membrane structure, and also the interactions of small molecules (transport substrates and their analogs, hormones, and so on) with membranous structures. Attempts have been made to study lipid-protein interactions in membranes and model systems using proton 2 and I3C 3. * nmr, but conventional high-resolution nmr suffers from many of the above mentioned difficulties associated with the study of insoluble systems: the majority of unperturbed membranes and lipid multilayers (liposomes) have broad, overlapping absorption lines compounded by poor signal to noise ratios, making this class of spectra of limited usefulness. Better spectra are, in general, obtained only after prolonged ultrasonic irradiation (sonication) of an aqueous suspension. This treatment results in the creation of smaller particles whose shortened rotational correlation times lead to aver- aging of the residual line-broadening dipolar interactions.5 However, there are indications that the native structure is essentially altered by this Spin labeling by covalent attachment of a paramagnetic group provides infor- mation which is still useful without sonication. The esr spectrum of the spin label can be analyzed in terms of the polarity of the environment of the label and the rate and anisotropy of its movement in the membrane.s-lo The general result of these studies has been that the labels are localized in portions of the membrane whose properties resemble those of a lipid bilayer, largely unaffected by the protein component of the system. This is in agreement with other studies such as differential scanning calorimetry and x-ray

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134 Annals New York Academy of Sciences

diffraction,I3--'" which indicate the presence of a lipid bilayer in the membrane structure. Very similar results have been obtained using fluorescent labels.17 However, there is definite evidence that the simple lipid bilayer is not the only important structure of the membrane and that its role is largely passive: the protein content in biological membranes on a weight for weight basis is 100400% of the lipid content.Iq Studies in model lipid systems (liposomes and "black" films) indicate that specific properties resembling those of biological membranes appear only after the lipid bilayer is modified by interaction with proteins, antibiotics, and so on,19 and there is much biochemical evidence that some interaction with lipids is necessary for the function of many membrane- bound enzymes,2n. 21 specific transport proteins,'?, 2 R membrane-bound ATP- aSeS 2 1 - 3 ; and hormone receptors.". 2h The specificity of this requirement is high both for the hydrophobic portion (acyl chains) and the polar group of the lipid, indicating that there are specific interactions of these proteins with those parts of the lipid molecules and that modification of the simple lipid bilayer is essential to biological function. The physical details of these inter- actions are not known, and they are apparently not revealed (as indicated above) by studies of spin- and fluorescent labels. This may be because the presence of the bulky reporting group inhibits the highly specified lipid-protein interactions which are needed for biological function.

The characteristic advantage of nmr, that it need not require any chemical modification of the system studied, and the advantage of labeling techniques in permitting the study of specific molecular sites can be combined by using isotopic rather than chemical labels and nmr techniques affording enhanced sensitivity sufficient to detect such labels. There has already been one 13C nmr study of membranes using I3C-enriched lipids,29 but low signal to noise ratio combined with the relative immobility and nonspecificity with respect to lipid-protein interaction of the labeled position (the carbonyl group of the fatty acid esterified to the glyceryl moiety) were such that very little new information was obtained. We will be concerned here with the possibility of more general labeling experiments which exploit the technique of proton-enhanced nuclear induction spectroscopy 30. 31 to obtain adequate sensi- tivity together with strong double irradiation to remove residual dipolar broadening by the protons. The technique is fully described e l ~ e w h e r e , ~ ~ therefore no detailed description will be given here. It suffices to say that the large magnetic polarization acquired by the abundant protons is transferred to the much rarer I3C spins, whose nmr signal is thereby greatly increased. Complete transfer depends on the existence of at least some residue of static dipolar interaction among the protons and between the protons and the nuclei, a situation which is most characteristic of rigid solids. As will be shown shortly, however, membrane structures apparently retain enough vestiges of solid-like5 behavior to exhibit a substantial degree of transfer of the required type. To obtain high resolution, it is also necessary to decouple the residual 13C-proton dipolar interaction during observation: it is not always realized that this generally requires decoupling fields sufficient to remove the static dipolar interaction in a rigid solid, i.e., the fact that the interaction has already been largely removed through random molecular motion does not necessarily help.3' Therefore, decoupling fields very much larger than those one is accustomed to in removing scalar electron-coupled spin-spin interactions must be employed.

In this paper we describe a particular labeling experiment which is now in

Urbina & Waugh: Proton-Enhanced Spectroscopy 735

progress in our laboratory. It exploits the fact that cyclopronane-containing fatty acids present in many bacterial membrane lipids :{3 seem to share specific functional properties with unsaturated fatty acids in the membrane, supporting the growth of unsaturated fatty acid a u x o t r o p h ~ . ~ ~ Like the unsaturated fatty acids they are necessary for the expression of the activities of the /3-galac- tosidase-permease "? and membrane-bound enzymes.Is In stationary cultures all the unsaturated fatty acids are substituted by cyclopropane fatty acids,3x presumably to maintain fatty acid residues of the same physical properties in the membrane while avoiding the problem of oxidation of the double bonds under unfavorable conditions.

We have prepared I3C labeled cyclopropane fatty acids, identical to those found in E. coli membranes (9,10-methylenehexadecanoic acid-17:O- and 11, 1%-methylene octadecanoic acid-19:O) ,19! m by reacting enriched methylene iodide with the corresponding unsaturated acid by a well-known procedure.:<" The structures and labelings of the resulting compounds were verified by infrared and 13C nmr high resolution spectra and by elementary analysis. The labeled fatty acids support the growth of an unsaturated fatty acid auxotroph (the 30-E derivative of E. coli K12)3G and allow the expression of the specific lipid-requiring /3-galactoside permease.

We intend to study the spin-lattice relaxation of the l3C in the labeled cell membranes, using an adaptation of the inversion-recovery method 3i to proton-enhanced nuclear induction spectroscopy. The physical states of the lipid molecules in interaction with membrane proteins and in the bilayer portions, and also the rate of exchange of lipid molecules between these states,22 can perhaps be studied from an analysis of the relaxation process for the nuclei in the membrane lipids and for lipids dispersed in excess water, which are known to exist in the bilayer configuration.38 The temperature dependence of the spin-lattice relaxation in these systems may illuminate the physical basis of the break in slope of the P-galactoside permease activity vs. temperature 22 as well as of other activities associated with the membrane."? This break depends on the physical properties of the fatty acids present in the membrane lipids, occurring as lower temperatures for higher degrees of unsaturation of the hydrocarbon chains or for chains of shorter length, paralleling the temperature dependence of the gel-liquid crystal transition of 1 i ~ i d s . l ~ This phenomenon seems to be related to a phase transition of the membrane lipids which has been observed by differential scanning calori- metry l 1 - l a and x-ray diffraction.1'-'" However, the physical details of how such a phase transition affects specific membrane activities 19, 26. 39 are not understood. The temperature dependence of T, for the 13C labels may give an indication of the kind of reorganization occurring at the transition point. We also intend to investigate the interaction between lipid and the p-galac- toside permease protein by labeling the latter with a maleimide spin label-" by essentially the same method used by Fox and Kennedy'? to identify the protein using radioactive markers. The effect of the unpaired electron on the 13C relaxation rates of the lipid may give information on the spatial organiza- tion of the lipid-protein complex and how this organization changes at the transition point of permease activity.

The foregoing remarks must be regarded largely as speculation and perhaps as wishful thinking, since the experiments outlined have not yet been performed at the time of this writing. It is, however, already reasonably clear that the 13C measurements envisioned will themselves be possible. For ex-

736 Annals New York Academy of Sciences

ample, we have performed pilot experiments, designed to test the major features of the technique, on unlabeled model lipid systems (liposomes) . The presence of incompletely averaged dipolar interactions in these systems," together with proton relaxation times at physiological temperatures of the order of hundreds of msec for both TIP4:! and T,,'.." create favorable conditions for enhance- ment of the 13C resonance. T,,, is sufficiently long to allow several proton- carbon cross-relaxation contacts, and T, is sufficiently short to allow the whole experiment to be repeated frequently, so that reasonable signal to noise ratios can be obtained in a short time. FIGURE 1 shows an enhanced spectrum (natural abundance) from a 200 mg sample of a 50% mixture of dimyristoyl- lecithin with 0.1 M NaCI, without sonication or the addition of surfactants.

I 1 I

-50 0 -2oa - 150 -too

FIGURE 1. Proton-enhanced I:% spectrum of dimyristoyllecithin, 50% in 0.1 M NaCl without sonication. These spectra could be improved in signal to noise ratio by averaging longer than the ca. 10 min runs used here ppm from external T M S .

This spectrum was obtained in ca. 10 min, using a proton-carbon cross- polarization time of ca. 10 min, using a proton-carbon cross-polarization time of ca. 15 msec and a repetition rate of 1 sec-I. Multiline spectra are observed both below and above the gel-liquid crystal transition of this lipid, which occurs at 22" C.'O Conventional nmr :'- does not give resolved spectra under these conditions. Our spectrum below the phase transition shows main peaks which correspond approximately with the principal absorption lines of the lipid sonicated in water or dissolved in organic solvents.' The line widths are independent of proton decoupling power above a few gauss, and are apparently not associated with residual dipolar interactions; nor are they associated with magnetic field inhornogeneity. They may originate in incompletely averaged chemical shift anisotropy. Above the transition point the absorption originating

Urbina & Waugh : Proton-Enhanced Spectroscopy 737

in the choline head group disappears. It is known from and lH relaxation studies of sonicated vesicles that this group undergoes a pronounced change in mobility at the transition. Since in our method the l3C obtains its mag- netization from the protons through a cross-relaxation process which depends upon the existence of a static component of the dipole-dipole interaction, it is possible that what we are observing is a breakdown of this cross-polarization above the transition. The same reasoning can explain the relatively low intensity of the resonance arising from the highly mobile hydrocarbon methyl groups both above and below the transition.

The interesting aspect of these spectra, apart from the fact that they indicate resolution and sensitivity adequate to the contemplated isotopic labeling studies, is that we are in a position to study unsonicated samples. This removes one source of uncertainty in the interpretation of nmr studies of membranes and of the interaction of lipids with proteins, antibiotics and other molecules.

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DISCUSSION

DR. POE: It has been suggested to me as a possiblc means for reducing dipolar coupling in model membranes that a very highly substituted, very highly purified model membrane be used, consisting of strictly isotopically substitued components in which, for example, all the hydrogen replaced the deuterium. Do you think that such a membrane would have sufficiently reduced dipolar interaction that the kind of spectra that you have described could be observed?

DR. WAUCH: I suspect that the problem is probably not that replacement of hydrogen with deuterium certainly will decrease the proton broadening of the carbon, but that it will leave some deuterium broadening. Of course there would also be some carbon-carbon interactions of one kind or another, which will also cause some difficulty. Besides, it sounds like a difficult thing to accomplish, just from the synthetic point of view, and results might be achieved more easily in another way. I suspect that it is easier to do specific labeling studies by the biosynthetic introduction of some isotopic label.

Urbina & Waugh: Proton-Enhanced Spectroscopy 739

Will bacteria grow when they are fully deuterated? DR. POE: Bacteria will grow at 100% D20 if they are adapted. DR. WAUGH: I think artificial membranes are certainly interesting as

prototype systems, but there is always the question in model membranes of whether you are really studying the interesting features, such as the ones associated with active and facilitated transport, which really require some intimate lipoprotein association that we don’t usually have in the model membrane.

DR. STERNLICHT: Would you comment on the temperature range at which your techniques are applicable?

DR. WAUGH: The technique is most applicable to rigid solids. A mem- brane is something between a solid and a liquid. When I say the technique is really adapted to solids, I mean that the maximum gain in sensitivity is obtained in rigid solids. That gain for I3C in natural abundance in an ordinary organic system is about a factor per thousand in power signal to noise over ordinary carbon Fourier transform spectroscopy. For larger abundance ratios the gain is correspondingly larger. That assumes that you have a rigid system in which the T,’s are long enough. That is not always so. In membrane it’s not the case; you are in an intermediate situation. The membrane we’ve looked at have T,’s and rotating frame T,’s of a few hundred milliseconds, which permits us to make several contacts between the protons and the carbons, but not the theoretically maximum number. So we get enough of a degree of enhancement to render the technique useful.

However, specifically in the case of membranes, it also appears possible that we’ll be able to learn more by doing the experiments at low temperatures. It is known that you can quench membranes to low temperatures either from above or below the gel liquid crystal transition, and that is a reversible process. You can recover the function by warming them up again. Therefore one can probably obtain structural information about the membrane either in the above-transition or below-transition region, simply by quenching it to liquid nitrogen temperature, thereby obtaining a much greater enhancement and sensitivity.

DR. FISK (Georgetown University, Washington, D.C.) : What are the modifications in the conventional pulse spectrometer necessary to do your experiments?

DR. WAUGH: The modification of a conventional spectrometer would involve increasing the decoupling power that is normally available to a few hundred watts. Although making power is easy and cheap, dissipating that power in the probe without melting anything is not so easy, especially with probes of current commercial spectrometers, which were not designed with this kind of thing in mind. At present it is necessary to make your own probe, which is not very difficult. All you need is a lot more decoupling power and a probe that will take it without blocking the receiver for the rare spins at the same time. You also need just a little bit of timing, a slightly different pulse sequence from the usual ones in order to turn these fields on and off at the appropriate times.