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Phospholipid headgroup dynamics in DOPG-d5-cytochrome c complexes as revealed by 2H and 31P NMR: The effects of a peripheral protein on collective lipid fluctuations
SOLID STATE Nuclear Magnetic Resonance
ELSEVIER
Solid State Nuclear Magnetic Resonance
8 (1997) 55-64
Phospholipid headgroup dynamics in DOPG-d,-cytochrome c complexes as revealed by *H and 31P NMR: The effects of a peripheral protein on collective lipid fluctuations Teresa J.T. Pinheiro a,*,Melinda J. Duer b, Anthony Watts a,* a Depurtment ofBiochemistry, Uniuersity ofOxford, South Purks Road, Oxford, OXI 3QU. UK b Depnrrmenf ofChemistry, University ofCambridge, Lensfield Road, Cambridge, CB2 IEW, UK Received 4 March 1996; accepted 4 June 1996
Abstract The dynamics of the glycerol headgroup of dioleoylphosphatidylglycerol (DOPG) in hydrated bilayers were studied by and the effects of binding a peripheral protein, cytochrome c, were evaluated. The fast headgroup segmental motions (TV, 10-‘“-lO- I3 s> of DOPG in fully hydrated bilayers were not affected upon binding of ‘H and 3’P NMR spectroscopy,
cytochrome c, as evaluated by the spin-lattice CT,) relaxation of deuterons in the DOPG glycerol headgroup. In contrast, the spin-spin (T&) relaxation is strongly affected, indicating that slow cooperative bilayer motions (TV,, 10-3-10-6 s) are enhanced upon the interaction with cytochrome c. *H and 3’P NMR spectral lineshape analysis reveal details of the nature of these motions. The importance of these effects are discussed in terms of a possible mechanism for modulating membrane-associated processes. 0 1997 Elsevier Science B.V. All rights reserved. Keywords: ’ H Spin-lattice relaxation; *H Spin-spin relaxation; Collective lipid motions; Membranes
1. Introduction Diacylphosphatidylglycerol (PG) is a major phospholipid found in higher plants, algae, and bacteria [l-3]. In mammalian systems PG occurs in minor amounts only, notably in lung surfactants [4] and in
Abbreviations: DMPC, dimyristoylphosphatidylcholine; DMPG, dimyristoylphosphatidylglycerol; DOPG, dioleoylphosphatidylglycerol; EFG, electric field gradient; MAS, magic angle spinning; T,, spin-lattice relaxation time; T2e. spin-spin relaxation time * Corresponding author. Tel.: + 44 1865 275 268; fax: +44 1865 275 234; e-mail: [email protected] ’ Present address: Department of Biological Sciences, Unviversity of Warwick, Coventry CV4 7AL, United Kingdom.
mitochondrial membranes [5], where it can function as a precursor of cardiolipin 161. Because of its negatively charged headgroup ( pK, = 3-5.5, depending on the ionic strength [2,7]) PG is a good candidate for the study of the interaction of membrane with ions [8], and peptides or proteins [9- 111. The dynamics of lipids in a bilayer structure involve a variety of motional modes. Diffusive intramolecular fast motions, such as bond vibration, bond rotation, and rruns-gauche isomerisation, occur at rates faster than 1 X 10’ s- ’ [ 12,131. In addition, collective motions, involving more than one lipid molecule, have been shown to exist in lipid membranes [ 143. These motions, also referred to as order director fluctuations, occur at lower frequencies as
0926-2040/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO926-2040(96)01255-6
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T.J.T. Pinheiro et al. / Solid State Nuclear Magnetic Resonance 8 (1997) 55-64
low as 103-lo6 Hz, depending on the number of molecules involved [15], and were thought to be confined to the lipid acyl chains. The headgroup and C,-C 3 segment in the acyl chains were found to be uncoupled from these slow motions, serving as an anchor around which those fluctuations occur [16]. An increase in the density of slow cooperative motions has been postulated upon the binding of proteins to a lipid bilayer [17-201. In particular, the effects of cytochrome c in cardiolipin bilayers has been shown to induce the occurrence or propagation of low frequency motions in the glycerol headgroup region of cardiolipin bilayers [21]. It was postulated that the glycerol headgroup in cardiolipin was particularly sensitive to the slow cooperative motions due to its “rigid” structure (sterified from both ends to two diacylphosphoglycerol moieties), enabling its motion to become strongly coupled to those within the chain segments. In the present study it is shown that the glycerol headgroup in DOPG bilayers can also participate in the slow cooperative fluctuations in the lipid bilayer. The effects on the higher frequency molecular motions in the phospholipid headgroup on binding of cytochrome c were analyzed through the T, relaxation of deuterons in the glycerol headgroup of DOPG-d,, while the effects on the lower frequency motions were investigated using deuterium Tze relaxation measurements. It is found that on binding of cytochrome c to DOPG bilayers the slow collective lipid motions are propagated into the lipid headgroup region. 3’P and H NMR spectral lineshape analysis combine to lend weight to these ideas, and give further details of the nature of the motion.
2. Experimental 2. I. Lipid synthesis Headgroup deuterated 1,2-dioleoyl-sn-glycero-3phospho-rut-glycerol at the (Y-,p- and y-methylenes in the glycerol headgroup segment (DOPG-d,) (Fig. 1) was synthesised as described elsewhere [9]. Briefly, the sodium salt of DOPG-d, was prepared by the method of Harlos and Eibl [22] using perdeuterated rat-isopropylidenoglycerol in the esterification step. This later compound was synthe-
Fig. 1. Schematic representation of the chemical structure of dioleoylphosphatidyl-glycerol (DOPG-d,) showing the deuterated positions at the W, p-, and y-methylenes in the glycerol headgroup.
sised by dissolving perdeuterated glycerol in acetone and boiling under reflux for 6 h in the presence of catalytic amounts of p-toluenesulfonic acid. The perdeuterated glycerol was produced by exchange of protonated glycerol against D,O with Raney nickel as a catalyst. Lipid purification was performed by silica acid column chromatography as described previously [7]. Cytochrome c from horse heart, type VI, Sigma Chemical Co., St. Louis, was purified by ion exchange chromatography on Whatman CM-32, and eluted with 65 mM phosphate buffer, pH 7.0 [23]. The eluent containing the purified protein was concentrated by ultrafiltration using Amicon YM-5 ultrafiltration membranes, followed by extensive dialysis against cold distilled water (4°C) to remove phosphate. Cytochrome c was quantified spectrophotometrically using a molar absorptivity of 2.95 X lo4 at pH 7.0 for the protein reduced with sodium dithionite [24]. 2.2. Membrane preparation Multilamellar liposomes of headgroup deuterated DOPG-d, were formed by handshaking hydration with excess 20 mM cacodylate buffer at pH 6.0,
TJ.T. Pinheiro et al./Sdid
Stute Nuclear Mugnetic Resonmce
containing 0.1 M NaCl and 5 mM EDTA. Typically, a film of 80-100 mg of lipid was prepared under rotatory evaporation, left for a minimum of 8 h under high vacuum to remove all traces of organic solvent, and hydrated with 200-300 ml buffer. Lipid-cytochrome c complexes were formed by addition of increasing amounts of protein in buffer solution to the pre-hydrated phospholipid bilayers, and followed with three cycles of freeze-thawing. Lipid-protein complexes were separated by ultracentrifugation (75 000 X g; 1 h; 4°C). The clear supematant was removed for protein analysis. Bound cytochrome c in the lipid-protein complexes was calculated from the free protein remaining in the supematant, as determined spectrophotometrically [24]. Lipid hydration and the preparation of lipid-protein complexes were carried under nitrogen atmosphere, using predeoxygenated buffer to prevent oxidation of the unsaturated acyl chains within the lipid.
2.4. Spectral lineshape analysis
57
coupling constants used in the simulations were 13.11, 11.67, 14.11, and 12.55 kHz for the four different (Y sites; 4.875 kHz for the B site; and 750 and 1250 Hz for the two y sites. The rate of hopping between sites is given with each simulation. Two hundred FID points were calculated, and 9800 molecular orientations were used in producing the powder average. For the simulations of the phosphorus-31 NMR spectra, the chemical shift anisotropy used was 5.25 kHz; 3200 molecular orientations and 200 FID points were considered for each calculation. Many different motional models were investigated. The molecular motion is defined by hopping at a given rate between N discrete sites with a given probability of occupation. Each site is defined by the relative orientation of the *H quadrupole coupling tensor or 3’P chemical shift tensor in that site, and a T2 value. The (4
2.3. NMR spectroscopy All NMR experiments were performed on a Bruker MSL 400 spectrometer operating at 61.42 MHz for deuterium and 161.98 MHz for phosphorus-3 1. Deuterium spectra were recorded using a quadrupolar echo pulse sequence, n/2.V-r-n/2y-rr-acquisitionD, [25], with quadrature phase cycling, n/2 pulse widths of 4 ps, recycle delay time CD,), 200 ms, and between 25000 to 50000 scans were accumulated. Transverse ‘H-spin relaxation was measured with the same echo sequence by varying the interpulse delays CT), from where a time constant for the exponential decay in the echo intensity referred as Tze was evaluated. Deuterium spin-lattice (T,) relaxation times were measured by the inversion-recovery technique with quadrupolar echo pulse sequence, n,-n/2.,-r-T,-r’-acquisition-DO, in which the recycle time, D,, was at least 5 X T,. Phosphorus-31 NMR spectra were recorded using the Hahn echo pulse sequence, n/2,,-T-7r,-T-acquisition-DO, with 7r/2 pulse widths of 5 pus, and proton decoupling during acquisition; Da, 4 s; number of scans, 80-264.
8 (1997) 55-64
2
1 O 1'
2
3
3
w 2 1 3 (W
Fig.
2. (a) Schematic
representation
of the molecular
motion
described in motional model (1). The motion assumed occurs by hops between neighbouring sites labelled 0, I, 2, 3 and I’, 2’, 3’. In fact I5 sites are used in the calculations, arranged in similar manner to the 7 shown here. The different sites represent different possible orientations of the molecule. The populations of the sites are varied as described in the text. In this model, the molecule changes orientation within a plane. The diagram
indicates the
direction of the unique axis of a nuclear interaction tensor in each site involved in the motion. There is a constant angular displacement between
each site. (b)
Schematic
representation
of the
molecular motion described in motional model (2). The motion is considered to occur in a similar manner to that in figure 2(a), except now the molecular orientation can change in three dimensions. The hopping is between 4 sites (labelled
I to 4)
and can
occur between any pair of sites. The populations of the sites are
Spectral FORTRAN
simulations were carried out using a program, CARLA [26]. The quadrupole
equal. In both models, the hopping frequency is the same for all hops.
58
TJ.T. Pinheiro et ul./Solid
State Nuclear Mqnetic
motional models considered may be summarised as follows. (1) Small angle diffusion in one plane. Motion is defined by 15 sites. The unique axis of the nuclear interaction tensor in the sites are shown in Fig. 2a. The overall amplitude of the motion, i.e. angle i -+ i’ was varied between 15 and 60”. Gaussian distribution of the various widths were considered for the probability of occupation of the sites. Hopping is between neighbouring sites only. (2) Large angle hopping within a cone. Four sites are defined in which the unique axes of the interaction tensor are equally angularly spaced around the surface of a cone, as illustrated in Fig. 2b. The cone angle used was varied between 80 and 140”. Hopping can occur between any pair of sites at an equal rate. There is equal probability of occupation of the sites. Between them, these two motional models described above represent the extremes of the likely motion of the order director axis. Other motions are possible but represent situations of intermediate between these limiting cases. These motions thus suffice for initial analysis; refinements can be made at a later stage once the broad nature of the motion has been determined. A value of T2 of 8 ms was used in all simulations.
3. Results and discussion 3.1. The ‘H NMR spectrum of DOPG-d,
in hydrated
bilayers
*H NMR spectra recorded from headgroup deuterated lipids in randomly oriented bilayers are spherically averaged powder patterns dominated by an axially symmetric electric field gradient (EFG) tensor. Molecules whose 2H EFG tensors have their unique axis oriented at 90” relative to the applied magnetic field originate intense resonance lines in the powder pattern spectrum [27]. The separation between these maxima provides a direct measure of the quadrupole splitting (Av,). The 2H NMR spectrum of DOPG-d, in hydrated bilayers at 25°C is presented in Fig. 3, where the assignment for the various quadrupole splittings is shown [28]. The outer most splittings where four resolved components of 10.5, 11.3, 11.8, and 12.7 kHz can be identified, are attributed to the (r-CD,
Resonance 8 (1997) 55-64 y-CD,OH
P-CDOH
1
1
5000
I
0 HERTZ
I
I
-5000
Fig. 3. Assignment of the various quadrupole splittings for the deuterium NMR spectrum of DOPG-d, in hydrated bilayers (see text for complete description).
deuterons. This is in agreement with the 2H NMR spectrum reported previously for bilayers of dimyristoylphosphatidylglycerol (DMPG-d,) [9], where the four resolved quadrupole splittings have been ascribed to possible magnetic inequivalence of the two deuterons in the racemic mixture of both D and L diasteromers of the glycerol headgroup B-carbon. A single quadrupole splitting of 4.4 kHz was observed for the deuteron in the B-methylene group, as reported by others for DMPG-d, in hydrated bilayers [9,10,28]. The smallest quadrupole splitting of 440 Hz is assigned to the deuterons of the y-CD, segment (Fig. 1). Two quadrupole splittings have been reported for the y-deuterons in the glycerol headgroup of DMPG-d, in hydrated bilayers [28]. A second quadrupole splitting, although less resolved, can also be identified for DOPG-d, bilayers (see arrows in Fig. 3). Indeed none of the *H NMR lineshapes can be successfully simulated without inclusion of inequivalent y sites, as discussed later in this section. These two quadrupole splittings have been attributed to motional inequivalence of the two deuterons in this segment [lo], as a result of restricted internal trans-gauche isomerisation, due to
TJ.T. Pinheiro et al./Solid
State Nuclear Mugnrtic Resonance 8 (1997) 55-64
an extensive hydrogen bonding network at the bilayer surface [29]. The quadrupole splitting for the a-CD, deuterons in the glycerol headgroup of DOPG-d, in hydrated bilayers is reduced over tenfold compared with that of a static methylene bond (= 170 kHz). This is a result of motional averaging due to the long axis rotation of the entire lipid molecule in the bilayer structure and additional fast segmental motions within the headgroup moiety. Furthermore, Av, for the P-CD group is about three times smaller than that for the (Y-CD,, and y-A ho is about ten times smaller than p-A yo. The decrease in quadrupole splitting between the CY-and y-methylenes could simply be associated with an increase in the motional freedom towards the terminal headgroup segments. Altematively, the difference could reflect changes in the C-D bond orientation relative to the long axis of rotation. The C-D vector generally describes the unique axis of the ‘H quadrupole tensor for alkyl 2H. Rapid rotation of this tensor axis about an axis oriented at an angle 13 to it, reduces the quadrupole coupling constant x (and hence A v,) to an effective value, xeff given by (x,rr/x) = #2 cos*8 - 111. Thus, the different quadrupole splittings for the IXCD,, P-CD, and y-CD, may reflect the different orientation of these C-D bonds relative to the long rotation axis. Given the rigidity of the headgroup of DOPG, this latter explanation is plausible, and if correct, would allow the long axis of rotation to be located. Deuterium spin-lattice CT,) relaxation measurements are particularly useful for determining the cause of the Av~ variation, since deuterium relaxation rates (l/T,) are dominated essentially by the rate of reorientation of the segment involved [30,31]. While the deuterium T, relaxation gives information about the fast local segmental motions (7, z lo- lo s) affecting the headgroup deuterated moiety in phospholipid bilayers, the spin-spin (T,,) relaxation times are determined, principally, by the slow motions of the deuterated segment CT,, 103-lo8 Hz). The T, relaxation times for the deuterons at the (Y-, p-, and y-positions in the glycerol headgroup of DOPG-d, in protein-free bilayers have all very similar values, T, (IxCD,) = 11.8 ms; T, (P-CD) = 10.5 ms; and T, (y-CD,) = 13.4 ms, in good agreement with reported values for the headgroup deuterated segments
59
of the saturated analogue DMPG-d, in hydrated bilayers [9]. This suggests that all these deuterons suffer the same motions. It therefore seems likely that the headgroup itself is relatively “rigid”, i.e. no reorientation of groups within the headgroup, and that the motion is confined to that about the long molecular axis. This could further suggest that differences in Av, for the headgroup deuterons arise from the different orientation of the C-D bonds relative to this long molecular axis as discussed previously. In contrast, T,, relaxation times increase appreciably from (Y- to y-methylene, T,, (a-CD*) z 500 /_Ls; Tze (P-CD) = 710 /.Ls; and T,, (y-CD,) % 1050 /_Ls. 3.2. The effects of cytochrome
c on DOPG-d,
head-
group “P NMR. Phosphorus-31 NMR of DOPG-d, in hydrated bilayers alone show the typical powder pattern spectrum for phospholipids in liquid-crystalline bilayers [32], with a lineshape dominated by an axially symmetric chemical shift tensor (bottom spectrum Fig. 4a). This spectrum results, principally, from the averaging of the phosphorus chemical shielding tensor by the rotation of the whole phospholipid molecule around its long axis, i.e., perpendicular to the bilayer surface [33]. The separation between the edges of a 3’ P NMR spectrum (v,, - v I > defines the residual chemical shift anisotropy bilayers of (A VCSA1, which for the protein-free DOPG-d, is about 40 ppm. On binding of cytochrome c notable spectral changes occur, including the appearance of a central spectral component in the 3’ P NMR spectra at around the isotropic position, and collapse of the low intensity end of the spectrum (Fig. 4a). The additional isotropic-like component increases with increasing amounts of bound cytochrome c, while the low-field intensities are progressively reduced. Narrow components have been observed in static 3’P NMR spectra of cardiolipin-cytochrome c complexes [34-361, and with other anionic phospholipid-cytochrome c complexes [37]. Additionally, in MAS “P NMR experiments these isotropic-like components are shifted from the isotropic central bands of mixed phospholipid bilayers [20,38]. The interpretation of such isotropic components is still unclear. In some stud-
TJ.T. Pinheiro et al./ Solid State Nuclear Magnetic Resonunce 8 (1997) 55-64
60
(4
(W
I
I
I
I
I
I
-15
-10
-5
0 kHz
5
10
I 15
Fig. 4. (a) 161.98 MHz 3’P NMR spectra of DOPG-d, in hydrated bilayers alone (lower spectrum), and with increasing amounts of bound cytochrome c. Lipid/protein ratio in molar stoichiometry; temperature. 25T. (b) Calculated spectral lineshapes using the model (2) described in Section 2. Hopping frequencies between the sites for each simulation are (top to bottom) 6.4, 5.0, 3.8 and 0.6 kHz, respectively.
ies, they have been attributed to vesicular or micellar structures on or within the bilayers, possibly associated with precursors of hexagonal-H,, phases [34,39].
Others have attributed them to the presence of smaller diameter (I 500 nm) vesicles induced upon protein binding, or as a result of motional or orientational averaging without involvement of non-bilayer structures [21,36-381. Alternatively, the lineshapes may arise from the 3’ P species which undergoes molecular motion so as to produce a feature (amongst others) at the isotro ic R chemical shift. It is to be noted that none of the P NMR spectra of protein containing samples can be successfully simulated by simply adding a Lorenzian line (of varying width) at the isotropic chemical shift of the protein-free powder pattern. In addition to the appearance of the feature at the isotropic chemical shift, the low intensity end of the powder pattern (v,,) collapses and the high intensity end (v I> broadens. Many of the features of the 3’P NMR spectra can be reproduced with the same motional model as was used to simulate the 2H NMR lineshapes (see below), albeit with slightly different hopping frequencies for a given lipid-protein ratio (Fig. 4b). Clearly, there is plenty of scope for refinement of the model, by including more sites, and varying their angular disposition. The simulations presented here are however sufficient to demonstrate that the 3’P NMR lineshapes are likely to arise from fluctuations of the whole lipid molecule at the rates of l-10 kHz. 2H NMR. Deuterium NMR spectra of DOPG-d, bilayers containing increasing amounts of bound cytochrome c are shown in Fig. 5a. There is an overall change in the 2H NMR spectral lineshape upon binding of cytochrome c. The resolution of the ‘H NMR spectra seems to decrease as cytochrome c is added. However, the 2H NMR spectra for high concentrations of protein cannot be simulated by simply broadening that for the protein-free sample. Clearly, there is some change in the motional process affecting the DOPG molecules upon the addition of cytochrome c. We have performed extensive spectral lineshape calculations for many different motional models. Calculated spectra are shown in Fig. 5b for increasing values of hopping frequency which most closely follows the experimental lineshapes changes induced by increasing the concentration of cytochrome c in the lipid-protein complexes. The motional model used to produce these simulations is the model (2) described in the Section 2, i.e., large amplitude
TJ.T. Pinheiro et al./ Solid State Nuclear Magnetic Resonance 8 (1997) 55-64
hopping motions of the *H nuclei within a cone. In this case the cone angle is 140” for all the *H nuclei in the headgroup (a, p, and -y>.It is to be noted that no other motional model gave acceptable fits to the experiment. This model may be interpreted as a large angle fluctuation of the long molecular axis, with the head groups held essentially rigid during the motion, i.e., all ‘H nuclei suffer the same motion. Clearly. this motion may be part of the “rippling ” of the phospholipid bilayer. The variation of deuterium spin-lattice CT,) and spin-spin (T,,) relaxation times for headgroup deuterated DOPG-d5 bilayers as a function of the concentration of bound cytochrome c is presented in Fig. 6. The T, relaxation times are essentially not affected on binding of cytochrome c for any of the deuterated segments, suggesting that rotation about the long molecular axis is little affected. However, T2e relaxation times decrease for all of the segments upon binding of cytochrome c. Furthermore, this reduction increases progressively from the outer most headgroup segment (y-CD,) to the inner most methylene group (OL-CD*). T2e relaxation times are reduced of about 50, 60, and 70% for y-CD,, p-CD and o-CD,, respectively, over the range of protein to lipid concentrations studied here. Similar effects have been observed on the interaction of bacteriophage Ml 3 coat protein with DMPC bilayers [ 17,191, where essentially no effect on the T, relaxation of deuterons in the lipid headgroup or in the acyl chains was observed, but profound effects were encountered in the T,, relaxation. Also the interaction of the pulmonary surfactant protein SP-C with deuterated DMPC bilayers was found not to affect significantly the T, relaxation and to affect the Tze relaxation, suggesting that the protein only influences the slow lipid motions in the bilayer [ 181. The spin-lattice relaxation processes in *H NMR are only sensitive to spectral densities, J(w), of the
I
10
5
0
-5
-10
kHz
(b)
I -15
I -10
61
I
I
I
I
I
-5
0 kHz
5
10
15
Fig. 5. (a) Experimental 61.42 MHz 2 H NMR spectra of LIOPG-cl, in hydrated bilayers alone (lower spectrum) and with increasing amounts of bound cytochrome c. Lipid/protein ratio in molar stoichiometry; temperature 25°C. (b) Calculated NMR spectral lineshapes using the model (2) described in Section 2. Hopping frequencies between the sites for the simulations are (top to bottom) 3.3, 2.5, 1.6, 0.8 and 0.4 kHz, respectively.
TJ.T. Pinheiro et al./ Solid State Nuclear Magnetic Resonance 8 (1997) 55-64
62
01 0.000
0.025
0.050
0.075
0100
PROTEtN/LIPlDRATIO Fig. 6. Deuterium relaxation behaviour of the o-, p-, and -y-deuterated segments in the glycerol headgroup of DGPG-d, in hydrated bilayers as a function of bound cytochrome c. (a) Spin-lattice CT,) and (b) spin-spin (T& relaxation times. o-CD,, triangles; P-CD, filled circles; and y-CD,, open circles. Protein/lipid ratio in molar stoichiometry; temperature 25°C. The lines arc for guidance only and have no theoretical significance.
quadrupolar interactions at w = w0 and where f0 = wa/2~r is the Larmor freo= 2wa, quency, while the spin-spin relaxation processes are also affected by J(O) [40]. Investigations by Stohrer et al. [41] have shown that colkctiue lipid motions constitute a dominant mechanism for the *H spinspin relaxation of deuterated phospholipids in bilayers. These motions may be visualised as cooperative fluctuations of the lipid acyl chains in a direction perpendicular to the bilayer normal, and are often referred to as order director jlucruations. Watnick and co-workers [ 161 have analyzed the spin-spin relaxation of deuterons placed at various locations within dimyristoylphosphatidylcholine bilayers, and have concluded that the low-frequency collective motions were confined to the hydrocarbon interior of the bilayer. The headgroup and the CT-C, bond of the acyl chains would serve as an anchor about which those fluctuations can propagate. These observations have suggested that lipid headgroups can be fluctuating
uncoupled from the cooperative acyl chains slow motions, possibly due to restrictions in the molecular packing in the bilayer arrangement imposed by the chemical structure of the headgroup and headgroup-headgroup interactions. However, the reduction in T2e of the deuterons in DOPG-d, headgroup when in complexes with cytochrome c observed in the present study, are indicative of an increase of slow cooperative motions in the lipid bilayer which involve the headgroup. This is supported by a recent *H NMR study by Spooner et al. [21] on cardiolipin-cytochrome c complexes, in which the protons of the sn-1 position in the headgroup glycerol segment of cardiolipin have been replaced by deuterons. Here no effects were observed on the spin-lattice relaxation, while the spin-spin relaxation was strongly affected. An identical decrease m Tze of about 70% is observed for both the sn-1 deuterons in the cardiolipin headgroup and the cx-deuterons in the headgroup of DOPG-d,, over the same protein concentration range. DOPG and cardiolipin have in common a glycerol moiety for headgroup. While in cardiolipin this glycerol is bridging two diacylphosphoglycerol moieties, in DOPG the glycerol headgroup is only esterified from one end (the cl-methylene) to the diacylglycerophosphate moiety (Fig. 11, leaving the other segments (p and r> with a larger motional freedom when compared with that of the glyceryl headgroup in cardiolipin. However, the T, values for the three segments are all very similar (see above), which suggests no major differences in the fast headgroup motions. Assuming that the fast motions constitute the dominant mechanism for the deuterium T, relaxation, an average correlation time CT,) for the fast segmental motions within the DOPG glyceryl headgroup can be calculated [42], being 2.0 X lo-” s. For the sn-1 CD, deuterons in the cardiolipin glyceryl headgroup TV is of the same order of magnitude, 2.9 X lo-” s, revealing identical rates of fast segmental motions within the glycerol headgroup for both lipids despite of their distinct chemical structures. It was thought [21] that the glycerol headgroup in cardiolipin bilayers, due to its more “rigid” structure, would be specially sensitive to the lowfrequency collective motions within the bilayer. However, the present study indicates that the glyc-
TJ.T. Pinheiro et al. /Solid
State Nuclear Mugnetic Resonance 8 (1997) 55-64
erol headgroup moiety in DOPG is also sensitive to the collective lipid motions in membranes. This may arise from the fact that the headgroup glycerol in this lipid is quite well “anchored” at the bilayer surface through an extended network of hydrogen bonding [29,43-451. The increase of order director fluctuations in lipid bilayers seems to be a common feature upon binding of peptides, such as gramicidin [46], and melittin [47], or peripheral proteins [21,48,49]. These results suggest that the binding of peripheral proteins, such as cytochrome c, to a membrane surface can establish the coupling between the lipid acyl chains and the headgroup segments, therefore stimulating or enhancing the cooperative bilayer lipid motions. Those effects may be of genera1 importance in biological membranes, whereby through the coupling of cooperative low-frequency fluctuations, protein active conformations can be enhanced or inhibited, and can therefore serve as a modulating mechanism for membrane-associated processes.
63
cooperative bilayer lipid motions by establishing the coupling between the lipid acyl chains and the headgroup segments. It is very plausible that the proteins themselves are also involved in these cooperative slow motions, and these can constitute a general mechanisms for modulating protein activity at the membrane interface. Through the coupling of cooperative low-frequency fluctuations, protein active conformations can be enhanced or inhibited, and therefore this could serve as a modulating mechanism for membrane-associated processes.
Acknowledgements This work was supported by the EC Grant B/89 000 154/893 and Programa CiEncia JNICT, Portugal, Grant BD/1793/91-ID (to T.J.T.P), SERC (Grants GR/H/51552), and an MRC studentship to T.J.T.P. (fees only). We thank Dr P.J.R. Spooner for useful discussions.
4. Conclusions We have shown that the slow collective molecular motions occurring in lipid membranes are strongly influenced by the binding of a peripheral protein, cytochrome c, at the membrane interface. On protein-free lipid bilayers these slow lipid collective motions appear to be confined to the acyl chains in the hydrophobic core of the membrane, leaving the headgroup moiety on the membrane surface uncoupled from such motions. In the current paper we have observed a strong reduction in the spin-spin relaxation times CT,,) of lipid headgroup deuterons upon the interaction with cytochrome c. The reduction of T,, reveals an enhancement of slow motions (r,, 10-3-10-6 s) at the membrane interface. These motions are mostly associated with the collective molecular fluctuations, which are known to exists in membrane systems and also referred to as “bilayer Increase in low-frequency collective undulations”. motions in the lipid bilayers and propagation to the lipid headgroup region seems to be a general effect induced by binding peripheral proteins to lipid membranes, as described in the Section 3. This observations led us to suggest that peripheral proteins at a membrane interface can stimulate or enhance the
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T.J.T. Pinheiro er al./ Solid State Nuclear Magnetic Resonance 8 (1997) 55-64
[15] R.L. Vold, R.R. Void and M. Warner, J. Chem. Sot. Faraday Trans. II, 84 (1988) 997. [16] PI. Watnick, P. Dea and S.I. Chan, Proc. Natl. Acad. Sci. USA, 87 (1990) 2082. [17] K.P. Datema, R.B. Spruijt, C.J.A.M. Wolfs and M.A. Hemminga, B&him. Biophys. Acta, 944 (1988) 507. [18] G.A. Simatos, K.B. Forward, M.R. Morrow and K.M.W. Keough, Biochemistry, 29 (1990) 5807. [19] J.C. Sanders, T.W. Poile, R.B. Spruijt, N.A.J. Van Nuland, A. Watts and M.A. Hemminga, Biochim. Biophys. Acta, 1066 (1991) 101. [20] P.J.R. Spooner and A. Watts, Biochemistry, 31 (1992) 10129. [21] P.J.R. Spooner, A.A. Duralski, S.E. Rankin, T.J.T. Pinheiro and A. Watts, Biophys. J., 65 (1993) 106. [22] K. Harlos and H. Eibl, Biochemistry, 19 (1980) 895. [23] D.L. Brautigan, S. Ferguson-Miller and E. Margoliash, Methods Enzym., 53 (1978) 128. [24] E. Margoliash and O.F. Walasek, Methods Enzym., 10 (1967) 339. [25] J.H. Davis, K.R. Jeffrey, M. Bloom, M.I. Valic and T.P. Higgs, Chem. Phys. Letters, 42 (1976) 390. [26] M.J. Duer, J. Rocha and J. Klinowski, J. Am. Chem. Sot., 114 (1992) 6867. [27] J.H. Davis, Biochim. Biophys. Acta, 737 (1983) 117. [28] R. Wohlgemuth, N. Waespe-Sarcevic and J. Seelig, Biochemistry, 19 (1980) 3315. [29] D. Marsh and A. Watts, FEBS Lett., 85 (1978) 124. [30] M.F. Brown, J. Mag. Reson., 35 (1979) 203. [31] J. Seelig and A. Seelig, Q. Rev. Biophys., 13 (1980) 19. [32] P.R. Cullis and B. de Kruijff, B&him. Biophys. Acta, 559 ( 1979) 399.
[33] I.C.P. Smith and I.H. Ekiel, in G.G. Gorenstein (Ed.), Phosphorus-3 I NMR, Principles and Applications, Academic Press, New York, 1984, pp. 447. [34] B. de Kruijff and P.R. Cullis, B&him. Biophys. Acta, 602 ( 1980) 477. [35] A. Rietveld, P. Sijens, A.J. Verkleij and B. de Kruijff, EMBO J., 2 (1983) 907. [36] P.J.R. Spooner and A. Watts, Biochemistry, 30 (1991) 3880. [37] T.J.T. Pinheiro and A. Watts, Biochemistry, 33 (1994) 2451. [38] T.J.T. Pinheiro and A. Watts, Biochemistry, 33 (1994) 2459. [39] S.B. Farren and P.R. Cullis, Biochem. Biophys. Res. Commun., 97 (1980) 182. [40] M. Bloom and E. Stemin, Biochemistry, 26 (1987) 2101. [41] J. Stohrer, G. Grobner, D. Reimer, K. Weisz, C. Mayer and G. Kothe, J. Chem. Phys., 95 (1991) 672. [42] A. Abragam, in W.C. Marchall and D.H. Wilkinson (Eds.), The Principles of Nuclear Magnetism, Oxford University Press, London, 1961. [43] F. Six1 and A. Watts, Biochemistry, 21 (1982) 6446. [44] I. Pascher, S. Sundel, K. Harlos and H. Eibl, B&him. Biophys. Acta, 896 (1987) 77. [45] J.M. Boggs, Biochim. Biophys. Acta, 906 (1987) 353. [46] D. Rice and E. Oldfield, Biochemistry, 18 (1979) 3272. [47] R. Smith, F. Sparovic, F.C. Bennett and B.A. Cornell, Biophys. J., 63 (1992) 469. [48] B.A. Cornell, R.G. Hiller, J. Raison, F. Separovic, R. Smith, J.C. Vary and C. Morris, Biochim. Biophys. Acta, 732 (1983) 473. [49] P.F. Devaux, G.L. Hoatson, E. Favre, P. Fellman, B. Farren, A. McKay and M. Bloom, Biochemistry, 18 (1986) 3272.
ELSEVIER
Solid State Nuclear Magnetic Resonance
8 (1997) 55-64
Phospholipid headgroup dynamics in DOPG-d,-cytochrome c complexes as revealed by *H and 31P NMR: The effects of a peripheral protein on collective lipid fluctuations Teresa J.T. Pinheiro a,*,Melinda J. Duer b, Anthony Watts a,* a Depurtment ofBiochemistry, Uniuersity ofOxford, South Purks Road, Oxford, OXI 3QU. UK b Depnrrmenf ofChemistry, University ofCambridge, Lensfield Road, Cambridge, CB2 IEW, UK Received 4 March 1996; accepted 4 June 1996
Abstract The dynamics of the glycerol headgroup of dioleoylphosphatidylglycerol (DOPG) in hydrated bilayers were studied by and the effects of binding a peripheral protein, cytochrome c, were evaluated. The fast headgroup segmental motions (TV, 10-‘“-lO- I3 s> of DOPG in fully hydrated bilayers were not affected upon binding of ‘H and 3’P NMR spectroscopy,
cytochrome c, as evaluated by the spin-lattice CT,) relaxation of deuterons in the DOPG glycerol headgroup. In contrast, the spin-spin (T&) relaxation is strongly affected, indicating that slow cooperative bilayer motions (TV,, 10-3-10-6 s) are enhanced upon the interaction with cytochrome c. *H and 3’P NMR spectral lineshape analysis reveal details of the nature of these motions. The importance of these effects are discussed in terms of a possible mechanism for modulating membrane-associated processes. 0 1997 Elsevier Science B.V. All rights reserved. Keywords: ’ H Spin-lattice relaxation; *H Spin-spin relaxation; Collective lipid motions; Membranes
1. Introduction Diacylphosphatidylglycerol (PG) is a major phospholipid found in higher plants, algae, and bacteria [l-3]. In mammalian systems PG occurs in minor amounts only, notably in lung surfactants [4] and in
Abbreviations: DMPC, dimyristoylphosphatidylcholine; DMPG, dimyristoylphosphatidylglycerol; DOPG, dioleoylphosphatidylglycerol; EFG, electric field gradient; MAS, magic angle spinning; T,, spin-lattice relaxation time; T2e. spin-spin relaxation time * Corresponding author. Tel.: + 44 1865 275 268; fax: +44 1865 275 234; e-mail: [email protected] ’ Present address: Department of Biological Sciences, Unviversity of Warwick, Coventry CV4 7AL, United Kingdom.
mitochondrial membranes [5], where it can function as a precursor of cardiolipin 161. Because of its negatively charged headgroup ( pK, = 3-5.5, depending on the ionic strength [2,7]) PG is a good candidate for the study of the interaction of membrane with ions [8], and peptides or proteins [9- 111. The dynamics of lipids in a bilayer structure involve a variety of motional modes. Diffusive intramolecular fast motions, such as bond vibration, bond rotation, and rruns-gauche isomerisation, occur at rates faster than 1 X 10’ s- ’ [ 12,131. In addition, collective motions, involving more than one lipid molecule, have been shown to exist in lipid membranes [ 143. These motions, also referred to as order director fluctuations, occur at lower frequencies as
0926-2040/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO926-2040(96)01255-6
56
T.J.T. Pinheiro et al. / Solid State Nuclear Magnetic Resonance 8 (1997) 55-64
low as 103-lo6 Hz, depending on the number of molecules involved [15], and were thought to be confined to the lipid acyl chains. The headgroup and C,-C 3 segment in the acyl chains were found to be uncoupled from these slow motions, serving as an anchor around which those fluctuations occur [16]. An increase in the density of slow cooperative motions has been postulated upon the binding of proteins to a lipid bilayer [17-201. In particular, the effects of cytochrome c in cardiolipin bilayers has been shown to induce the occurrence or propagation of low frequency motions in the glycerol headgroup region of cardiolipin bilayers [21]. It was postulated that the glycerol headgroup in cardiolipin was particularly sensitive to the slow cooperative motions due to its “rigid” structure (sterified from both ends to two diacylphosphoglycerol moieties), enabling its motion to become strongly coupled to those within the chain segments. In the present study it is shown that the glycerol headgroup in DOPG bilayers can also participate in the slow cooperative fluctuations in the lipid bilayer. The effects on the higher frequency molecular motions in the phospholipid headgroup on binding of cytochrome c were analyzed through the T, relaxation of deuterons in the glycerol headgroup of DOPG-d,, while the effects on the lower frequency motions were investigated using deuterium Tze relaxation measurements. It is found that on binding of cytochrome c to DOPG bilayers the slow collective lipid motions are propagated into the lipid headgroup region. 3’P and H NMR spectral lineshape analysis combine to lend weight to these ideas, and give further details of the nature of the motion.
2. Experimental 2. I. Lipid synthesis Headgroup deuterated 1,2-dioleoyl-sn-glycero-3phospho-rut-glycerol at the (Y-,p- and y-methylenes in the glycerol headgroup segment (DOPG-d,) (Fig. 1) was synthesised as described elsewhere [9]. Briefly, the sodium salt of DOPG-d, was prepared by the method of Harlos and Eibl [22] using perdeuterated rat-isopropylidenoglycerol in the esterification step. This later compound was synthe-
Fig. 1. Schematic representation of the chemical structure of dioleoylphosphatidyl-glycerol (DOPG-d,) showing the deuterated positions at the W, p-, and y-methylenes in the glycerol headgroup.
sised by dissolving perdeuterated glycerol in acetone and boiling under reflux for 6 h in the presence of catalytic amounts of p-toluenesulfonic acid. The perdeuterated glycerol was produced by exchange of protonated glycerol against D,O with Raney nickel as a catalyst. Lipid purification was performed by silica acid column chromatography as described previously [7]. Cytochrome c from horse heart, type VI, Sigma Chemical Co., St. Louis, was purified by ion exchange chromatography on Whatman CM-32, and eluted with 65 mM phosphate buffer, pH 7.0 [23]. The eluent containing the purified protein was concentrated by ultrafiltration using Amicon YM-5 ultrafiltration membranes, followed by extensive dialysis against cold distilled water (4°C) to remove phosphate. Cytochrome c was quantified spectrophotometrically using a molar absorptivity of 2.95 X lo4 at pH 7.0 for the protein reduced with sodium dithionite [24]. 2.2. Membrane preparation Multilamellar liposomes of headgroup deuterated DOPG-d, were formed by handshaking hydration with excess 20 mM cacodylate buffer at pH 6.0,
TJ.T. Pinheiro et al./Sdid
Stute Nuclear Mugnetic Resonmce
containing 0.1 M NaCl and 5 mM EDTA. Typically, a film of 80-100 mg of lipid was prepared under rotatory evaporation, left for a minimum of 8 h under high vacuum to remove all traces of organic solvent, and hydrated with 200-300 ml buffer. Lipid-cytochrome c complexes were formed by addition of increasing amounts of protein in buffer solution to the pre-hydrated phospholipid bilayers, and followed with three cycles of freeze-thawing. Lipid-protein complexes were separated by ultracentrifugation (75 000 X g; 1 h; 4°C). The clear supematant was removed for protein analysis. Bound cytochrome c in the lipid-protein complexes was calculated from the free protein remaining in the supematant, as determined spectrophotometrically [24]. Lipid hydration and the preparation of lipid-protein complexes were carried under nitrogen atmosphere, using predeoxygenated buffer to prevent oxidation of the unsaturated acyl chains within the lipid.
2.4. Spectral lineshape analysis
57
coupling constants used in the simulations were 13.11, 11.67, 14.11, and 12.55 kHz for the four different (Y sites; 4.875 kHz for the B site; and 750 and 1250 Hz for the two y sites. The rate of hopping between sites is given with each simulation. Two hundred FID points were calculated, and 9800 molecular orientations were used in producing the powder average. For the simulations of the phosphorus-31 NMR spectra, the chemical shift anisotropy used was 5.25 kHz; 3200 molecular orientations and 200 FID points were considered for each calculation. Many different motional models were investigated. The molecular motion is defined by hopping at a given rate between N discrete sites with a given probability of occupation. Each site is defined by the relative orientation of the *H quadrupole coupling tensor or 3’P chemical shift tensor in that site, and a T2 value. The (4
2.3. NMR spectroscopy All NMR experiments were performed on a Bruker MSL 400 spectrometer operating at 61.42 MHz for deuterium and 161.98 MHz for phosphorus-3 1. Deuterium spectra were recorded using a quadrupolar echo pulse sequence, n/2.V-r-n/2y-rr-acquisitionD, [25], with quadrature phase cycling, n/2 pulse widths of 4 ps, recycle delay time CD,), 200 ms, and between 25000 to 50000 scans were accumulated. Transverse ‘H-spin relaxation was measured with the same echo sequence by varying the interpulse delays CT), from where a time constant for the exponential decay in the echo intensity referred as Tze was evaluated. Deuterium spin-lattice (T,) relaxation times were measured by the inversion-recovery technique with quadrupolar echo pulse sequence, n,-n/2.,-r-T,-r’-acquisition-DO, in which the recycle time, D,, was at least 5 X T,. Phosphorus-31 NMR spectra were recorded using the Hahn echo pulse sequence, n/2,,-T-7r,-T-acquisition-DO, with 7r/2 pulse widths of 5 pus, and proton decoupling during acquisition; Da, 4 s; number of scans, 80-264.
8 (1997) 55-64
2
1 O 1'
2
3
3
w 2 1 3 (W
Fig.
2. (a) Schematic
representation
of the molecular
motion
described in motional model (1). The motion assumed occurs by hops between neighbouring sites labelled 0, I, 2, 3 and I’, 2’, 3’. In fact I5 sites are used in the calculations, arranged in similar manner to the 7 shown here. The different sites represent different possible orientations of the molecule. The populations of the sites are varied as described in the text. In this model, the molecule changes orientation within a plane. The diagram
indicates the
direction of the unique axis of a nuclear interaction tensor in each site involved in the motion. There is a constant angular displacement between
each site. (b)
Schematic
representation
of the
molecular motion described in motional model (2). The motion is considered to occur in a similar manner to that in figure 2(a), except now the molecular orientation can change in three dimensions. The hopping is between 4 sites (labelled
I to 4)
and can
occur between any pair of sites. The populations of the sites are
Spectral FORTRAN
simulations were carried out using a program, CARLA [26]. The quadrupole
equal. In both models, the hopping frequency is the same for all hops.
58
TJ.T. Pinheiro et ul./Solid
State Nuclear Mqnetic
motional models considered may be summarised as follows. (1) Small angle diffusion in one plane. Motion is defined by 15 sites. The unique axis of the nuclear interaction tensor in the sites are shown in Fig. 2a. The overall amplitude of the motion, i.e. angle i -+ i’ was varied between 15 and 60”. Gaussian distribution of the various widths were considered for the probability of occupation of the sites. Hopping is between neighbouring sites only. (2) Large angle hopping within a cone. Four sites are defined in which the unique axes of the interaction tensor are equally angularly spaced around the surface of a cone, as illustrated in Fig. 2b. The cone angle used was varied between 80 and 140”. Hopping can occur between any pair of sites at an equal rate. There is equal probability of occupation of the sites. Between them, these two motional models described above represent the extremes of the likely motion of the order director axis. Other motions are possible but represent situations of intermediate between these limiting cases. These motions thus suffice for initial analysis; refinements can be made at a later stage once the broad nature of the motion has been determined. A value of T2 of 8 ms was used in all simulations.
3. Results and discussion 3.1. The ‘H NMR spectrum of DOPG-d,
in hydrated
bilayers
*H NMR spectra recorded from headgroup deuterated lipids in randomly oriented bilayers are spherically averaged powder patterns dominated by an axially symmetric electric field gradient (EFG) tensor. Molecules whose 2H EFG tensors have their unique axis oriented at 90” relative to the applied magnetic field originate intense resonance lines in the powder pattern spectrum [27]. The separation between these maxima provides a direct measure of the quadrupole splitting (Av,). The 2H NMR spectrum of DOPG-d, in hydrated bilayers at 25°C is presented in Fig. 3, where the assignment for the various quadrupole splittings is shown [28]. The outer most splittings where four resolved components of 10.5, 11.3, 11.8, and 12.7 kHz can be identified, are attributed to the (r-CD,
Resonance 8 (1997) 55-64 y-CD,OH
P-CDOH
1
1
5000
I
0 HERTZ
I
I
-5000
Fig. 3. Assignment of the various quadrupole splittings for the deuterium NMR spectrum of DOPG-d, in hydrated bilayers (see text for complete description).
deuterons. This is in agreement with the 2H NMR spectrum reported previously for bilayers of dimyristoylphosphatidylglycerol (DMPG-d,) [9], where the four resolved quadrupole splittings have been ascribed to possible magnetic inequivalence of the two deuterons in the racemic mixture of both D and L diasteromers of the glycerol headgroup B-carbon. A single quadrupole splitting of 4.4 kHz was observed for the deuteron in the B-methylene group, as reported by others for DMPG-d, in hydrated bilayers [9,10,28]. The smallest quadrupole splitting of 440 Hz is assigned to the deuterons of the y-CD, segment (Fig. 1). Two quadrupole splittings have been reported for the y-deuterons in the glycerol headgroup of DMPG-d, in hydrated bilayers [28]. A second quadrupole splitting, although less resolved, can also be identified for DOPG-d, bilayers (see arrows in Fig. 3). Indeed none of the *H NMR lineshapes can be successfully simulated without inclusion of inequivalent y sites, as discussed later in this section. These two quadrupole splittings have been attributed to motional inequivalence of the two deuterons in this segment [lo], as a result of restricted internal trans-gauche isomerisation, due to
TJ.T. Pinheiro et al./Solid
State Nuclear Mugnrtic Resonance 8 (1997) 55-64
an extensive hydrogen bonding network at the bilayer surface [29]. The quadrupole splitting for the a-CD, deuterons in the glycerol headgroup of DOPG-d, in hydrated bilayers is reduced over tenfold compared with that of a static methylene bond (= 170 kHz). This is a result of motional averaging due to the long axis rotation of the entire lipid molecule in the bilayer structure and additional fast segmental motions within the headgroup moiety. Furthermore, Av, for the P-CD group is about three times smaller than that for the (Y-CD,, and y-A ho is about ten times smaller than p-A yo. The decrease in quadrupole splitting between the CY-and y-methylenes could simply be associated with an increase in the motional freedom towards the terminal headgroup segments. Altematively, the difference could reflect changes in the C-D bond orientation relative to the long axis of rotation. The C-D vector generally describes the unique axis of the ‘H quadrupole tensor for alkyl 2H. Rapid rotation of this tensor axis about an axis oriented at an angle 13 to it, reduces the quadrupole coupling constant x (and hence A v,) to an effective value, xeff given by (x,rr/x) = #2 cos*8 - 111. Thus, the different quadrupole splittings for the IXCD,, P-CD, and y-CD, may reflect the different orientation of these C-D bonds relative to the long rotation axis. Given the rigidity of the headgroup of DOPG, this latter explanation is plausible, and if correct, would allow the long axis of rotation to be located. Deuterium spin-lattice CT,) relaxation measurements are particularly useful for determining the cause of the Av~ variation, since deuterium relaxation rates (l/T,) are dominated essentially by the rate of reorientation of the segment involved [30,31]. While the deuterium T, relaxation gives information about the fast local segmental motions (7, z lo- lo s) affecting the headgroup deuterated moiety in phospholipid bilayers, the spin-spin (T,,) relaxation times are determined, principally, by the slow motions of the deuterated segment CT,, 103-lo8 Hz). The T, relaxation times for the deuterons at the (Y-, p-, and y-positions in the glycerol headgroup of DOPG-d, in protein-free bilayers have all very similar values, T, (IxCD,) = 11.8 ms; T, (P-CD) = 10.5 ms; and T, (y-CD,) = 13.4 ms, in good agreement with reported values for the headgroup deuterated segments
59
of the saturated analogue DMPG-d, in hydrated bilayers [9]. This suggests that all these deuterons suffer the same motions. It therefore seems likely that the headgroup itself is relatively “rigid”, i.e. no reorientation of groups within the headgroup, and that the motion is confined to that about the long molecular axis. This could further suggest that differences in Av, for the headgroup deuterons arise from the different orientation of the C-D bonds relative to this long molecular axis as discussed previously. In contrast, T,, relaxation times increase appreciably from (Y- to y-methylene, T,, (a-CD*) z 500 /_Ls; Tze (P-CD) = 710 /.Ls; and T,, (y-CD,) % 1050 /_Ls. 3.2. The effects of cytochrome
c on DOPG-d,
head-
group “P NMR. Phosphorus-31 NMR of DOPG-d, in hydrated bilayers alone show the typical powder pattern spectrum for phospholipids in liquid-crystalline bilayers [32], with a lineshape dominated by an axially symmetric chemical shift tensor (bottom spectrum Fig. 4a). This spectrum results, principally, from the averaging of the phosphorus chemical shielding tensor by the rotation of the whole phospholipid molecule around its long axis, i.e., perpendicular to the bilayer surface [33]. The separation between the edges of a 3’ P NMR spectrum (v,, - v I > defines the residual chemical shift anisotropy bilayers of (A VCSA1, which for the protein-free DOPG-d, is about 40 ppm. On binding of cytochrome c notable spectral changes occur, including the appearance of a central spectral component in the 3’ P NMR spectra at around the isotropic position, and collapse of the low intensity end of the spectrum (Fig. 4a). The additional isotropic-like component increases with increasing amounts of bound cytochrome c, while the low-field intensities are progressively reduced. Narrow components have been observed in static 3’P NMR spectra of cardiolipin-cytochrome c complexes [34-361, and with other anionic phospholipid-cytochrome c complexes [37]. Additionally, in MAS “P NMR experiments these isotropic-like components are shifted from the isotropic central bands of mixed phospholipid bilayers [20,38]. The interpretation of such isotropic components is still unclear. In some stud-
TJ.T. Pinheiro et al./ Solid State Nuclear Magnetic Resonunce 8 (1997) 55-64
60
(4
(W
I
I
I
I
I
I
-15
-10
-5
0 kHz
5
10
I 15
Fig. 4. (a) 161.98 MHz 3’P NMR spectra of DOPG-d, in hydrated bilayers alone (lower spectrum), and with increasing amounts of bound cytochrome c. Lipid/protein ratio in molar stoichiometry; temperature. 25T. (b) Calculated spectral lineshapes using the model (2) described in Section 2. Hopping frequencies between the sites for each simulation are (top to bottom) 6.4, 5.0, 3.8 and 0.6 kHz, respectively.
ies, they have been attributed to vesicular or micellar structures on or within the bilayers, possibly associated with precursors of hexagonal-H,, phases [34,39].
Others have attributed them to the presence of smaller diameter (I 500 nm) vesicles induced upon protein binding, or as a result of motional or orientational averaging without involvement of non-bilayer structures [21,36-381. Alternatively, the lineshapes may arise from the 3’ P species which undergoes molecular motion so as to produce a feature (amongst others) at the isotro ic R chemical shift. It is to be noted that none of the P NMR spectra of protein containing samples can be successfully simulated by simply adding a Lorenzian line (of varying width) at the isotropic chemical shift of the protein-free powder pattern. In addition to the appearance of the feature at the isotropic chemical shift, the low intensity end of the powder pattern (v,,) collapses and the high intensity end (v I> broadens. Many of the features of the 3’P NMR spectra can be reproduced with the same motional model as was used to simulate the 2H NMR lineshapes (see below), albeit with slightly different hopping frequencies for a given lipid-protein ratio (Fig. 4b). Clearly, there is plenty of scope for refinement of the model, by including more sites, and varying their angular disposition. The simulations presented here are however sufficient to demonstrate that the 3’P NMR lineshapes are likely to arise from fluctuations of the whole lipid molecule at the rates of l-10 kHz. 2H NMR. Deuterium NMR spectra of DOPG-d, bilayers containing increasing amounts of bound cytochrome c are shown in Fig. 5a. There is an overall change in the 2H NMR spectral lineshape upon binding of cytochrome c. The resolution of the ‘H NMR spectra seems to decrease as cytochrome c is added. However, the 2H NMR spectra for high concentrations of protein cannot be simulated by simply broadening that for the protein-free sample. Clearly, there is some change in the motional process affecting the DOPG molecules upon the addition of cytochrome c. We have performed extensive spectral lineshape calculations for many different motional models. Calculated spectra are shown in Fig. 5b for increasing values of hopping frequency which most closely follows the experimental lineshapes changes induced by increasing the concentration of cytochrome c in the lipid-protein complexes. The motional model used to produce these simulations is the model (2) described in the Section 2, i.e., large amplitude
TJ.T. Pinheiro et al./ Solid State Nuclear Magnetic Resonance 8 (1997) 55-64
hopping motions of the *H nuclei within a cone. In this case the cone angle is 140” for all the *H nuclei in the headgroup (a, p, and -y>.It is to be noted that no other motional model gave acceptable fits to the experiment. This model may be interpreted as a large angle fluctuation of the long molecular axis, with the head groups held essentially rigid during the motion, i.e., all ‘H nuclei suffer the same motion. Clearly. this motion may be part of the “rippling ” of the phospholipid bilayer. The variation of deuterium spin-lattice CT,) and spin-spin (T,,) relaxation times for headgroup deuterated DOPG-d5 bilayers as a function of the concentration of bound cytochrome c is presented in Fig. 6. The T, relaxation times are essentially not affected on binding of cytochrome c for any of the deuterated segments, suggesting that rotation about the long molecular axis is little affected. However, T2e relaxation times decrease for all of the segments upon binding of cytochrome c. Furthermore, this reduction increases progressively from the outer most headgroup segment (y-CD,) to the inner most methylene group (OL-CD*). T2e relaxation times are reduced of about 50, 60, and 70% for y-CD,, p-CD and o-CD,, respectively, over the range of protein to lipid concentrations studied here. Similar effects have been observed on the interaction of bacteriophage Ml 3 coat protein with DMPC bilayers [ 17,191, where essentially no effect on the T, relaxation of deuterons in the lipid headgroup or in the acyl chains was observed, but profound effects were encountered in the T,, relaxation. Also the interaction of the pulmonary surfactant protein SP-C with deuterated DMPC bilayers was found not to affect significantly the T, relaxation and to affect the Tze relaxation, suggesting that the protein only influences the slow lipid motions in the bilayer [ 181. The spin-lattice relaxation processes in *H NMR are only sensitive to spectral densities, J(w), of the
I
10
5
0
-5
-10
kHz
(b)
I -15
I -10
61
I
I
I
I
I
-5
0 kHz
5
10
15
Fig. 5. (a) Experimental 61.42 MHz 2 H NMR spectra of LIOPG-cl, in hydrated bilayers alone (lower spectrum) and with increasing amounts of bound cytochrome c. Lipid/protein ratio in molar stoichiometry; temperature 25°C. (b) Calculated NMR spectral lineshapes using the model (2) described in Section 2. Hopping frequencies between the sites for the simulations are (top to bottom) 3.3, 2.5, 1.6, 0.8 and 0.4 kHz, respectively.
TJ.T. Pinheiro et al./ Solid State Nuclear Magnetic Resonance 8 (1997) 55-64
62
01 0.000
0.025
0.050
0.075
0100
PROTEtN/LIPlDRATIO Fig. 6. Deuterium relaxation behaviour of the o-, p-, and -y-deuterated segments in the glycerol headgroup of DGPG-d, in hydrated bilayers as a function of bound cytochrome c. (a) Spin-lattice CT,) and (b) spin-spin (T& relaxation times. o-CD,, triangles; P-CD, filled circles; and y-CD,, open circles. Protein/lipid ratio in molar stoichiometry; temperature 25°C. The lines arc for guidance only and have no theoretical significance.
quadrupolar interactions at w = w0 and where f0 = wa/2~r is the Larmor freo= 2wa, quency, while the spin-spin relaxation processes are also affected by J(O) [40]. Investigations by Stohrer et al. [41] have shown that colkctiue lipid motions constitute a dominant mechanism for the *H spinspin relaxation of deuterated phospholipids in bilayers. These motions may be visualised as cooperative fluctuations of the lipid acyl chains in a direction perpendicular to the bilayer normal, and are often referred to as order director jlucruations. Watnick and co-workers [ 161 have analyzed the spin-spin relaxation of deuterons placed at various locations within dimyristoylphosphatidylcholine bilayers, and have concluded that the low-frequency collective motions were confined to the hydrocarbon interior of the bilayer. The headgroup and the CT-C, bond of the acyl chains would serve as an anchor about which those fluctuations can propagate. These observations have suggested that lipid headgroups can be fluctuating
uncoupled from the cooperative acyl chains slow motions, possibly due to restrictions in the molecular packing in the bilayer arrangement imposed by the chemical structure of the headgroup and headgroup-headgroup interactions. However, the reduction in T2e of the deuterons in DOPG-d, headgroup when in complexes with cytochrome c observed in the present study, are indicative of an increase of slow cooperative motions in the lipid bilayer which involve the headgroup. This is supported by a recent *H NMR study by Spooner et al. [21] on cardiolipin-cytochrome c complexes, in which the protons of the sn-1 position in the headgroup glycerol segment of cardiolipin have been replaced by deuterons. Here no effects were observed on the spin-lattice relaxation, while the spin-spin relaxation was strongly affected. An identical decrease m Tze of about 70% is observed for both the sn-1 deuterons in the cardiolipin headgroup and the cx-deuterons in the headgroup of DOPG-d,, over the same protein concentration range. DOPG and cardiolipin have in common a glycerol moiety for headgroup. While in cardiolipin this glycerol is bridging two diacylphosphoglycerol moieties, in DOPG the glycerol headgroup is only esterified from one end (the cl-methylene) to the diacylglycerophosphate moiety (Fig. 11, leaving the other segments (p and r> with a larger motional freedom when compared with that of the glyceryl headgroup in cardiolipin. However, the T, values for the three segments are all very similar (see above), which suggests no major differences in the fast headgroup motions. Assuming that the fast motions constitute the dominant mechanism for the deuterium T, relaxation, an average correlation time CT,) for the fast segmental motions within the DOPG glyceryl headgroup can be calculated [42], being 2.0 X lo-” s. For the sn-1 CD, deuterons in the cardiolipin glyceryl headgroup TV is of the same order of magnitude, 2.9 X lo-” s, revealing identical rates of fast segmental motions within the glycerol headgroup for both lipids despite of their distinct chemical structures. It was thought [21] that the glycerol headgroup in cardiolipin bilayers, due to its more “rigid” structure, would be specially sensitive to the lowfrequency collective motions within the bilayer. However, the present study indicates that the glyc-
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State Nuclear Mugnetic Resonance 8 (1997) 55-64
erol headgroup moiety in DOPG is also sensitive to the collective lipid motions in membranes. This may arise from the fact that the headgroup glycerol in this lipid is quite well “anchored” at the bilayer surface through an extended network of hydrogen bonding [29,43-451. The increase of order director fluctuations in lipid bilayers seems to be a common feature upon binding of peptides, such as gramicidin [46], and melittin [47], or peripheral proteins [21,48,49]. These results suggest that the binding of peripheral proteins, such as cytochrome c, to a membrane surface can establish the coupling between the lipid acyl chains and the headgroup segments, therefore stimulating or enhancing the cooperative bilayer lipid motions. Those effects may be of genera1 importance in biological membranes, whereby through the coupling of cooperative low-frequency fluctuations, protein active conformations can be enhanced or inhibited, and can therefore serve as a modulating mechanism for membrane-associated processes.
63
cooperative bilayer lipid motions by establishing the coupling between the lipid acyl chains and the headgroup segments. It is very plausible that the proteins themselves are also involved in these cooperative slow motions, and these can constitute a general mechanisms for modulating protein activity at the membrane interface. Through the coupling of cooperative low-frequency fluctuations, protein active conformations can be enhanced or inhibited, and therefore this could serve as a modulating mechanism for membrane-associated processes.
Acknowledgements This work was supported by the EC Grant B/89 000 154/893 and Programa CiEncia JNICT, Portugal, Grant BD/1793/91-ID (to T.J.T.P), SERC (Grants GR/H/51552), and an MRC studentship to T.J.T.P. (fees only). We thank Dr P.J.R. Spooner for useful discussions.
4. Conclusions We have shown that the slow collective molecular motions occurring in lipid membranes are strongly influenced by the binding of a peripheral protein, cytochrome c, at the membrane interface. On protein-free lipid bilayers these slow lipid collective motions appear to be confined to the acyl chains in the hydrophobic core of the membrane, leaving the headgroup moiety on the membrane surface uncoupled from such motions. In the current paper we have observed a strong reduction in the spin-spin relaxation times CT,,) of lipid headgroup deuterons upon the interaction with cytochrome c. The reduction of T,, reveals an enhancement of slow motions (r,, 10-3-10-6 s) at the membrane interface. These motions are mostly associated with the collective molecular fluctuations, which are known to exists in membrane systems and also referred to as “bilayer Increase in low-frequency collective undulations”. motions in the lipid bilayers and propagation to the lipid headgroup region seems to be a general effect induced by binding peripheral proteins to lipid membranes, as described in the Section 3. This observations led us to suggest that peripheral proteins at a membrane interface can stimulate or enhance the
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