Values of isotropic hyperfine splitting constant
Lipid spin labels containing nitroxide groups at different positions in the fatty acid chain, such as 1-palmitoyl-2-stearoyl-(
It is generally accepted, that, unlike bulky fluorescent labels7, 8, nitroxides are well incorporated into fluid lipid bilayers9 and not excluded from them. However, it has been shown by NMR that although the most probable location of the nitroxide group for 5-, 10- and 16- PC spin labels in the fluid POPC membrane corresponds to the fully extended conformation, the distribution is relatively broad and other conformations should also be present10. Bent conformations were previously found for doxylstearic acids in monomolecular films11, water/hydrocarbon emulsion particles12 and micellar systems13. In fluid membranes the fluidity, polarity and accessibility parameters reported by ESR using PC spin labels and
In this chapter we focus on the behavior of PC spin labels in the gel phase and frozen membranes. We show how the superior
2. Resolution of different hydrogen-bond states by 240GHz ESR in bulk organic solvents
It has been well established that the g-tensor and hyperfine components of nitroxide radicals are very sensitive to the local environment, to polarity and proticity in particular. In general, changing the local environment of the nitroxide moiety from water (polar) to hydrocarbon (non-polar) causes an increase in the
On the other hand, superior g-factor resolution of HF ESR allows for observation of two resolved spectral components corresponding to two (Smirnova et al.19 at 130GHz) or possibly more (Bordignon et al.20 at 95, 275 and 360GHz ) states of hydrogen-bonding. Two hydrogen bonding states coexisting in frozen deuterated alcohols were previously demonstrated for perdeuterated TEMPONE by X-band ESR21.
As one sees from Fig.1, whereas at X-band one sees a continuous increase in the 14N hyperfine splitting with change of the local environment from non-polar/aprotic to polar/protic, the 240 GHz ESR shows three distinct values of the gxx parameter. Although the presence/ratio of these components strongly depends on the polarity-proticity of the solvent, there is little variation in the gxx measured for each such component. For all four spin labels studied three distinct components could be detected: (1) “non-polar”, as in toluene, DBPh or the minor component in alcohols, (2) “polar”, the major component for ethanol and major or minor component in TFE and water/glycerol, depending on the nitroxide used, and (3) “very polar” component observable in TFE and water/glycerol. Although components 2 and 3 cannot be separated at 240GHz as two distinct peaks, their presence is quite obvious (compare Figs.1A-D for different nitroxides). We assign these components to different hydrogen-bonding states of nitroxide radicals and speculate that state 2 corresponds to a single hydrogen bond, while state 3 is double-bonded. Existence of multiple hydrogen bonding to a nitroxide has been predicted theoretically22-24 and later suggested as an explanation for complex ESR lineshapes observed in spin labeled proteins20. Interestingly, the gxx value of the non-hydrogen bonded component for all four spin labels studied shows little dependence on the polarity of the frozen glass-forming solvent (Table 3). This contrasts with some theoretical predictions for the g-factor25, as well as some room temperature measurements for
|Solvent, dielectric constant at room temperature51||Oxo-TEMPO||TEMPO||4-Hydroxy TEMPO||3-carboxy-2,2,5,5 tetramethylpyrrolidine - 1-oxyl|
|Solvent, dielectric constant||Oxo-TEMPO||TEMPO||4-Hydroxy TEMPO||3-carboxy-2,2,5,5 tetramethylpyrrolidine - 1-oxyl|
|10||69.6||66.6||69.5 (more broadening)||66.5|
3. Membrane environment
3.1. X-band ESR
The full extent of the 9 GHz spectrum is determined by the largest of the principal values of the 14N hyperfine constant, which is Azz. Hence at X-band, the distance between outer extrema for a well-resolved spectrum in the rigid limit is exactly 2Azz. This value is known to increase ~ 2G if nitroxide is transferred from a non-polar solvent like hydrocarbons to water and, as mentioned above, can be considered a measure for local polarity. Table.3 shows the 2Azz determined by X-band ESR as the outer splitting of the rigid limit spectra at 77K. As seen from the data, DMPC/Cholesterol (Fig. 2B) and DPPC/Cholesterol (Fig. 2D) membranes, consistent with previous observations3, show abrupt drop in the 2Azz value, between
The dependencies of relaxation enhancement by 10 mM of Ni(ClO4)2 on the spin-labeling position for the gel and liquid crystal phase of DMPC are given in Fig. 3. Here P is the relaxation parameter
3.2. High field/ High frequency ESR
High frequency ESR and PC spin labels were previously used to study polarity profiles in phospholipid membranes14, 26, 27. For example, in a detailed ESR study at 250GHz 5,7,10,12,14 and 16 PC were studied in DPPC and DPPC/gramicidin systems26. It was found that in pure DPPC most spins are strongly aggregated and the spectrum consists mostly (especially for 7-12 PCs) of a singlet-like signal. Although the g-factor values for the resolved rigid-limit component seems to indicate increasing immersion of the nitroxide into the hydrophobic core of the membrane with increasing
3.2.1. Frozen DMPC/DPPC membranes with cholesterol: Partition-like depth distribution of spin labels
Several W-band (94GHz) studies by Marsh and coworkers14, 27, 28 on DMPC/Cholesterol membranes utilized all n-PC spin labels in the 4-16 range (except 15-PC). Based on the gxx values detailed polarity profiles for these systems were suggested. These polarity profiles appeared to be similar to the polarity profiles previously obtained by rigid limit X-band ESR from the hyperfine splitting3 values. However, a close inspection of the spectra of 27 shows two partially resolved components, which likely correspond to hydrogen-bonded and non-hydrogen-bonded states of the nitroxide radical. These two components are discernible not only for the area of abrupt “polarity change” (PC 5-10), but also for PC14-16. Higher resolution of 240GHz ESR allows for complete separation and identifying these components. Fig.4 A, B show the spectra for DMPC and DPPC in the presence of cholesterol. The ESR spectra in the two different lipids are very similar, nearly identical. There are two components discernible for n7, which are completely resolved for n10, with gxx= 2.009435 and gxx= 2.008820 (with gzz taken as 2.00233, see 29). The two g values are nearly the same for all n. For 5-PC only the polar component is present.
Indeed, theoretical estimates32 show that the concentration of water in the middle of the DPPC membrane is ~ 1 mM. It should be even lower for membranes containing cholesterol, since cholesterol is known to substantially decrease water permeability across lipid membranes33, 34. For the equilibrium constant between hydrogen bonded/unbonded forms of 16-sasl in toluene - trifluoroethanol mixtures Marsh30 gives a value ~ 1M-1. This value is determined from the isotropic hyperfine splitting at room temperature. Our estimates for this constant for various nitroxides based on measurements of 2Azz values in ethanol – DBPH mixtures by 9GHz ESR at 77K or on the ratio of hydrogen-bonded and non-hydrogen bonded components in frozen ethanol (~ 17M hydroxyl concentration) measured by 240GHz ESR give values of the same order, between 0.2M-1 and 0.5M-1. A 1:1 ratio of the two spectral components for a nitroxide located in the middle of the bilayer would thus yield a water concentration in the membrane core of ~0.5M, about three orders of magnitude higher than expected from theoretical estimates. Also, consistent with the above estimates of the equilibrium constant, in our test experiments we did not see any appearance of a hydrogen bonded component for nitroxides dissolved in frozen nearly saturated (~16 mM) solutions of water in toluene.
|PC label||Ratio HB/non-HB in DMPC||Ratio HB/non-HB in DPPC|
And finally, a strong point in favor of considering bent conformations of n-PC spin labels is the non-monotonic dependence of the hydrogen-bonded fraction on n (Table 4). In a fluid membrane the membrane depth distribution of nitroxide moieties for each spin-labeled lipid correlates to its n-value10. Higher n show deeper average immersion although the distribution is broad and even high n numbers show a substantial fraction of conformations with the nitroxide touching the carbonyl area. In the much denser packed gel phase the situation can be different and the spin labels may prefer defects in the lipid structure35, 36. One of the areas with such defects is just above the cholesterol rings and this should correspond to a hydrophobic local environment. It can be reached by the nitroxide of 10PC, but not 7PC. This would explain the jump in the fraction of non-polar component between 7 and 10PC, while further decrease in this fraction could be attributed to U-shaped conformations for higher values of n. These conformations put the nitroxide moiety back to the surface region with high water content, while the hydrocarbon chain mostly remains located in the hydrophobic part of the membrane.
Indeed, if we assume that the spin-labeled sn-chain takes on mostly the fully extended conformation, we would observe a similar jump after the nitroxide moiety reaches the hydrophobic core of the membrane, but with further increase in the n number the hydrogen-bonded fraction would decrease and quickly disappear.
Another limiting case suggests that the acyl-chain hydrocarbon tether connecting the nitroxide to the lipid head group can take all possible coil conformations of the chain. It will put the possible position of the nitroxide of an n-PC spin label anywhere within a half sphere with a radius of the all-stretched conformation for the nitroxide tethers. This half sphere would rest on the membrane surface (cf. Fig.5). The ratio of hydrogen bonded/unbonded components would be opposite to the volume ratio of the spherical cap, which is cut off by the border of the hydrophobic core of the membrane, and the rest of the half sphere. If the cap height is half of the sphere radius, the ratio will be 2.2, similar to what we see for 7PC (Table 4). This observation can be then used to set (in a rather arbitrary fashion) the cutoff of the hydrophobic core at “3.5” PC, half of the acyl tether for 7PC in the fully extended conformation. The predictions of the components ratio obtained with this cutoff value by further applying the formula for the spherical cap are given in Table 5.
Although this model is qualitatively better when compared to Table 4, it does not reproduce the observed abrupt drop between positions 7 and 10. Also, no model assuming some random distribution of nitroxide depth position would reproduce the decrease in the hydrophobic fraction with further increase in n beyond 10.
To better explain the observed effects one could assume some set of preferential depth positions in the membrane to be occupied by the nitroxide ring. These positions may be areas of defects in the membrane structure, which can more easily accommodate a structure-disturbing nitroxide moiety than areas with compact alignment of hydrocarbon chains. One of the areas of such defects could start above the end of the rigid fused-ring system of cholesterol37, which is about the level reachable by n-PC labels starting from n=9. Once the tether length is sufficient, nitroxides start populating these favorable locations causing the change in the component ratio. However, the preference of the nitroxide ring for the location in the hydrophobic part of a DMPC/Cholesterol or DPPC/Cholesterol membrane apparently does not completely overwhelm its affinity to some sites close to the membrane surface. It gives a partition-like distribution between the two sites which is observable in the spectrum of all n-PC labels with n>7 as two components.
A gradual increase in the hydrogen-bonded fraction between PC10 and PC16 can be also explained using this partition model. For an n-PC spin label, the same carbon atom of the nitroxide ring that is connected to the acyl tether also has a hydrocarbon tail with a length of 18-n carbons attached. Bringing the nitroxide label of 10PC to the membrane surface will require, on the average, placing more hydrophobic CH2 groups to the polar area than for 16PC. This will be associated with some energy penalty preventing the nitroxide of 10PC from leaving the hydrophobic core and affecting the partition. Note also a slight drop in the accessibility of the 10PC position for Ni2+ ions for cholesterol-free DMPC membranes, which may have the same origin.
3.2.2. DMPC and DPPC without cholesterol. Nitroxide moiety as a foreign body in the bilayer
It has been previously observed that in the absence of cholesterol, PC spin labels in DPPC26 and DMPC27 at rigid limit conditions have a strong broad background signal. This signal points at aggregation of spin labels and is most pronounced for the7-12 PC positions. Since no significant signs of such aggregation are seen in the fluid phase of the membrane or in the gel phase in the presence of cholesterol, this aggregation can be specifically attributed to the gel or crystalline (sub-gel) state of pure-lipid membranes. Also, since diffusion in the gel phase is slow, one can expect hysteresis effects and effects of the sample-treatment procedure, see38, 39.
Fig. 6 shows 240 GHz ESR spectra of PC spin labels in DMPC membranes without cholesterol. These spectra were recorded after the sample was slowly cooled from 295 to 85K within ~ 2 hours. As in the presence of cholesterol, two components with different gxx are clearly discernible. However, another broad unresolved component, which is present for all spin labels but most pronounced for 7-12PC, follows a previously observed26 pattern for DPPC at 250GHz. Our 240 GHz data for DPPC obtained under the same conditions as those for DMPC are very similar to the 250 GHz results by Earle et al.26 and shown in Fig.7. A non-polar (non -hydrogen bonded) component similar to the non-polar component in membranes containing cholesterol can be identified by its characteristic gxx value. However, there is an important and obvious difference between DMPC and DPPC at first inspection. In DPPC there is only one resolved component which shows the gxx value corresponding to the non-hydrogen bonded state of the nitroxide, while in DMPC two components are clearly
discernible in the spectrum. This observation is in good accord with the X-band results, which indicate a less polar environment reported by high values of n in DPPC compared to DMPC (cf. Table 3, Figs. 2A, 2C).
It is very unlikely that small structural differences between the gel phase bilayers of DMPC and DPPC40, 41 cause dramatically different water penetration into these membranes and explain the presence of the hydrogen-bonded component in DMPC, but not DPPC. Moreover, it has been recently shown that water penetration into the membrane depends rather on the surface area of the lipid (which is nearly identical for DMPC and DPPC) than on the membrane thickness42. Thus, it makes the explanation of the difference between DMPC and DPPC through dramatically different water penetration very unlikely. Even more intriguing, the average environment of the nitroxide in DPPC looks, at the first glance, less polar in the absence of cholesterol, since no component with a clear peak at smaller gxx can be detected, cf. Fig 8.
However, the most salient feature of all spectra, both DMPC and DPPC, in the absence of cholesterol, is a broad singlet-like background signal which accounts for most of the spins. The location of spins responsible for this background is unclear. In Fig. 8 one can see an apparent broadening of the hydrogen-bonded component in DMPC and complete absence of this component in DPPC, concomitant with an increase in the background. A likely explanation for these observations is that the hydrogen-bonded and non hydrogen- bonded forms of PC spin labels aggregate differently. If the hydrogen bonded form is more prone to aggregation than the non- bonded one, it likely becomes broader due to more interactions between spins. In the superposition, spectral peaks from this broad component are less intense and eventually, with an increase in broadening, not discernible at all. To test this hypothesis we cooled our samples very rapidly in an attempt to trap the hydrogen-bonded component before aggregation could occur.
Quantitative study of aggregation in the gel or subgel phases is difficult because of the slow diffusion rates and various hysteresis effects. For example, even though the gel phase of DPPC does not favor formation of gramicidin channels, it takes between an hour and several days for these channels to dissociate39. There are also indications pointing to a relatively slow time scale for aggregation of PC spin labels in the gel-phase. Fig. 9 shows saturation curves for 0.5 mol. % of 7PC in DMPC. At 190C, in the Pβ phase, this system show good saturation, with a P parameter of ~ 19. Saturation measurements performed immediately after cooling to 20C give a similarly high P value. However, after longer exposure at this temperature the P value starts to decrease and after several hours drops below 2. This increase in relaxation is very likely due to aggregation, and this aggregation appears to be a relatively slow process at 20C. Also, this increase in relaxation is reversible; return to 190C reverses the aggregation and the P value. Although a temperature of 20C should correspond to the subgel phase, the exact phase state of the lipid at this condition is not obvious due to likely supercooling, see below. It usually takes days at this temperature to form the LC phase, which then can be characterized by X-Ray diffraction.
Previously, similar observations of exclusion of 5PC spin labels from DPPC after exposure at 00C by using Saturation Transfer ESR43 were attributed to formation of a sub-gel phase.
We expected that quick freezing (within ~ 0.2s) of the sample by instantly submerging into liquid nitrogen should substantially eliminate these slow lipid rearrangement effects compared to the standard slow cooling procedure using a flow cryostat. Indeed, spectra recorded after this simple quick-freeze procedure were different from the spectra obtained by gradual cooling in a flow of gaseous nitrogen. As seen in Fig. 10A the spectrum of 14PC in DPPC does not have a broad singlet-like component and can be well simulated by a simple superposition of two rigid limit spectra with gxx of 2.00943 and gxx= 2.00882.
Importantly, most 14PC spins are present in the form of the hydrogen bonded component, which has a fraction greater than the non-bonded one by a 2.2:1 ratio.
As seen from Fig 11, quick freezing helps to dramatically decrease the amount of the broad background signal and yields well resolved rigid-limit spectra even when a standard cooling procedure results in nearly unresolvable, broad-feature spectra.
One of the possible artifacts of using molecular probes as reporters in biological and model membranes is their exclusion from the membrane interior to the membrane surface. Such exclusion of certain molecules or structural units is a natural feature of membrane biological function. For example, exclusion of tryptophans and their affinity to the membrane interface is an important factor in lipid-protein interactions and stabilization of certain conformations of transmembrane protein/peptides44. Exclusion from the bilayer and formation of some U-shaped conformations have been known for a long time for a number of fluorescent labels7, 9, 11.
Spin labels are usually considered as more adequate molecular probes for lipid bilayers, as evidenced by studies in fluid membranes (see Introduction). However, our current study shows that in the gel phase the situation is different. Several ESR parameters (accessibility for Ni2+ ions absorbed on the membrane surface, hyperfine tensor and g-tensor components) unambiguously indicate that in the gel phase a substantial fraction of nitroxide is located in the membrane region with high water content. Pure lipids DMPC and DPPC form the L΄β “tilted gel” phase with densely packed and relatively well ordered hydrocarbon chains. The ability of nitroxide groups on spin labeled stearic acids to be excluded from hydrocarbon environments and form U-shaped conformations has been previously reported12, 13. In principle, formation of the L΄β phase can be compared with freezing of a bulk three-dimensional solvent. In this case solutes are excluded from crystallizing solvent and form some regions with high local concentration. If the solute is a nitroxide radical, freezing of its solution in non glass-forming media is well known to yield a broad signal similar to one observed in DMPC or DPPC without cholesterol (Figs 6, 7).
In the case of a lipid in the gel phase the complete exclusion of spin labels into a separate phase with a high spin concentration appears to take two steps. The first one, exclusion of nitroxides from the hydrophobic area of the membrane, occurs once the lipid forms an Lβ΄or even a Pβ phase (cf. Fig 3 for 190C, the Pβ phase of DMPC, also the quick-freeze spectra in Fig. 10). The second stage, which is a formation of separate phase by these bent-conformation molecules, can be attributed to lateral aggregation in the supercooled gel, possibly at the gel/subgel phase transition.
Although formation of subgel phases was initially observed after storing a multilamellar suspension of DPPC at ~00C for several days45, there are reports of quicker gel/subgel transformations in DPPC and DMPC. For example, in one protocol46 the subgel (SGII) phase in DPPC forms at about 70C upon cooling at 20C/min. These conditions are very similar to cooling conditions observed in our “slow cooling” experiment – and, in general, the cooling conditions usually existing in standard helium/nitrogen cryostats. For DMPC, formation of the subgel phase was reported after incubation at temperatures of -50C or lower for 2h47. But, the kinetics of subgel formation is complex48 and in some cases the final subgel phase contains spectroscopic features characteristic for the Lβ΄ phase49. It could be possible that aggregation of spin labels does not require a complete transformation into the subgel phase, in the sense of giving a clean X-ray pattern, and can happen sooner during cooling.
Again, we would like to stress that exclusion of nitroxides from the hydrophobic membrane core occurs in all gel phases and is a separate effect from the succeeding lateral aggregation in the supercooled gel phase.
Our quick freeze vs. slow cooling experiments can also help answer an important question about the nature of the two components in the 240GHz spectrum. Does the component with a larger gxx correspond to the location of the nitroxide in the hydrophobic part of the membrane? Or both components arise from equilibrium of the hydrogen-bonded/non-hydrogen bonded forms in the same location, like TEMPONE in ethanol (cf. Fig. 1A)? The different aggregation behavior of spins contributing into these components points to their different location. That is, the major hydrogen-bonded component comes from nitroxides excluded from the hydrophobic part into the area with high water content, while the minor non-polar component may arise from spins somehow trapped in the defects of the hydrophobic membrane core.
3.3. What about biological membranes?
The gel phase rarely exists in biological membranes and the subgel phase is unknown for them. However, polarity measurements in frozen natural membranes using PC spin labels can also be affected by the affinity of spin labels to some structural defects, especially in the presence of proteins. For example, all PC labels in mixtures of DPPC with gramicidin A show about the same gxx value, indicative of the same polarity26, most likely due to an interaction with the peptide’s -helical structure. Also, natural membranes usually have complex lipid head group composition and variety of lengths and unsaturation in acyl chains. This can affect the free volume available for spin labels both in the hydrophobic core and in the membrane interface. Fig. 12 shows 240 GHz spectra for 14PC in egg-yolk lecithin with and without cholesterol.
One can see that, similar to DMPC and DPPC membranes, the fraction of the hydrogen- bonded component drops with addition of cholesterol (cf. Figs 4 and the quick-freeze spectra in Fig.10 ). However, compared to DMPC and DPPC membranes this fraction in EYL is lower. Actually, in EYL/Cholesterol only a non-polar component can be observed in the spectrum. It can be explained by the presence of a double bond in the acyl chain of EYL. This unsaturated bond has a kink, which disrupts the packing of lipids. It creates extra free space in the hydrophobic core of the membrane50, and thus may shift the partition of nitroxides in favor of the membrane interior. Also, a variety of headgroups may improve packing in the polar head area, additionally forcing nitroxides into the hydrophobic core.
The ESR parameters of PC spin labels in frozen membranes do not simply represent the membrane polarity or water penetration profile. Instead, they show a distribution between hydrogen-bonded (HB) and non-hydrogen bonded (non-HB) states, which is affected by a number of factors in the membrane composition. In frozen lipid membranes at 240 GHz n-PC labels with n > 7 usually show two-component ESR spectra corresponding to HB and non-HB states of the nitroxide. In DMPC and DPPC membranes without cholesterol the ESR spectra recorded after gradual sample cooling show a broad background signal as well. But if the sample is instantly frozen, the broad background signal is absent and one observes mainly the HB component. We attribute the HB component for n10 to bent conformations of the nitroxide tethers. Such conformations at 9 GHz ESR manifest themselves in apparent “polar” Azz values and increased accessibility of the nitroxide to aqueous Ni2+ ions. These bent conformations prevail in the absence of cholesterol due to the tendency of the pure gel phase to exclude nitroxides, similar to the exclusion of solutes from crystallizing solvents. The formation of a separate phase of spin-labeled lipid (i.e. precipitate) manifests itself in the broad background ESR signal and occurs slowly in the supercooled gel. In membranes with cholesterol the observed HB/ non-HB ratio can best be described by a partition-like equilibrium between nitroxides located in defects of lipid structure in the hydrophobic core of the membrane and those close to the membrane surface.
HF ESR, HFHF ESR: High Field ESR, High Field/High Frequency ESR
EYL: egg yolk lecithin
n-PC spin labels: 1-palmitoyl-2-stearoyl-(n-doxyl)-sn-glycero-3-phosphocholines
TEMPOL, 4-Hydroxy-TEMPO: 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl
TEMPONE, Oxo-TEMPO: 4-oxy-2,2,6,6-tetramethylpiperidine-N-oxyl
This work was supported by the National Institute of Health, grants NIH/NIBIB R010EB003150 and NIH/NCRR P41-RR 016292 and NIH/NIGMS P41GM103521.
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