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Comments On Wet Phospholipid Bilayers As Disclosed by the Nearest-Neighbor Recognition Method
The so-called “nearest-neighbor recognition” method, developed in the Regen laboratory,1-8 utilizes dimeric phospholipids connected by an S-S linkage (Chart 1). If two different homodimers, designated as A-A and B-B in the chart, are mixed in equal amounts within a vesicle bilayer, then an equilibrium with the heterodimer A-B (or its equivalent, B-A) can be established catalytically. In the absence of a perturbing effect, one would expect a statistical ratio for A-A:A-B:B-B of 1:2:1. In such a case, the equilibrium constant K ) [A-B]2/[A-A][B-B] ) 4.0. If the equilibrium favors the homodimers, then K would be less than 4. K
A-A + B-B y\z 2A-B
(1)
The lipids in Chart 1 presumably form vesicles although their size, morphology, and monodispersity were not specified as a function of K.8 Experiments were carried out at 60 °C where the bilayers are “fluid”. Regen et al. reported that K decreases from 3.92 in H2O to 1.80 or 1.56 in D2O (both values being presented in their table of data).8 Remarkably, when as little as 2% H2O was added to the D2O, the K shot back up to 4.00. When cholesterol (29 mol %) was mixed in with the lipids, K decreased to 2.0 in both H2O and D2O. The above results were explained by invoking a “wet” bilayer interior. In pure D2O, or in the presence of cholesterol, “the hydrocarbon interior is dehydrated and more tightly packed.”8 Tight packing ostensibly amplifies homodimer selectivity, although the structural basis of this effect was not made clear. (A single experiment at a temperature below 60 °C, where the bilayer is no longer “fluid”, would have been useful in this regard.) Nor is it clear why the hydration perturbations were relegated to the hydrocarbon chains as opposed to the more polar headgroup region. And Regen et al.8 provided no specific information on the water concentration (i.e., the “wetness”) within the membrane core that ostensibly gives rise to the solvent isotope effect. Finally, it was not made clear why the vesicle prefers H2O over D2O to such an extent that the bilayer interior can “extract” only H2O molecules from a mixture containing 2% H2O plus 98% D2O. To support their thesis, Regen et al.8 cite prominently a 1994 paper of Subczynski et al.9 in which spin-label (1) Krisovitch, S. M.; Regen, S. L. J., Am. Chem. Soc. 1992, 114, 9828. (2) Davidson, S. M. K.; Liu, Y.; Regen, S. L. J. Am. Chem. Soc. 1993, 115, 10104. (3) Dewa, T.; Vigmond, S. J.; Regen, S. L. J. Am. Chem. Soc. 1996, 118, 3435. (4) Sugahara, M.; Uragami, M.; Regen, S. L. J. Am. Chem. Soc. 2002, 124, 4253. (5) Sugahara, M.; Uragami, M.; Regen, S. L. J. Am. Chem. Soc. 2003, 125, 13040. (6) Cao, H.; Tokutake, N.; Regen, S. L. J. Am. Chem. Soc. 2003, 125, 16182. (7) Zhang, J.; Jing, B.; Tokutake, N.; Regen, S. L. J. Am. Chem. Soc. 2004, 126, 10856. (8) Tokutake, N.; Jing, B.; Regen, S. L. Langmuir 2004, 20, 8958. (9) Subczynski, W. K.; Wisniewska, A.; Yin, J.-J.; Hyde, J. S.; Kusumi, A. Biochemistry 1994, 33, 7670.
Figure 1. Schematics of the Regen lipids: (A) reproduced from ref 1; (B) redrawn to include all-anti conformations in the headgroup loop. Note the poor chain/chain overlap in both cases. Note also in A the energetic s-cis amide bond and the O-/OdC interaction. Chart 1
data suggest that the core region of bilayer membranes have polarities comparable to 2-propanol and that, therefore, “saturated phospholipid membranes provide poor hydrophobic barriers.”9 On the other hand, Regen et al.8 declined a clear opportunity to cite opposing conclusions. For example, we ourselves have shown by 13C NMR that static water is absent at five locations along bilayer chains (in sharp contrast to the situation with micelles).10 And molecular dynamics calculations11 (also not mentioned8) detect no water penetration in bilayers inside of the ester carbonyls. Given the uncertain status of internal membrane hydration, and given Regen’s creative new method for (10) Menger, F. M.; Aikens, P.; Wood, M., Jr. J. Chem. Soc., Chem. Commun. 1988, 180. (11) Tieleman, D. P.; Marrink, S. J.; Berendsen, H. J. C. Biochim. Biophys. Acta 1997, 1331, 235.
10.1021/la047638z CCC: $30.25 © 2005 American Chemical Society Published on Web 02/02/2005
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studying the subject, it seemed fitting to scrutinize “nearest-neighbor recognition” method in greater detail. First and foremost, one must examine the data themselves. Regen’s K values span a factor of only about 2.8 Thus, the aforementioned disparity of 1.56 and 1.80 for K in D2O represents an appreciable uncertainty. Of even greater concern, the K for vesicles with 29% cholesterol was given as 2.0,8 whereas the value for the same system in an earlier publication was 2.9.2 Let us assume a K ) 1.80 in D2O from which it is possible to calculate a heterodimer-to-homodimer ratio of 1.34. Thus, switching from H2O to D2O decreases this ratio from 2.00 to 1.34. Cholesterol (29 mol %) causes a similar decrease. The main question relates, of course, to the physical-chemical origin of the ratio changes. Consider the cholesterol effect, for example. As just mentioned, Regen et al. ascribed the cholesterol-enhanced preference for homodimer to dehydration.8 But in a previous paper they cited “nearest neighbor recognition” as the mechanism for the cholesterol effect.5 That is to say, it was proposed that cholesterol favors longer chain lipids (e.g., B-B) as its nearest neighbors, thereby lowering K.5 This is an interesting explanation but quite different from their subsequently published mechanism involving cholesterolinduced expulsion of internalized water. A final point bears on the interpretation of the data. The dimeric lipids used in the Regen method are by no means “normal”. For one thing, Regen’s lipids are dianionic, as opposed to the more prevalent zwitterionic state, and it is not known how molecular packing in a vesicle (12) Fraser, R. R.; Boussard, G.; Saunders: J. K.; Lambert, J. B.; Mixan, C. E. J. Am. Chem. Soc. 1971, 93, 3822.
Comments
bilayer would be affected by an anionic surface charge. More importantly, schematics of the dimeric lipids (as given by Regen in Figure 1A8 and redrawn by us in Figure 1B to include all-anti conformations in the headgroup loop) both show decided problems in achieving optimal chainoverlap. Exacerbating the problem is a dihedral angle about the RS-SR bond of 90° with a rotational barrier of 7-9 kcal mol.12 It is difficult to speculate how the Regen lipids might self-assemble in a bilayer so as to maximize chain-chain interactions while minimizing “unfilled” space. Interdigitation and/or introduction of energetic headgroup conformations are two possibilities, but whatever Nature’s solution to this problem, one can reasonably conclude that the resulting bilayers are not faithful replications of the real thing. In effect, Regen’s vesicles are composed entirely of probe molecules. Both inner and outer vesicle surfaces are abnormally carpeted with long loops linking phospholipids into molecular couplets. The transient and static water content of biological membranes remains an important and as yet unresolved issue. Acknowledgment. This work was supported by the National Institutes of Health. Fredric M. Menger,* Hailing Zhang, and Ashley L. Galloway
Department of Chemistry, Emory University, Atlanta, Georgia 30322 Received September 22, 2004 In Final Form: December 6, 2004 LA047638Z
