Article pubs.acs.org/Langmuir
“Staying Out” Rather than “Cracking In”: Asymmetric Membrane Insertion of 12:0 Lysophosphocholine Helen Y. Fan,*,† Dew Das,† and Heiko Heerklotz*,†,‡,§ †
Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada Institute for Pharmaceutical Sciences, University of Freiburg, Freiburg, Germany § BIOSS Centre for Biological Signalling Studies, Freiburg, Germany ‡
S Supporting Information *
ABSTRACT: Interactions between detergents and model membranes are well described by the three-stage model: saturation and solubilization boundaries divide bilayer-only, bilayer−micelle coexistence, and micelle-only ranges. An underlying assumption of the model is the equilibration of detergent between the two membrane leaflets. However, many detergents partition asymmetrically at room temperature due to slow flip-flop, such as sodium dodecyl sulfate (SDS) and lysolipids. In this work, we use isothermal titration calorimetry (ITC) and dynamic light scattering (DLS) to investigate the solubilization of unilamellar POPC vesicles by 12:0 lysophosphocholine (12:0 LPC). Flip-flop of 12:0 LPC occurs beyond the time scale of our experiments, which establish a characteristic nonequilibrated state with asymmetric distribution: 12:0 LPC partitions primarily into the outer leaflet. Increasing asymmetry stress in the membrane does not lead to membrane failure, i.e., “cracking in” as seen for alkyl maltosides and other surfactants; instead, it reduces further membrane insertion which leads to the “staying out” of 12:0 LPC in solution. At above the critical micellar concentration of 12:0 LPC in the presence of the membrane, micelles persist and accommodate further LPC but take up lipid from vesicles only very slowly. Ultimately, solubilization proceeds via the micellar mechanism (Kragh-Hansen et al., 1995). With a combination of demicellization and solubilization experiments, we quantify the molar ratio partition coefficient (0.6 ± 0.1 mM−1) and enthalpy of partitioning (6.1 ± 0.3 kJ·mol−1) and estimate the maximum detergent/lipid ratio reached in the outer leaflet ( −3.7 >0 6 (10 °C)a
set set DDM a
>0 >0
These values are defined for different conditions, shown here for comparison only.
intercept of 0.82 ± 0.07 kJ·mol−1 and −1.88 ± 0.07 kJ·mol−1· mM, respectively (R2 = 0.99). The detergent/lipid ratio in the outer leaflet reaches its aq maximum of Rout, b * when cD approximately reaches the CMC. out, w→b Rb * may thus be estimated through eq 2 and Rout, b *ΔHD,e from above 0.82 kJ ·mol−1 w→b ΔHD,e
the inner leaflet (either by flipping or cracking in) so that solubilization can proceed quickly through membrane disruption. The titration experiments were designed to minimize the progress of micellar solubilization. By excluding all alternatives, we could thus shed light on the conditions required to render the micellar mechanism relevant for a system. The rate and precise mechanism of the subsequent, slow lipid transfer to micelles (e.g., by collisions or monomer transfer) have not been addressed here. The other possible mechanism of solubilization resulting from asymmetry stress is “shedding”. It has been described as the growth and detachment of mixed micelles out of the outer leaflet over a wide range of concentrations.46 This process is expected to be exothermic overall with the transfer of lipids to micelles. However, solubilization would likely proceed relatively quickly beyond its onset, since the half-time of lysolipid partitioning into the outer leaflet is ∼50−500 ms.59 It is likely that the solubilization profile involving shedding would have a similar coexistence range, i.e., a similar ITC pattern, as those for detergents with fast flip-flop. In addition, shedding would likely involve mixed micelles with low detergent/lipid ratios or vesicles with a considerable Rout, b *. The mixed micelles would typically be worm-like, giving rise to a large hydrodynamic size and significant contribution to the DCR. The above properties are incongruent with the premise of “staying out”, and do not appear to be important for the system studied here. An additional process that may result from asymmetric detergent distributions is “budding off”, or exovesiculation of very small vesicles (with substantially larger area in their outer leaflet than in the inner). This is another means of relaxing asymmetry stress which allows for an overall higher Rout, b *. The wider size distribution of post-ITC vs pre-ITC in Figure 2B indicates that budding off does occur to some extent in the case of 12:0 LPC. However, budding off is limited to the stretching out of undulations or nonspherical shapes by osmotic effects: Many small spheres have a much smaller total volume than a large sphere with the same total surface area. Budding off from smooth, spherical liposomes would therefore increase the internal salt concentration and give rise to enormous osmotic pressures, which is prohibited at physiological salt concentrations. The fluorinated surfactant 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro-n-octylphosphocholine (F6OPC) has also been shown to partition very weakly into the membrane, with Rsat e of 0.031.60 In contrast to 12:0 LPC, F6OPC micelles have a poor tolerance for lipid and do not solubilize POPC vesicles even with extended exposure, high concentrations, and elevated temperature. While F6OPC may become asymmetrically distributed in the membrane upon partitioning, the coexistence of F6OPC micelles with vesicles should not be confused with “staying out” as described here, which arises from hindered bilayer flip-flop rather than an inability to solubilize vesicles. Functional Consequences of “Staying Out”. We have attempted to shed light on the partitioning and solubilizing
= R bout, * = K *·CMC (5)
where K* is the partition coefficient at the point where reached. In addition, 0, * ΔGstress = RT ln
K0 K*
Rout, b *
is
(6)
Given that asymmetry stress leads to an unfavorable, nonequilibrium state that relaxes as kinetics permit, we may assume w→b ΔG0,stress * > 0 so that K* < K0. This limits Rout, b * and ΔHD,e , as illustrated in the first row of Table 2. Assuming further that, as seen for alkyl maltosides, the stress causes a penalty to enthalpy (i.e., ΔHstress * > 0), we obtain a limiting ratio of 0.13 in the outer leaflet and a penalty of >2.7 kJ·mol−1 to the free energy. Why 12:0 LPC Stays Out and DDM Cracks In. It is intriguing that dodecyl maltoside (DDM) and 12:0 LPC follow two fundamentally different pathways in response to asymmetry stress. Despite having the same number of carbons in their tail and head groups of comparable size, the former “cracks in” and the latter “stays out”. We propose that the response of a system to asymmetry stress is governed by two parameters: i. the standard free energy of membrane insertion (related to the partition coefficient), which limits the maximal asymmetry stress induced by the surfactant, and ii. the activation free energy of surfactant translocation to the inner leaflet, which determines the stress threshold for cracking in (i.e., the Re limit for asymmetric insertion). DDM has a roughly 10-fold larger partition coefficient (∼5 mM−1) than 12:0 LPC. At the same time, one would expect the nonionic maltoside group to require less activation free energy to be forced through the hydrophobic core of the membrane (i.e., crack in) than the zwitterionic PC group. This suggests that a hypothetical Rout b of cracking in for 12:0 LPC may be even higher than the 0.34 of DDM. However, this “cracking in” limit is never reached by 12:0 LPC because weak partitioning makes it “stay out” already at a weaker asymmetry (see Table 2). Micellar Solubilization, Shedding, and Budding Off. “Staying out” can be considered as a prerequisite for solubilization via the micellar mechanism, which is defined here as the recruitment of lipids from the outer membrane leaflet into pre-existing micelles that are originally virtually lipid-free. This transfer is generally very slow (typically needing tens of hours); it is negligible if the surfactant is able to access F
DOI: 10.1021/acs.langmuir.6b03292 Langmuir XXXX, XXX, XXX−XXX
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properties of lysolipids by improving the quantitative and mechanistic understanding of interactions between 12:0 LPC and a model POPC membrane. In doing so, we hope to have clarified the roles of lysolipids in processes such as the gating of MscL channels in vitro61 and the slow release of drugs from lipid-based drug delivery solutions. Asymmetry in the number of phospholipids and thus in membrane tension between the two leaflets is observed to drive endocytic vesiculation in living cells; this endocytosis is inhibited by the addition of lysolipids which reduce membrane tension asymmetry.62 Endocytosis is also inhibited under external hypoosmotic pressure and does not recover with the addition of lysolipids.63 These phenomena arise through the asymmetric distribution of lysolipids in the absence of flippase transport. Membrane permeant molecules such as cholesterol may buffer asymmetry stress by redistributing in favor of the underpopulated leaflet, whereas antibiotic peptides and pharmaceutical permeation enhancers may utilize “cracking in” to permeate or permeabilize biological membranes.36,39 The staying out phenomenon discussed here raises the question of whether the biological control of membrane asymmetry stress might also contribute to a regulation of the membrane binding of amphiphilic drugs and biomolecules (including lysolipids). At the same time, weakly partitioning amphiphiles such as LPC might act as “pressure valves” that are released from the membrane when the lateral pressure in the respective leaflet is too high. Thus, they could also oppose the activity of certain membrane permeabilizers.
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
We are grateful to Dr. Sandro Keller and Johannes Klingler of the University of Kaiserslautern for helpful discussions. We also thank the Natural Sciences and Engineering Research Council of Canada for funding to H.H. and scholarship to H.Y.F.
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CONCLUSIONS Surfactants are known to translocate in the membrane through spontaneous flip-flop or transient defects induced by asymmetry stress (“cracking in”). In contrast, 12:0 LPC does not translocate across a lipid membrane (here POPC) at room temperature. Instead, the stress induced by the overpopulation of the outer (i.e., accessible) leaflet relative to the inner opposes further uptake and substantially reduces the partition coefficient. Consequently, added 12:0 LPC remains mostly in the aqueous phase; once it reaches the CMC, pure surfactant micelles persist. Further uptake into the membrane halts (“staying out”) as long as no lipid is extracted. The transfer of lipid to micelles is very slow (negligible for over 6 h up to 3 mM POPC) and drives what has been referred to as “micellar solubilization”. Liposomes can therefore resist high detergent/ lipid ratios (e.g., 15) over several hours, although much less 12:0 LPC suffices to solubilize all lipid into mixed micelles under equilibrated conditions. The “staying out” scenario must be expected to have significant biological consequences: for changes in membrane shape that accompany processes such as fusion or endo- and exocytosis and for the regulation of the partitioning of drugs and biomolecules by changes in the local pressure of the accessible leaflet.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b03292. ITC solubilization experiments and DLS size distributions (PDF) G
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