The importance of the phospholipid bilayer and the length of the cholesterol molecule in membrane structure

The properties of mixtures of phosphatidylcholine and analogues of cholesterol bearing side chains of varying lengths were examined by a variety of methods. The incorporation of the analogues into sonicated liposomes and their effect on the rate of osmotic shrinking of multilamellar liposomes were determined. The ordering of a steroid spin label was studied in an oriented multibilayer system and the effect of the analogues on the phase transition of dipalmitoyl phosphatidylcholine monitored using the spin label TEMPO ($\text{2,2,6,6-}\text{tetramethylpiperidine}\text{-N-}\text{oxyl}$). Mixtures of analogues and phospholipid were also studied in monolayers.In all the bilayer systems studied cholesterol caused the greatest ‘rigidifying’ effect, the analogues with shorter or longer side chains being less effective. However, in the monolayer experiments the length of the sterol molecule was found to be much less critical. It is suggested that cholesterol is anchored in position in a phospholipid bilayer by virtue of the molecule being the precise length required to maximise interactions between neighbouring molecules without disturbing the bilayer structure.     

Alignment of cholesterol in the membrane bilayer

Freeze-fracture replicas of filipin-treated samples of guinea pig colon mucosa reveal areas in the membrane of the goblet cell granules labeled by filipin-cholesterol complexes (FCC) intermingled with regions patterned by "lines." The FCC and "lines" are arranged in an approximately rhombic pattern. Other membranes of the same cell or of other cells display either FCC only, aligned and occasionally ordered in "rhombs," "lines" only, with a similar pattern, or randomly distributed FCC. Optical diffraction was used to analyze and compare replicas of membranes with ordered FCC and "lines", as well as randomly distributed FCC. The results demonstrate that all these structures are reciprocally related through a common distribution pattern in the membrane. This observation supports the assumption that cholesterol has a preferential ordered distribution within the membrane bilayer.     

Effect of the orientation of dipole fragments of the phospholipid molecule on the bilayer structure

By the method of minimization of phospholipid bilayers free energy it was shown that at a decrease of inclination angle of dipole fragments of phospholipid molecules to the bilayer surface there take place: a) an increase of bilayer density, b) a decrease of bilayer thickness caused by a decrease of dipole repulsive forces, c) an increase of the number of molecules in the bilayer due to hydrophobization of the latter. The existence of upper and lower critical angles of dipoles inclination to the bilayer surface were found: theta upcrit, theta lowcrit, if theta greater than or equal to the layers must be destroyed, and if theta less than or equal to theta lowcrit, transition of bilayers to the crystalline state must take place.     

The immiscible cholesterol bilayer domain exists as an integral part of phospholipid bilayer membranes

Electron paramagnetic resonance (EPR) spin-labeling methods were used to study the organization of cholesterol and phospholipids in membranes formed from Chol/POPS (cholesterol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine) mixtures, with mixing ratios from 0 to 3. It was confirmed using the discrimination by oxygen transport and polar relaxation agent accessibility methods that the immiscible cholesterol bilayer domain (CBD) was present in all of the suspensions when the mixing ratio exceeded the cholesterol solubility threshold (CST) in the POPS membrane. The behavior of phospholipid molecules was monitored with phospholipid analogue spin labels (n-PCs), and the behavior of cholesterol was monitored with the cholesterol analogue spin labels CSL and ASL. Results indicated that phospholipid and cholesterol mixtures can form a membrane suspension up to a mixing ratio of ~2. Additionally, EPR spectra for n-PC, ASL, and CSL indicated that both phospholipids and cholesterol exist in these suspensions in the lipid-bilayer-like structures. EPR spectral characteristics of n-PCs (spin labels located in the phospholipid cholesterol bilayer, outside the CBD) change with increase in the cholesterol content up to and beyond the CST. These results present strong evidence that the CBD forms an integral part of the phospholipid bilayer when formed from a Chol/POPS mixture up to a mixing ratio of ~2. Interestingly, CSL in cholesterol alone (without phospholipids) when suspended in buffer does not detect formation of bilayer-like structures. A broad, single-line EPR signal is given, similar to that obtained for the dry film of cholesterol before addition of the buffer. This broad, single-line signal is also observed in suspensions formed for Chol/POPS mixtures (as a background signal) when the Chol/POPS ratio is much greater than 3. It is suggested that the EPR spin-labeling approach can discriminate and characterize the fraction of cholesterol that forms the CBD within the phospholipid bilayer.     

Abrupt modifications of phospholipid bilayer properties at critical cholesterol concentrations.

The fluorescence generalized polarization (GP) of 2-dimethylamino-6-lauroylnaphthalene (Laurdan) reveals different effects of cholesterol on the phase behavior of phospholipid bilayers. Phospholipid vesicles composed of gel, liquid-crystalline, and coexisting domains of the two phases have been studied at temperatures from 1 to 65 degrees C, without cholesterol and with cholesterol concentrations of 3-50 mol %. Laurdan GP measurements show the general effect of cholesterol of increasing the molecular dynamics of the gel and of decreasing the molecular dynamics of the liquid-crystalline phase. In the liquid-crystalline phase, the increased order yields Laurdan GP values close to those obtained in the gel phase. At cholesterol concentrations > 15 mol % a phase transition cannot be detected. Using the wavelength dependence of the excitation and emission GP spectra we determine that differences between the two phospholipid phases cannot be detected. In particular, in vesicles composed of coexisting gel and liquid-crystalline phases the GP wavelength dependence characteristic of coexisting domains cannot be observed at cholesterol concentrations > or = 15 mol %. Cholesterol causes the decrease in both the polarity and the dipolar relaxation effects on the neighborhood of the fluorescent naphthalene moiety of Laurdan. Probably because of a cholesterol-induced increase in the bilayer packing, these effects do not occur continuously with the increase of cholesterol concentration in the bilayer. Cholesterol concentrations inducing higher Laurdan GP values have been determined at about 5, 10, 15, 30, and 45 mol % with respect to phospholipids. We propose that the formation of ordered molecular microdomains at critical cholesterol concentrations can explain the occurrence of the observed discontinuities.     

Enhanced fluctuations in small phospholipid bilayer vesicles containing cholesterol

Ultrasonic and calorimetric studies of small homogenously-sized DMPC unilamellar vesicles showed two thermal transitions at temperatures T _c1 and T _c2 (T _c2 T _c1 ); T _c2 is close to the phase transition temperature, T _c , of large vesicles. The process at T _c2 is not a fusion of vesicles and is interpreted as characterizing an order-disorder transition essentially similar to that of large vesicles. The temperatures T _c1 and T _c2 become increasingly similar as the cholesterol content is increased, while the clusters at T _c2 (85 lipid molecules in pure DMPC) increase in size up to approximately 180 lipid molecules at 12 mol% cholesterol. Incorporation of cholesterol thus brings about enhanced fluctuations in this model system of a membrane.Abbreviations DMPC dimyristoylphosphatidylcholine - SUV small unilamellar vesicles - LUV large unilamellar vesicles - MLV multilamellar vesicles     

Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II. Incorporation of a vesicular membrane marker into the planar membrane

Fusion of multilamellar phospholipid vesicles with planar phospholipid bilayer membranes was monitored by the rate of appearance in the planar membrane of an intrinsic membrane protein present in the vesicle membranes. An essential requirement for fusion is an osmotic gradient across the planar membrane, with the cis side (the side containing the vesicles) hyperosmotic to the opposite (trans) side; for substantial fusion rates, divalent cation must also be present on the cis side. Thus, the low fusion rates obtained with 100 mM excess glucose in the cis compartment are enhanced orders of magnitude by the addition of 5-10 mM CaCl2 to the cis compartment. Conversely, the rapid fusion rates induced by 40 mM CaCl2 in the cis compartment are completely suppressed when the osmotic gradient (created by the 40 mM CaCl2) is abolished by addition of an equivalent amount of either CaCl2, NaCl, urea, or glucose to the trans compartment. We propose that fusion occurs by the osmotic swelling of vesicles in contact with the planar membrane, with subsequent rupture of the vesicular and planar membranes in the region of contact. Divalent cations catalyze this process by increasing the frequency and duration of vesicle-planar membrane contact. We argue that essentially this same osmotic mechanism drives biological fusion processes, such as exocytosis. Our fusion procedure provides a general method for incorporating and reconstituting transport proteins into planar phospholipid bilayer membranes.     

Conformational transitions in the membrane scaffold protein of phospholipid bilayer nanodiscs

Phospholipid bilayer nanodiscs are model membrane systems that provide an environment where membrane proteins are highly stable and monodisperse without the use of detergents or liposomes. Nanodiscs consist of a discoidal phospholipid bilayer encircled by two copies of an amphipathic alpha helical membrane scaffold protein, which is modeled from apolipoprotein A-1. Hydrogen exchange mass spectrometry was used to probe the structure and dynamics of the scaffold protein in the presence and absence of lipid. On nanodisc self-assembly, the entire scaffold protein gained significant protection from exchange, consistent with a large, protein-wide, structural rearrangement. This protection was short-lived and the scaffold protein was highly deuterated within 2 h. Several regions of the scaffold protein, in both the lipid-free and lipid-associated states, displayed EX1 unfolding kinetics. The rapid deuteration of the scaffold protein and the presence of correlated unfolding events both indicate that nanodiscs are dynamic rather than rigid bodies in solution. This work provides a catalog of the expected scaffold protein peptic peptides in a nanodisc-hydrogen exchange mass spectrometry experiment and their deuterium uptake signatures, data that can be used as a benchmark to verify correct assembly and nanodisc structure. Such reference data will be useful control data for all hydrogen exchange mass spectrometry experiments involving nanodiscs in which transmembrane or lipid-associated proteins are the primary molecule(s) of interest.     

Stopped-flow fluorometric study of the interaction of melittin with phospholipid bilayers: importance of the physical state of the bilayer and the acyl chain length.

Stopped-flow fluorometry has been employed to study the effects of melittin, the major protein component of bee venom, on dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) small unilamellar vesicles (SUVs) on the millisecond time scale, before melittin-induced vesicle fusion takes place. Use is made of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), which is an oriented fluorescent probe that anchors itself to the bilayer-water interface and is aligned parallel to the normal to the bilayer surface; its fluorescence anisotropy reports on the "fluidity" of the bilayer. For DMPC bilayers, melittin is found to decrease their fluidity only at their melting transition temperature. This perturbation appears to be exerted almost instantaneously on the millisecond time scale of the measurements, as deduced from the fact that its rate is comparable to that obtained by following the change in the fluorescence of the single tryptophan residue of melittin upon inserting itself into the bilayer. The perturbation is felt in the bilayer over a distance of at least 50 A, with measurements of transfer of electronic energy indicating that the protein is not sequestered in the neighborhood of TMA-DPH. The length of the acyl chains is found to be an important physical parameter in the melittin-membrane interaction: unlike the case of DMPC SUVs, melittin does not alter the fluidity of DPPC SUVs and has a considerably greater affinity for them. These results are discussed in terms of the concept of elastic distortion of the lipids, which results from a mismatch between the protein and the acyl chains that are attempting to accommodate it. Melittin is also found to cause a small (approximately 10%) enhancement in the total fluorescence intensity of TMA-DPH, which is interpreted as indicating a reduction in the degree of hydration of the bilayer.     

Interaction of amphotericin B and nystatin with phospholipid bilayer membranes:effect of cholesterol.

Sodium-22 efflux was measured in multilamellar liposomes, exposed to one of the two polyene antibiotics amphotericin B or nystatin. Polyene mediated 22Na transport progressively rises with membrane sterol concentrations up to about 20 mol %, but falls with higher cholesterol concentrations. The polyene induced 22Na movement in cholesterol rich liposomes could be 'restored' by the addition of either dibucaine or propranolol (two local anesthetics) to the aqueous solution. These observations are interpreted in terms of the model of De Kruijff and Demel (Biochim. Biophys. Acta, 339, 57-70, 1974). In this model, nystatin and amphotericin B first complex with cholesterol and then these complexes aggregate to form transmembrane channels. It is here proposed that the aggregation of these complexes is inhibited by a high cholesterol content (decreased membrane fluidity) but that the two local anesthetics, by disrupting phospholipid-sterol interactions (increased membrane fluidity), can 'restore' this process of aggregation.     

Applications of phospholipid bilayer nanodiscs in the study of membranes and membrane proteins

Phospholipid bilayer Nanodiscs are novel model membranes derived from high-density lipoprotein particles and have proven to be useful in studies of membrane proteins. Membrane protein enzymology has been hampered by the inherent insolubility of membrane proteins in aqueous environments and has necessitated the use of model membranes such as liposomes and detergent-stabilized micelles. Current model membranes display a polydisperse particle size distribution and can suffer from problems of inconsistency and instability. It is also unclear how well they mimic biological lipid bilayers. In contrast, Nanodiscs, the particle size of which is constrained by a coat of scaffold proteins, are relatively monodisperse, stable model membranes with a "nativelike" lipid bilayer. Nanodiscs have already been used to study a variety of membrane proteins, including cytochrome P450s, seven-transmembrane proteins, and bacterial chemoreceptors. These proteins are simultaneously monomerized, solubilized, and incorporated into the well-defined membrane environment provided by Nanodiscs. Nanodiscs may also provide useful insights into the thermodynamics and biophysics of biological membranes and binding of small molecules to membranes.     

The permeability and the effect of acyl-chain length for phospholipid bilayers containing cholesterol: theory and experiment.

The model of Cruzeiro-Hansson et al. (Biochim. Biophys. Acta (1989) 979, 166-1176) for lipid-cholesterol bilayers at low cholesterol concentrations is used to predict the thermodynamic properties and the passive ion permeability of lipid bilayers as a function of acyl-chain length and cholesterol concentration. Numerical simulations based on the Monte Carlo method are used to determine the equilibrium state of the system near the main gel-fluid phase transition. The permeability is calculated using an ansatz which relates the passive permeability to the amount of interfaces formed in the bilayer when cholesterol is present. The model predicts at low cholesterol contents an increase in the membrane permeability in the transition region both for increasing cholesterol concentration and for decreasing chain length at a given value of the reduced temperature. This is in contrast to the case of lipid bilayers containing high cholesterol concentrations where the cholesterol strongly suppresses the permeability. Experimental results for the Na+ permeability of C15PC and DPPC (C16PC) bilayers containing cholesterol are presented which confirm the theoretical predictions at low cholesterol concentrations.     

Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. I. Discharge of vesicular contents across the planar membrane

Multilamellar phospholipid vesicles are introduced into the cis compartment on one side of a planar phospholipid bilayer membrane. The vesicles contain a water-soluble fluorescent dye trapped in the aqueous phases between the lamellae. If a vesicle containing n lamellae fuses with a planar membrane, an n-1 lamellar vesicle should be discharged into the opposite trans compartment, where it would appear as a discernible fluorescent particle. Thus, fusion events can be assayed by counting the number of fluorescent particles appearing in the trans compartment. In the absence of divalent cation, fusion does not occur, even after vesicles have been in the cis compartment for 40 min. When CaCl2 is introduced into the cis compartment to a concentration of greater than or equal to 20 mM, fusion occurs within the next 20 min; it generally ceases thereafter because of vesicle aggregation in the cis compartment. With approximately 3 x 10(8) vesicles/cm3 in the cis compartment, about 25-50 fusion events occur following CaCl2 addition. The discharge of vesicular contents across the planar membrane is the most convincing evidence of vesicle-membrane fusion and serves as a model for that ubiquitous biological phenomenon--exocytosis.     

Non-isothermal potential of phospholipid bilayer films Influence of cholesterol and macrocyclic carrier effects

The effect of cholesterol on the ion selective behavior of phospholipid (phosphatidylcholine or phosphatidylethanolamine) bilayer films is studied through the measurement of the membrane non-isothermal potential.It is shown how the mixed phosphatidylcholine-cholesterol membrane can be either cation of anion permselective according to the film composition (cationic behavior is met in the 0–10% cholesterol composition range while anionic selectivity appears in the 20–50% range).On the contrary, mixed phosphatidylethanolamine-cholesterol membranes show the absence of ionic selectivity already met with pure phosphatidylethanolamine films.The presence of a cationic carrier as Dibenzo-18-crown-6 in the film transforms all the studied films (cationic, anionic and no selective bilayers) into ideally cationic selective membranes.These results are discussed on the basis of the current ideas on the charge distribution through the bilayer membranes. Moreover, the role of the permeating ions as potential determining species is stressed.     

Effects of lipophilic dications on planar bilayer phospholipid membrane and mitochondria

In this paper, we studied effects of phosphonium dications P2C5 and P2C10 on bilayer planar phospholipid membrane (BLM) and rat liver mitochondria. In line with our previous observations [M.F. Ross, T. Da Ros, F.H. Blaikie, T.A. Prime, C.M. Porteous, I.I. Severina, V.P. Skulachev, H.G. Kjaergaard, R.A. Smith, M.P. Murphy, Accumulation of lipophilic dications by mitochondria and cells, Biochem. J. 400 (2006) 199-208], we showed both P2C5 and P2C10 are cationic penetrants for BLM. They generated transmembrane diffusion potential (Delta Psi), the compartment with a lower dication concentration positive. However, the Delta Psi values measured proved to be lower that the Nernstian. This fact could be explained by rather low BLM conductance for the cations at their small concentrations and by induction of some BLM damage at their large concentrations. The damage in question consisted in appearance of non-Ohmic current/voltage relationships which increased in time. Such a non-Ohmicity was especially strong at Delta Psi >100 mV. Addition of penetrating lipophilic anion TPB, which increases the BLM conductance for lipophilic cations, yielded the Nernstian Delta Psi, i.e. 30 mV per ten-fold dication gradient. In the State 4 mitochondria, dications stimulated respiration and lowered Delta Psi. Moreover, they inhibited the State 3 respiration with succinate or glutamate and malate (but not with TMPD and ascorbate) in an uncoupler-sensitive fashion. Effect on the in State 4 mitochondria, similarly to that on BLM, was accounted for by a time-dependent membrane damage. On the other hand, the State 3 effect was most probably due to inhibition of the respiratory chain Complex I and/or Complex III. The damaging and inhibitory activities of lipophilic dications should be taken into account when one considers a possibility to use them as a vehicle to target antioxidants or other compounds to mitochondria.     

Effects of lipophilic dications on planar bilayer phospholipid membrane and mitochondria

In this paper, we studied effects of phosphonium dications P2C5 and P2C10 on bilayer planar phospholipid membrane (BLM) and rat liver mitochondria. In line with our previous observations [M.F. Ross, T. Da Ros, F.H. Blaikie, T.A. Prime, C.M. Porteous, I.I. Severina, V.P. Skulachev, H.G. Kjaergaard, R.A. Smith, M.P. Murphy, Accumulation of lipophilic dications by mitochondria and cells, Biochem. J. 400 (2006) 199-208], we showed both P2C5 and P2C10 are cationic penetrants for BLM. They generated transmembrane diffusion potential (ΔΨ), the compartment with a lower dication concentration positive. However, the ΔΨ values measured proved to be lower that the Nernstian. This fact could be explained by rather low BLM conductance for the cations at their small concentrations and by induction of some BLM damage at their large concentrations. The damage in question consisted in appearance of non-Ohmic current/voltage relationships which increased in time. Such a non-Ohmicity was especially strong at ΔΨ > 100 mV. Addition of penetrating lipophilic anion TPB, which increases the BLM conductance for lipophilic cations, yielded the Nernstian ΔΨ, i.e. 30 mV per ten-fold dication gradient. In the State 4 mitochondria, dications stimulated respiration and lowered ΔΨ. Moreover, they inhibited the State 3 respiration with succinate or glutamate and malate (but not with TMPD and ascorbate) in an uncoupler-sensitive fashion. Effect on the in State 4 mitochondria, similarly to that on BLM, was accounted for by a time-dependent membrane damage. On the other hand, the State 3 effect was most probably due to inhibition of the respiratory chain Complex I and/or Complex III. The damaging and inhibitory activities of lipophilic dications should be taken into account when one considers a possibility to use them as a vehicle to target antioxidants or other compounds to mitochondria.