Anthony Watts

Control of the structure and fluidity of phosphatidylglycerol bilayers by pH titration

Abstract Titration of the single dissociable proton in phosphatidylglycerol bilayers not only shifts the ordered-fluid phase transition but also changes the bilayer fluidity in the region above the phase transition, and gives rise to a quite different bilayer structure in the region below the phase transition: 1. 1. The ordered-fluid phase transition temperatures of dimyristoyl and dipalmitoyl phosphatidylglycerol bilayers have been measured as a function of bulk pH in 0.1 M salt using water-lipid partitioning spin labels. From the dissociation curve obtained, it is found that the one titrable proton has an apparent p K a of 2.9 for bilayers of both lipids. 2. 2. In the fully ionized state, these phosphatidylglycerols not only give very similar transition temperatures (23°C for dimyristoyl and 40°C for dipalmitoyl chains) and pre-transition temperatures to the similar chain length phosphatidylcholines, but also show identical bilayer structures at corresponding temperatures when examined by freeze-fracture electron microscopy: i.e. defects, ripples and jumbled patterns, in the ordered, pre-transitional and fluid phases, respectively. 3. 3. When the phosphatidylglycerols are fully protonated, the bilayer pre-transition, as monitored by the spin labels, is absent and the main transition is somewhat broader and increased in temperature by approx. 17°C. Exclusively smooth bilayers are observed by electron microscopy at all temperatures. This suggests that, contrary to the situation in the charged bilayers, the phosphatidylglycerol molecules are not tilted relative to the bilayer normal in the ordered phase. 4. 4. In fluid bilayers, above the main transition temperature, the fluidity is found to be greater when the phosphatidylglycerol molecules are charged than when uncharged, due to the increased intermolecular separation caused by electrostatic repulsion. The results demonstrate that the structure and fluidity of charged lipid bilayer membranes can be changed isothermally without the mediation of the ordered-fluid phase transition.

Cooperativity of the phase transition in single- and multibilayer lipid vesicles

The effect of membrane morphology on the cooperativity of the ordered-fluid, lipid phase transition has been investigated by comparing the transition widths in extended, multibilayer dispersons of dimyristoyl phosphatidyl-choline, and also of dipalmitoyl phosphatidylcholine, with those in the small, single-bilayer vesicles obtained by sonication. The electron spin resonance spectra of three different spin-labelled probes, 2,2,6,6-tetramethylpiperdine-N-oxyl, phosphatidylcholine and stearic acid, and also 90 degrees light scattering and optical turbidity measurements were used as indicators of the phase transition. In all cases the transition was broader in the single-bilayer vesicles than in the multibilayer dispersions, corresponding to a decreased cooperativity on going to the small vesicles. Comparison of the light scattering properties of centrifuged and uncentrifuged, sonicated vesicles suggests that these are particularly sensitive to the presence of intermediate-size particles, and thus the spin label measurements are likely to give a more reliable measure of the degree of cooperativity of the small, single-bilayer vesicles. Application of the Zimm and Bragg theory ((1959) J. Chem. Phys. 31, 526-535) of cooperative transitions to the two-dimensional bilayer system shows that the size of the cooperative unit, 1/square root sigma, is a measure of the mean number of molecules per perimeter molecule, in a given region of ordered or fluid lipid at the centre of the transition. From this result it is found that it is the vesicle size which limits the cooperativity of the transition in the small, single-bilayer vesicles. The implications for the effect of membrane structure and morphology on the cooperativity of phase transitions in biological membranes, and for the possibility of achieving lateral communication in the plane of the membrane, are discussed.

ESR spin-label studies of lipid-protein interactions in membranes.

Lipid spin labels have been used to study lipid-protein interactions in bovine and frog rod outer segment disc membranes, in (Na+, K+)-ATPase membranes from shark rectal gland, and in yeast cytochrome oxidase-dimyristoyl phosphatidylcholine complexes. These systems all display a two component ESR spectrum from 14-doxyl lipid spin-labels. One component corresponds to the normal fluid bilayer lipids. The second component has a greater degree of motional restriction and arises from lipids interacting with the protein. For the phosphatidylcholine spin label there are effectively 55 +/- 5 lipids/200,000-dalton cytochrome oxidase, 58 +/- 4 mol lipid/265,000 dalton (Na+, K+)-ATPase, and 24 +/- 3 and 22 +/- 2 mol lipid/37,000 dalton rhodopsin for the bovine and frog preparations, respectively. These values correlate roughly with the intramembrane protein perimeter and scale with the square root of the molecular weight of the protein. For cytochrome oxidase the motionally restricted component bears a fixed stoichiometry to the protein at high lipid:protein ratios, and is reduced at low lipid:protein ratios to an extent which can be quantitatively accounted for by random protein-protein contacts. Experiments with spin labels of different headgroups indicate a marked selectivity of cytochrome oxidase and the (Na+, K+)-ATPase for stearic acid and for cardiolipin, relative to phosphatidylcholine. The motionally restricted component from the cardiolipin spin label is 80% greater than from the phosphatidylcholine spin label for cytochrome oxidase (at lipid:protein = 90.1), and 160% greater for the (Na+, K+)-ATPase. The corresponding increases for the stearic acid label are 20% for cytochrome oxidase and 40% for (Na+, K+)-ATPase. The effective association constant for cardiolipin is approximately 4.5 times greater than for phosphatidylcholine, and that for stearic acid is 1.5 times greater, in both systems. Almost no specificity is found in the interaction of spin-labeled lipids (including cardiolipin) with rhodopsin in the rod outer segment disc membrane. The linewidths of the fluid spin-label component in bovine rod outer segment membranes are consistently higher than those in bilayers of the extracted membrane lipids and provide valuable information on the rate of exchange between the two lipid components, which is suggested to be in the range of 10(6)-10(7) s-1.

Charge-induced tilt in ordered-phase phosphatidylglycerol bilayers Evidence from x-ray diffraction

X-ray diffraction studies have been performed, as a function of water content, on dipalmitoyl phosphatidylglycerol bilayers, both in the charged state at pH 8.0 and in the protonated state at pH 1.5, using buffers of 1.5 M salt concentration. Measurements were made at 20 degrees C, and the high-angle reflections indicated that the bilayers were in the ordered phase at both pH values. Lamellar diffractions were observed under all conditions studied. THe lamellar repeat reached a limiting value of 62.4 A (6.24 nm) at a water/lipid ratio of 0.24 at pH 8.0, and a limiting value of 67.3 A (6.73 nm) at a water/lipid ratio of 0.22 at pH 1.5. The area per lipid molecule in the plane of the bilayer, deduced from the bilayer thickness and the lipid partial specific volume, is 48 A2 (0.48 nm2) at pH 8.0 and 37 A2 (0.37 nm2) at pH 1.5. The area per molecule in the plane perpendicular to the chain axes, deduced from the X-ray short spacings, is 40.5 A2 (0.405 nm2) at pH 8.0 and 39.2 A2 (0.392 nm2) at pH 1.5. Thus the lipid molecules are tilted by approx. 30 degrees relative to the bilayer normal at pH 8.0, but are essentially untilted at pH 1.5.

Non-electrostatic contribution to the titration of the ordered-fluid phase transition of phosphatidylglycerol bilayers.

The ordered-fluid phase transition of phospholipid bilayers is of interest not only because it yields information on the nature of the fluid bilayer regions in biomembranes, but also because it may provide a means for triggering lateral phase separation between the ordered and fluid lipids in membranes [ 1,2]. It has been shown [3-61 that the phase transition of negatively charged lipid bilayers shows strong temperature shifts on titrating the phosphate group of the lipid polar head. This provides a way of triggering the phase transition at constant temperature by varying the pH. The free energy of the bilayer made of protonated lipids, G* = H* X.7*, changes upon deprotonation to G* GPr and this change results in a shift of the phase transition temperature from q = LUYF/LU~ to Tt , given by:

Lipid-lipid and lipid-protein interactions in chromaffin granule membranes. A spin label ESR study.

The ESR spectra of six different positional isomers of a stearic acid and three of a phosphatidylcholine spin label have been studied as a function of temperature in chromaffin granule membranes from the bovine adrenal medulla, and in bilayers formed by aqueous dispersion of the extracted membrane lipids. Only minor differences were found between the spectra of the membranes and the extracted lipid, indicating that the major portion of the membrane lipid is organized in a bilayer arrangement which is relatively unperturbed by the presence of the membrane protein. The order parameter profile of the spin label lipid chain motion is less steep over the first half of the chain than over the section toward the terminal methyl end of the chain. This stiffening effect is attributed to the high proportion of cholesterol in the membrane and becomes less marked as the temperature is raised. The isotropic hyperfine splitting factors of the various positional isomers display a profile of decreasing polarity as one penetrates further into the interior of the membrane. No marked differences are observed between the effective polarities in the intact membranes and in bilayers of the extracted membrane lipids. The previously observed temperature-induced structural change occurring in the membranes at approx. 35 degrees C was found also in the extracted lipid bilayers, showing this to be a result of lipid-lipid interactions and not lipid-protein interactions in the membrane. A steroid spin label indicated a second temperature-dependent structural change occurring in the lipid bilayers at lower temperatures. This correspond to the onset of a more rapid rotation about the long axis of the lipid molecules at a temperature of approx. 10 degrees C. The lipid bilayer regions probed by the spin labels used in this study may be involved in the fusion of the chromaffin granule membrane leading to hormone release by exocytosis.

Distinct states of lipid mobility in bovine rod outer segment membranes Resolution of spin label results

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0 degrees C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24 degrees C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0 degrees C and a considerable temperature dependence. This component which is not resolved at high temperatures (24--35 degrees C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62--64 G at 0 degrees C, with very little temperature dependence (61--62 G at 35 degrees C), and is attributed to protein aggregation.

Membrane structure and dynamics.

Research into membrane structure and dynamics has been actively pursued for over live decades by physicists, chemists, biologists and biophysicists, of both a practical and a theoretical persuasion, yet there is still a wealth of new information being produced. The resolution of membrane structure is progressing on many fronts, but in the past most of the attention has been paid to the hydrocarbon core of the bilayer structural element and how changes here can modulate biological function. As a result of developments in both the technology and methodology for handling membranes, even a structural description of integral proteins is possible for some limited cases.

Protein-immobilized lipid in dimyristoylphosphatidylcholine-substituted cytochrome oxidase: Evidence for both boundary and trapped-bilayer lipid

Abstract Cytochrome oxidase-dimyristoyl phosphatidylcholine complexes have been prepared at defined lipid:protein ratios to study the effects of protein packing density on the lipid fluidity. All the complexes reveal a two-component ESR spectrum from an incorporated phosphatidylcholine spin label, corresponding to both an immobilized lipid boundary layer and fluid bilayer regions. Difference spectra, obtained by subtracting the same immobilized spectrum from the spectra of the various complexes, demonstrate a strong perturbation of the lipid bilayer fluidity which is quite distinct from the immobilized boundary layer formation.

Effect of cholesterol on rhodopsin stability in disk membranes

The effect of cholesterol on rhodopsin stability has been investigated in intact disk membranes. Because cholesterol readily equilibrates between membranes, the disk membrane cholesterol content can be altered by incubation with cholesterol/phospholipid vesicles. The effect of membrane cholesterol on rhodopsin was investigated using three independent techniques: thermal bleaching, differential scanning calorimetry (DSC) and activation of the cGMP cascade. Rhodopsin exhibited an increased resistance to thermally induced bleaching as the membrane cholesterol level was increased. DSC also indicated that the protein is stabilized by cholesterol in that the Tm increased in response to higher membrane cholesterol. A similar degree of stabilization was observed in both the unbleached and bleached states in the DSC experiments. These results suggest that cholesterol affects the disk membrane properties such that thermally induced unfolding is inhibited, thus stabilizing the rhodopsin structure. Furthermore, high membrane cholesterol inhibited the activation of the cGMP cascade. This is consistent with the stabilization of the metarhodopsin I photointermediate relative to the metarhodopsin II intermediate.