Interactions of proteins and cholesterol with lipids in bilayer membranes.

Mixtures of lipids and protein, the ATPase from rabbit sarcoplasmic reticulum, were studied by freeze-fracture electron microscopy and by measurement of the amount of fluid lipid with the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl (TEM-PO). In dimyristoyl phosphatidylcholine vesicles the protein molecules were randomly distributed above the transition temperature, Tt, of the lipid and aggregated below Tt. For mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine the existence of fluid and solid domains were shown in the temperature interval predicted from earlier TEMPO measurements. When protein was incorporated into this lipid mixture, freeze-fracture particles were randomly distributed in fluid lipids, or aggregated when only solid lipids were present. In mixtures of dimyristoyl phosphatidylcholine with cholesterol the protein was distributed randomly above the transition temperature of the phosphatidylcholine. Below that transition temperature the protein was excluded from a banded phase of solid lipid in the case of 10 mol% cholesterol. In mixtures containing 20 mol% cholesterol, protein molecules formed linear arrays, 50-200 nm in length, around smooth patches of lipid. Phase diagrams for lipid/cholesterol and lipid/protein systems are proposed which account for many of the available data. A model for increasing solidification of lipid around protein molecules or cholesterol above the transition temperature of the lipid is discussed.     

Interactions of proteins and cholesterol with lipids in bilayer membranes

Mixtures of lipids and proteins, the ATPase from rabbit sarcoplasmic reticulum, were studied by freeze-fracture electron microscopy and by measurement of the amount of fluid lipid with the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO). In dimyristoyl phosphatidylcholine vesicles the protein molecules were randomly distributed above the transition temperature, $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$, of the lipid and aggregated below $\text{T}{}_{\mathrm{<mtext>t</mtext>}}$. For mixtures af dimyristoyl and dipalmitoyl phosphatidylcholine the existence of fluid and solid domains was shown in the temperature interval predicted from earlier TEMPO measurements. When protein was incorporated into this lipid mixture, freeze-fracture particles were randomly distributed in fluid lipids, or aggregated when only solid lipids were present.In mixtures of dimyristoyl phosphatidylcholine with cholesterol the protein was distributed randomly above the transition temperature of the phosphatidylcholine. Below that transition temperature the protein was excluded from a banded phase of solid lipid in the case of 10 mol% cholesterol. In mixtures containing 20 mol% cholesterol, protein molecules formed linear arrays, 50–200 nm in length, around smooth patches of lipid.Phase diagrams for lipid/cholesterol and lipid/protein systems are proposed which account for many of the available data. A model for increasing solidification of lipid around protein molecules or cholesterol above the transition temperarture of the lipid is discussed.     

Low-frequency collective modes in dry and hydrated proteins.

We have observed Brillouin-like low frequency collective modes in the scattering of 1 A neutrons from a fully in vivo deuterated protein. These modes are tentatively interpreted as due to short-lived coherent excitations propagating with velocities between 2,000 and 4,000 m/s in elements of the secondary structure and patches of closely associated water.     

Lateral diffusion in inhomogeneous membranes. Model membranes containing cholesterol.

The problem of lateral diffusion in inhomogeneous membranes is illustrated by a theoretical calculation of the lateral diffusion of a fluorescent lipid probe in binary mixtures of phosphatidylcholine and cholesterol under conditions of temperature and composition such that this lipid mixture consists of alternating parallel domains of fluid and solid lipid, having separations that are small compared with the distance scale employed in photobleaching experiments. The theoretical calculations clearly illustrate how inhomogeneities in membrane composition affecting the lateral motion of membrane components on a small (10-100 nm) distance scale can give complex diffusive responses in experiments such as fluorescence photobleaching that employ comparatively macroscopic distances (10-100 micrometers) for the measurement of diffusive recovery. The theoretical calculations exhibit the unusual dependence of the apparent lateral diffusion coefficient of a fluorescent lipid probe on lipid composition in binary mixtures of cholesterol and phosphatidylcholines as reported by Rubenstein et al. (1979, Proc. Natl. Acad. Sci. U.S.A., 76:15-18).     

Dynamics and heterogeneity of bovine hippocampal membranes: Role of cholesterol and proteins

The structural and dynamic consequence of alterations in membrane lipid composition (specifically cholesterol) in neuronal membranes is poorly understood. Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors. In this paper, we have explored the role of cholesterol and proteins in the dynamics and heterogeneity of bovine hippocampal membranes using fluorescence lifetime distribution analysis of the environment-sensitive fluorescent probe Nile Red incorporated into such membranes by the maximum entropy method (MEM), and time-resolved fluorescence anisotropy measurements. The peak position and the width of the lifetime distribution of Nile Red show a progressive reduction with increasing cholesterol depletion from native hippocampal membranes indicating that the extent of heterogeneity decreases with decrease in membrane cholesterol content. This is accompanied by a concomitant decrease of the fluorescence anisotropy and rotational correlation time. Our results point out that the microenvironment experienced by Nile Red is relatively insensitive to the presence of proteins in hippocampal membranes. Interestingly, Nile Red lifetime distribution in liposomes of lipid extracts is similar to that of native membranes indicating that proteins do not contribute significantly to the high level of heterogeneity observed in native membranes. These results could be relevant in understanding the neuronal diseases characterized by defective membrane lipid metabolism.     

Dynamics and heterogeneity of bovine hippocampal membranes: role of cholesterol and proteins

The structural and dynamic consequence of alterations in membrane lipid composition (specifically cholesterol) in neuronal membranes is poorly understood. Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors. In this paper, we have explored the role of cholesterol and proteins in the dynamics and heterogeneity of bovine hippocampal membranes using fluorescence lifetime distribution analysis of the environment-sensitive fluorescent probe Nile Red incorporated into such membranes by the maximum entropy method (MEM), and time-resolved fluorescence anisotropy measurements. The peak position and the width of the lifetime distribution of Nile Red show a progressive reduction with increasing cholesterol depletion from native hippocampal membranes indicating that the extent of heterogeneity decreases with decrease in membrane cholesterol content. This is accompanied by a concomitant decrease of the fluorescence anisotropy and rotational correlation time. Our results point out that the microenvironment experienced by Nile Red is relatively insensitive to the presence of proteins in hippocampal membranes. Interestingly, Nile Red lifetime distribution in liposomes of lipid extracts is similar to that of native membranes indicating that proteins do not contribute significantly to the high level of heterogeneity observed in native membranes. These results could be relevant in understanding the neuronal diseases characterized by defective membrane lipid metabolism.     

Temperature effect of cholesterol association with synaptosomal plasma membranes of rabbit brain.

Association of exogenous cholesterol with rabbit brain synaptosomal plasma membranes follows an exponential path described by the general formula y = a X ebx. The co-operative nature of this association was shown when increasing amounts of unlabelled cholesterol glucoside (up to 0.5 mM) were added to a fixed amount (5 microM) of [14C]cholesterol, when a biphasic curve of the binding of [14C]cholesterol into the membranes was obtained. Arrhenius plots of this association revealed two break points which occur at 25 degrees C and 42 degrees C. The first break apparently corresponds to the transition from the crystalline to the gel phase. The second break may be due to the (continuously) increasing entropy of the system which creates at a certain point difficulties in the binding of cholesterol into the lipid 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.     

Transfer of cholesterol between liposomal membranes.

The transfer of cholesterol between liposomal membranes was examined. On incubation of liposomes compsoed of egg yolk phosphatidylcholine, phosphatidic acid and cholesterol (molar percentage, 65.8 : 1.3 : 32.9 or 65.5 : 6.3 : 31.2), almost complete equilibration of the cholesterol pools was achieved within 6 to 8 h at 37 degrees C. The rate of transfer of cholesterol from the liposomes, in which cholesterol was introduced by 'the exchange reaction', was not significantly different from that from liposomes prepared in the presence of cholesterol, in which the cholesterol was distributed homogenously. These findings indicate that half life for 'flip-flop' of cholesterol molecules in egg yolk phosphatidylcholine liposomes is less than 6 h at 37 degrees C. The transfer of cholesterol between liposomes was strongly dependent on temperature and was affected by the fatty acid composition of the phospholipid, suggesting that the 'fluidity' of the membranes strongly influences the transfer rate. A preferential distribution of cholesterol molecules was observed in heterogeneous liposomes with different classes of phospholipids. The 'affinity order' of cholesterol for phospholipid deduced from the present experiments is as follows: beef brain sphingomyelin greater than dipalmitoylglycerophosphocholine = dimyristoylglycerophosphocholine greater than egg yolk phosphatidylcholine.     

The influence of cholesterol on head group mobility in phospholipid membranes.

The dielectric dispersion in the MHz range of the zwitterionic dipolar phosphocholine head groups has been measured from 0--70 degrees C for various mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol. The abrupt change in the derived relaxation frequency f2 observed for pure DPPC at the gel-to-liquid crystalline phase transition at 42 degrees C reduces to a more gradual increase of frequency with temperature as the cholesterol content is increased. In general the presence of cholesterol increases the DPPC head group mobility due to its spacing effect. Below 42 degrees C no sudden changes in f2 are found at 20 or 33 mol% cholesterol, where phase boundaries have been suggested from other methods. Above 42 degrees C, however, a decrease in f2 at cholesterol contents up to 20--30 mol% is found. This is thought to be partly due to an additional restricting effect of the cholesterol on the number of hydrocarbon chain conformations and consequently on the area occupied by the DPPC molecules.     

Fast flip-flop of cholesterol and fatty acids in membranes: implications for membrane transport proteins

PURPOSE OF REVIEW: The rates by which unesterified fatty acids and cholesterol move through and desorb from membranes have been difficult to measure, in part because of the simple structures of these lipids but also because methods have generally not clearly distinguished the two steps of membrane transport. Lack of definitive knowledge has given rise to speculation about the mechanism(s) of membrane 'transport' proteins for fatty acids and cholesterol. RECENT FINDINGS: New biophysical and biochemical approaches have provided evidence that fatty acids and cholesterol exhibit rapid diffusion (flip-flop), as fast as milliseconds, across both protein-free phospholipid bilayers and cell membranes. In contrast, desorption of the cholesterol molecule from a membrane surface (hours) is much slower than that of common dietary fatty acids (milliseconds to seconds). SUMMARY: Knowledge of these properties provides a framework for understanding transport and metabolism of cholesterol and fatty acids and how their putative membrane and intracellular transporters might function.     

The incorporation of cholesterol into inner mitochondrial membranes and its effect on lipid phase transition

Incubations of rat liver inner mitochondrial membranes with liposomes prepared from $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$ (DPPC) and cholesterol resulted in a considerable enrichment of the cholesterol composition of these membranes. This enrichment is not accompanied by an alteration in the membrane phospholipid content or fatty acid composition. The exogenous cholesterol appears to be integrated into the membrane structure because it has effects consistent with the known properties of this sterol in other natural and artificial membrane systems.Differential scanning calorimetry on both intact membranes and extracted lipids showed that as the ratio of cholesterol to phospholipid was increased, the endotherm corresponding to the lipid phase transition was reduced. Freeze-fracture electron microscopy of the native membranes showed that intramembranous particles are randomly distributed above the phase transition temperature. Below this temperature large smooth areas, believed to correspond to lipid in the gel state from which proteins have been excluded, can be observed. In the presence of high concentrations of cholesterol the fracture faces observed below the lipid transition temperature show no regions of phase segregation, an observation consistent with previous studies using pure lipids where cholesterol was observed to prevent the lipid undergoing a cooperative phase transition.The results are discussed in terms of the observed low concentrations of cholesteorl in normal liver inner mitochondrial membranes and the distribution of cholesterol within the liver cells.