Effect Of Lipid Environment Research Articles

Effect of lipid environment on amyloid fibril formation of human serum amyloid A

Human serum amyloid A (SAA) is a precursor protein of AA amyloidosis and a component of high-density lipoproteins (HDLs), thus it is essential to investigate the amyloid fibril formation of SAA under a lipid environment. We used synthetic fragment peptides corresponding to the N-terminal (residues 1–27) and central (residues 43–63) regions of the SAA molecule, which are known to have amyloidogenic properties. Measurements of tryptophan fluorescence in conjunction with circular dichroism showed that SAA (1–27) peptide binds to neutral and acidic lysophospholipids, whereas SAA (43–63) peptide binds only to acidic lysophospholipids. For both these SAA peptides, binding to lysophospholipids inhibited heparin-induced amyloid-like fibril formation by stabilizing the α-helical structure. However, acidic lysophospholipids implied a possibility to promote fibril formation of SAA (1–27) peptide by themselves. These results suggest that the amyloid fibril formation of SAA may be modulated by altering the lipid head group composition of HDLs during metabolism.

Effects of lipid environment on the conformational changes of an ABC importer

In order to shuttle substrates across the lipid bilayer, membrane proteins undergo a series of conformation changes that are influenced by protein structure, ligands, and the lipid environment. To test the effect of lipid on conformation change of the ABC transporter MolBC, EPR studies were conducted in lipids and detergents of variable composition. In both a detergent and lipid environment, MolBC underwent the same general conformation changes as detected by site-directed EPR spectroscopy. However, differences in activity and the details of the EPR analysis indicate conformational rigidity that is dependent on the lipid environment. From these observations, we conclude that native-like lipid mixtures provide the transporter with greater activity and conformational flexibility as well as technical advantages such as reconstitution efficiency and protein stability.

Effects of Sensory Rhodopsin II Complexation with its Cognate Transducer HtrII on the Local Environment of Internal Water Molecules

Recent studies indicate that internal water molecules play a critical role in membrane protein function. Here we report evidence that the local environment of one or more internal water molecules in sensory rhodopsin II (SRII) is altered by interaction with its cognate transducer HtrII. The SRII-HtrII complex mediates blue-light repellent phototaxis in halophilic archaea, using a signaling pathway similar to that in bacterial chemotaxis. We studied the photocycle of SRII and a SRII-HtrII fusion complex from Natronobacterium pharaonis using low-temperature static and room temperature time-resolved Fourier transform infrared (FTIR) difference spectroscopy. When cooled to 80 K, and illuminated the protein is trapped in its K state. A shift of 2 cm−1 between SRII (3626 cm−1) and SRII-HtrII (3628 cm−1) is found for a negative OH stretching band assigned to an internal water molecule, most likely located near the active Schiff base. In contrast, the OH stretching band for this water in the K state appears at the same frequency (3619 cm−1) for both the free and complexed receptor. Similar shifts are observed upon hydration with H2O18 shifted to a lower frequency confirming these bands arise from the OH stretching mode of water. Data are also presented on the effects of lipid environment on structural changes of internal water molecules and the receptor-transducer interactions. This work was supported by National Institutes of Health GrantsR01GM069969 (to K.J.R.) and R37GM27750 (to J.L.S.), U.S. Department of Energy Grant DE-FG02-07ER15867, and the Robert A. Welch Foundation (to J.L.S.).

Dynamics of Ganglioside Headgroup in Lipid Environment: Molecular Dynamics Simulations of GM1 Embedded in Dodecylphosphocholine Micelle

Recognition of membrane-anchored glycosphingolipid receptors by various ligands is the key event in several biological phenomena. However, mere presence of these cell-surface receptors does not always ensure their recognition and binding by their respective ligands, a phenomenon termed “crypticity”. Earlier studies have suggested that different glycan headgroup orientations exposing and/or masking different epitopes may explain the crypticity of glycolipids. The effect of lipid environment on the orientation and conformation of GM1 ganglioside has been investigated in the present study by MD simulation technique in an attempt to understand the structural basis of crypticity. In addition, the effect of the length of the ceramide hydrocarbon tails on the headgroup conformation has also been investigated. MD simulations for 1 ns were performed with explicit water molecules for both GM1 headgroup (GM1-Os) and GM1 embedded in dodecylphosphocholine micelle. The simulations show that the conformations of the hyd...

The effect of lipid environment in purple membrane on bacteriorhodopsin

The decay rate of the Bacteriorhodopsin (BR) photocycle intermediate M 412 and proton, the proton pump efficiency (H +/M 412), the ratios of M 412 to other intermediates and the rotational correlation time ( τ c ) in purple membrane (PM) fragments treated by the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) with different concentrations were studied. The results show that: (1) The largest effect of CHAPS on M 412 decay rate and proton decay rate of BR, τ c of PM and the ratios of M 412 to other intermediates in BR photocycle is in the range of its critical micelle concentration (CMC). This indicates that changes of the ratios of M 412 to other intermediates, τ c , M 412 decay and proton decay occur and are due to the variation of the lipid environment. (2) The dependency of proton yield on CHAPS concentrations is basically consistent with that of M 412s%. This indicates the relation between proton pumping function and M 412. These studies show the importance of maintaining a native environment.

The Effect of Lipid Environment and Retinoids on the ATPase Activity of ABCR, the Photoreceptor ABC Transporter Responsible for Stargardt Macular Dystrophy

ABCR is a photoreceptor-specific ATP-binding cassette transporter that has been linked to various retinal diseases, including Stargardt macular dystrophy, and implicated in retinal transport across rod outer segment (ROS) membranes. We have examined the ATPase and GTPase activity of detergent-solubilized and reconstituted ABCR. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-solubilized ABCR had ATPase and GTPase activity (K(m) approximately 75 micrometer V(max) approximately 200 nmol/min/mg) that was stimulated 1.5-2-fold by all-trans-retinal and dependent on phospholipid and dithiothreitol. The K(m) for ATP decreased to approximately 25 micrometer after reconstitution, whereas the V(max) was strongly dependent on the lipid used for reconstitution. ABCR reconstituted in ROS phospholipid had a V(max) for basal and retinal activated ATPase activity that was 4-6 times higher than for ABCR in soybean or brain phospholipid. This enhanced activity was mainly due to the high phosphatidylethanolamine (PE) content of ROS membranes. PE was also required for retinoid-stimulated ATPase activity. ATPase activity of ABCR was stimulated by the addition of N-retinylidene-PE but not the reduced derivative, retinyl-PE. ABCR expressed in COS-1 cells also exhibited retinal-stimulated ATPase activity similar to that of the native protein. These results support the view that ABCR is an active retinoid transporter, the nucleotidase activity of which is strongly influenced by its lipid environment.

Size-exclusion chromatographic reconstitution of the bovine brain benzodiazepine receptor: Effects of lipid environment on the binding characteristics

The benzodiazepine receptor from calf brain was solubilized with sodium deoxycholate (2 mg/ml) in the presence of 0.5 M KCl and protease inhibitors, and bound flunitrazepam with an equilibrium dissociation constant (Kd) of 2.7±1.2 nM and with 0.40±0.04 pmol binding sites per mg protein (Bmax). Up to 60% of the benzodiazepine binding sites (average 25%) could be reconstituted in lipid vesicles, upon size-exclusion chromatography of protein–detergent–lipid mixtures on Sephadex G-50 Medium for detergent depletion. The flunitrazepam affinity for the reconstituted receptor varied with the lipid composition (Kd 1.4–4 nM). Freezing and thawing increased the size of the small proteoliposomes obtained by chromatographic reconstitution and, on the average, doubled the number of operative flunitrazepam binding sites. When the proteoliposomes were stored at −20°C or −80°C or in lyophilized state, the receptor retained its benzodiazepine binding affinity and Bmax over a period of 2 months.

Regulation of a candidate aminophospholipid-transporting ATPase by lipid.

The effect of lipid environment on the activation of a vanadate-sensitive Mg(2+)-ATPase purified from human erythrocytes was studied in detergent-lipid-protein mixed micelles. ATPase activity was stimulated maximally by phosphatidylserine. Other anionic diacylglycerophospholipids (phosphatidic acid, cardiolipin, phosphatidylglycerol, and phosphatidylinositol) supported 25-100% of the phosphatidylserine-stimulated activity. Another aminophospholipid, egg PE, supported 38% of the phosphatidylserine-stimulated activity. The phosphoinositides (phosphatidylinositol, phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate) also stimulated the ATPase; however, activity decreased with increasing lipid phosphorylation. Monoacyl negatively charged lipids (lysophosphatidylserine, fatty acids) and zwitterionic lipids (phosphatidylcholine and sphingomyelin) did not activate the enzyme. ATPase activation was dependent on phospholipid fatty acyl chain composition: ATPase activity increased with increasing PS acyl chain length, and the optimal fatty acid composition was one saturated and one unsaturated fatty acid. However, the long, unsaturated acyl chain requirement could be satisfied by nonactivating lipids. The characteristics of this ATPase are similar to those of the Mg(2+)-ATP-dependent aminophospholipid flippase, suggesting that it may be associated with the transporter.

Effects of Lipid Environment on Membrane Transport: The Human Erythrocyte Sugar Transport Protein/Lipid Bilayer System

Membrane transport proteins (carriers) catalyze transmembrane movements of hydrophilic molecular species. To perform this function, carriers employ a variety of mechanisms for transmembrane solute flux that are distinct from and more rapid than leakage or non-Stokesian transbilayer diffusion of solutes (37). The low permeability of the membrane lipid bilayer to most hydrophilic species prevents the rapid dissipation of transbilayer solute gradients co­ established by membrane transport systems and intracellular metabolism and thus assists in the maintenance of a cytosolic environment favorable to cellular homeostasis. In addition to providing a permeability barrier, the membrane lipid bilayer provides a matrix for the attachment of membrane proteins. Most integral membrane proteins contain extensive, hydrophilic (extramembranous) and hydrophobic (membrane spanning) sequences. As a consequence, transverse protein reorientations (flip-flop) within the lipid bilayer arc highly improbable

Effects of lipid environment on the light-induced conformational changes of rhodopsin. 2. Roles of lipid chain length, unsaturation, and phase state.

When rhodopsin is incorporated into the saturated short-chain phospholipid dimyristoylphosphatidylcholine, photolysis of the protein results in an abnormal sequence of spectral transitions, and the dominant product of metarhodopsin I decay is free retinal plus opsin [Baldwin, P. A., & Hubbell, W. L. (1985) Biochemistry (preceding paper in this issue)]. By incorporation of rhodopsin into a series of phosphatidylcholines of defined composition, we have determined the properties of the lipid environment that are responsible for the altered spectral behavior. Metarhodopsin II is not found in appreciable amounts in bilayers containing acyl chains that are too short (14 or fewer carbon atoms in length), in the presence of only n-alkyl chains, or below the characteristic phase-transition temperature of recombinant membranes. Double bonds are not required for the formation of the metarhodopsin II intermediate, as it is observed in diphytanoylphosphatidylcholine recombinants.

Effects of lipid environment on the light-induced conformational changes of rhodopsin. 1. Absence of metarhodopsin II production in dimyristoylphosphatidylcholine recombinant membranes.

Photolysis of bovine rhodopsin in dimyristoylphosphatidylcholine recombinant membranes results in the production of a relatively stable metarhodopsin I like photointermediate that decays slowly to a species with a broad absorbance maximum centered at about 380 nm [O'Brien, D. F., Costa, L. F., & Ott, R. A. (1977) Biochemistry 16, 1295-1303]. On the basis of the results of a variety of chemical and spectroscopic tests, we show that this process corresponds to the production of free retinal plus opsin and not to the slow production of metarhodopsin II. Electron spin resonance studies using a novel disulfide spin-label that is covalently linked to rhodopsin indicate that the apparent arrest of the protein at the metarhodopsin I stage is not due to simple aggregation of the protein in this short-chain, saturated lipid bilayer but must be understood in terms of the effect of the lipid host on the conformational energies of individual protein molecules. Limited production of metarhodopsin II is observed under acidic conditions. Thus, the rhodopsin-dimyristoylphosphatidylcholine recombinants offer a unique system for the study of the effect of the phospholipid bilayer environment on the conformation of an intrinsic membrane protein.

The Effects of Lipid Environment, Ion-Binding and Chemical Modifications on the Structure of the Gramicidin Transmembrane Channel

The weight-average mol wt was found to be 9,000, apparently themonomer of the DCCD-protein, suggesting that theconformation changes upon addition of DMSO. Small-angle x-ray scattering intensity curves obtained from theDCCD-proteinin watershowadiscreteinterferencemaxi-mumindicative of a considerable degree of regularity inthetwodimensional arrangementin theplaneperpendicu-lar to the DCCD-protein-axis. Awell-orientated diffrac-tion pattern of the DCCD-protein was obtained fromwhich cylindrically average intensity distribution curveswere calculated, revealing a

Control of the potassium ion-stimulated adenosine triphosphatase of pig gastric microsomes: Effects of lipid environment and the endogenous activator

The K +-stimulated ATPase activity associated with the purified gastric microsomes from the pig gastric mucosa can be completely inactivated by treatment with 15% ethanol for 60 s at 37 °C but not at 25 °C. Sequential exposure of the microsomes to 15% ethanol at 25 and 37 °C caused the release of 2.9 and 4.3% of the total membrane phospholipids, respectively, consisting entirely of phosphatidyl choline and phosphatidyl ethanolamine. The ethanol-treated (37 °C) membrane had high basal (with Mg 2+ as the only cation in the assay mixture) activity, which was further enhanced during reconstitution with phosphatidyl choline or phosphatidyl ethanolamine. The high basal activities could be reduced to the normal control level by assaying the enzyme in presence of the “activator protein,” partially purified from the soluble supernatant of the pig gastric cells. Phosphatidyl choline was somewhat more effective than phosphatidyl ethanolamine in the restoration of the activity of the ethanol-treated enzyme while phosphatidyl serine, phosphatidyl inositol, and sphingomyelin were without any effect. Synthetic phosphatidyl choline with various fatty acid substitutions were tested for their effectiveness in the restoration of the ethanol-inactivated enzyme. The distearoyl (18:0), dioleoyl (18:1), and dilinoleoyl (18:2) derivatives of phosphatidyl choline were almost equally effective while dipalmitoyl (16:0) phosphatidyl choline was somewhat less effective in the reconstitution process. Cholesterol appeared to interfere with phosphatidyl choline in the restoration of the activity of ethanol-treated enzyme. The fatty acid composition of phosphatidyl choline and phosphatidyl ethanolamine extracted by 15% ethanol at 37 °C was clearly different than those of the total microsome. Our data suggest that the phospholipids extracted by 15% ethanol at 37 °C are derived primarily from the immediate lipid environment of the enzyme and ATP together with Mg 2+ and K + help the partially delipidated enzyme to retain the appropriate conformation for the subsequent reconstitution. Furthermore, ethanol appears to either release or inactivate the membrane-associated activator protein, demonstrated to be essential for the K +-stimulated activity of the pig gastric ATPase.

Effect of lipid environment on the motion of a spin-label covalently bound to myelin basic protein.

Effect of lipid environment on the motion of a spin-label covalently bound to myelin basic protein.