The effect of chloroplast coupling factor ATP synthetase (CF1 · CF0) reconstitution on fluidity properties of isolated thylakoid lipid vesicles

The reconstitution of chloroplast coupling factor ATP synthetase (CF₁ · CF₀) with thylakoid lipids by cholate dialysis produced vesicles that displayed higher steady-state anisotropy (r_s) values for both 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-diphenyl hexatriene fluorescence than the pure lipid alone. This is interpreted as meaning that the insertion of protein into the lipid bilayer brings about an increase in the ordering of acyl chains. This ordering effect became more obvious as the protein-to-lipid ratio was increased. Time-resolved decay analyses of DPH fluorescence anisotropy confirmed the conclusion drawn from the steady-state measurements, but further indicated that the dynamic motion of the probe was also slightly restricted after CF₁ · CF₀ incorporation. The restriction of DPH motion and the change in the half-angle for its cone of rotation was observed at relatively low protein-to-lipid ratios as compared with other reconstituted or biological membranes, suggesting that perhaps lipid-protein interactions occur with the inserted CF₁ · CF₀ complex.     

Alterations to plasma membrane lipid contents affect the biophysical properties of erythrocytes from individuals with hypertension

Genetic and environmental factors may contribute to high blood pressure, which is termed essential hypertension. Hypertension is a major independent risk factor for cardiovascular disease, stroke and renal failure; thus, elucidation of the etiopathology of hypertension merits further research. We recently reported that the platelets and neutrophils of patients with hypertension exhibit altered biophysical characteristics. In the present study, we assessed whether the major structural elements of erythrocyte plasma membranes are altered in individuals with hypertension. We compared the phospholipid (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingosine) and cholesterol contents of erythrocytes from individuals with hypertension (HTN) and healthy individuals (HI) using LC/MS-MS. HTN erythrocytes contained higher phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine contents and a lower cholesterol content than HI erythrocytes. Furthermore, atomic force microscopy revealed important morphological changes in HTN erythrocytes, which reflected the increased membrane fragility and fluidity and higher levels of oxidative stress observed in HTN erythrocytes using spectrophotofluorometry, flow cytometry and spectrometry. This study reveals that alterations to the lipid contents of erythrocyte plasma membranes occur in hypertension, and these alterations in lipid composition result in morphological and physiological abnormalities that modify the dynamic properties of erythrocytes and contribute to the pathophysiology of hypertension.     

Interaction of the N-terminal segment of HCV protein NS5A with model membranes

We have identified a membrane-active region in the HCV NS5A protein by performing an exhaustive study of membrane rupture induced by a NS5A-derived peptide library on model membranes having different phospholipid compositions. We report the identification in NS5A of a highly membranotropic region located at the suggested membrane association domain of the protein. We report the binding and interaction with model membranes of two peptides patterned after this segment, peptides 1A and 1B, derived from the strains 1a_H77 and 1b_HC-4J respectively. We show that they insert into phospholipid membranes, interact with them, and are located in a shallow position in the membrane. The NS5A region where this segment resides might have an essential role in the membrane replication and/or assembly of the viral particle through the modulation of the replication complex, and consequently, directly implicated in the HCV life cycle.     

Structure of the C-terminal domain of the pro-apoptotic protein Hrk and its interaction with model membranes

The protein harakiri (Hrk) is a pro-apoptotic BH3-only protein which belongs to the Bcl-2 family. Hrk appears associated to the mitochondrial outer membrane, apparently by a putative transmembrane domain, where it exerts its function. In this work we have identified a 27mer peptide supposed to be the putative membrane domain of the protein at the C-terminal region, and used infrared and fluorescence spectroscopies to study its secondary structure as well as to characterize its effect on the physical properties of phospholipid model membranes. The results presented here showed that the C-terminal region of Hrk adopts a predominantly α-helical structure whose proportion and destabilization capability varied depending on phospholipid composition. Moreover it was found that the orientation of the α-helical component of this C-terminal Hrk peptide was nearly perpendicular to the plane of the membrane. These results indicate that this domain is able of inserting into membranes, where it adopts a transmembrane α-helical structure as well as it considerably perturbs the physical properties of the membrane.     

H2O2 induces rapid biophysical and permeability changes in the plasma membrane of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the diffusion rate of hydrogen peroxide (H₂O₂) through the plasma membrane decreases during adaptation to H₂O₂ by means of a mechanism that is still unknown. Here, evidence is presented that during adaptation to H₂O₂ the anisotropy of the plasma membrane increases. Adaptation to H₂O₂ was studied at several times (15min up to 90min) by applying the steady-state H₂O₂ delivery model. For wild-type cells, the steady-state fluorescence anisotropy increased after 30min, or 60min, when using 2-(9-anthroyloxy) stearic acid (2-AS), or diphenylhexatriene (DPH) membrane probe, respectively. Moreover, a 40% decrease in plasma membrane permeability to H₂O₂ was observed at 15min with a concomitant two-fold increase in catalase activity. Disruption of the ergosterol pathway, by knocking out either ERG3 or ERG6, prevents the changes in anisotropy during H₂O₂ adaptation. H₂O₂ diffusion through the plasma membrane in S. cerevisiae cells is not mediated by aquaporins since the H₂O₂ permeability constant is not altered in the presence of the aquaporin inhibitor mercuric chloride. Altogether, these results indicate that the regulation of the plasma membrane permeability towards H₂O₂ is mediated by modulation of the biophysical properties of the plasma membrane.     

Premicellar complexes of sphingomyelinase mediate enzyme exchange for the stationary phase turnover

During the steady state reaction progress in the scooting mode with highly processive turnover, Bacillus cereus sphingomyelinase (SMase) remains tightly bound to sphingomyelin (SM) vesicles (Yu et al., Biochim. Biophys. Acta 1583, 121-131, 2002). In this paper, we analyze the kinetics of SMase-catalyzed hydrolysis of SM dispersed in diheptanoylphosphatidyl-choline (DC₇PC) micelles. Results show that the resulting decrease in the turnover processivity induces the stationary phase in the reaction progress. The exchange of the bound enzyme (E*) between the vesicle during such reaction progress is mediated via the premicellar complexes (E_i^#) of SMase with DC₇PC. Biophysical studies indicate that in E_i^# monodisperse DC₇PC is bound to the interface binding surface (i-face) of SMase that is also involved in its binding to micelles or vesicles. In the presence of magnesium, required for the catalytic turnover, three different complexes of SMase with monodisperse DC₇PC (E_i^# with i = 1, 2, 3) are sequentially formed with Hill coefficients of 3, 4 and 8, respectively. As a result, during the stationary phase reaction progress, the initial rate is linear for an extended period and all the substrate in the reaction mixture is hydrolyzed at the end of the reaction progress. At low mole fraction (X) of total added SM, exchange is rapid and the processive turnover is limited by the steps of the interfacial turnover cycle without becoming microscopically limited by local substrate depletion or enzyme exchange. At high X, less DC₇PC will be monodisperse, E_i^# does not form and the turnover becomes limited by slow enzyme exchange. Transferred NOESY enhancement results show that monomeric DC₇PC in solution is in a rapid exchange with that bound to E_i^# at a rate comparable to that in micelles. Significance of the exchange and equilibrium properties of the E_i^# complexes for the interpretation of the stationary phase reaction progress is discussed.     

Mechanistic role of ergosterol in membrane rigidity and cycloheximide resistance in Saccharomyces cerevisiae

Mutants of Saccharomyces cerevisiae defective in the late steps of ergosterol biosynthesis are viable but accumulate structurally altered sterols within the plasma membrane. Despite the significance of pleiotropic abnormalities in the erg mutants, little is known about how sterol alterations mechanically affect the membrane structure and correlate with individual mutant phenotypes. Here we demonstrate that the membrane order and occurrence of voids are determinants of membrane rigidity and hypersensitivity to a drug. Among five ergΔ mutants, the erg2Δ mutant exhibited the most marked sensitivity to cycloheximide. Notably, measurement of time-resolved anisotropy indicated that the erg2Δ mutation decreased the membrane order parameter (S), and dramatically increased the rotational diffusion coefficient (D_w) of 1-[4-(trimethylamino)pheny]-6-phenyl-1,3,5-hexatriene (TMA-DPH) in the plasma membrane by 8-fold, providing evidence for the requirement of ergosterol for membrane integrity. The IC₅₀ of cycloheximide was closely correlated with S/D_w in these strains, suggesting that the membrane disorder and increasing occurrence of voids within the plasma membrane synergistically enhance passive diffusion of cycloheximide across the membrane. Exogenous ergosterol partially restored the membrane properties in the upc2-1erg2Δ strain. In this study, we describe the ability of ergosterol to adjust the dynamic properties of the plasma membrane, and consider the relevance of drug permeability.     

The fidelity of response by 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene in time-resolved fluorescence anisotropy measurements on lipid vesicles

Time-resolved fluorescence anisotropy measurements on 1⁰-[4-(tri-methylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules in lipid vesicles of palmitoyloleoylphosphatidylcholine (POPC), PC extracted from egg yolk (EggPC), dioleoyl-PC (DOPC), dilinoleoyl-PC (DLPC), phosphatidylglycerol extracted from egg yolk (EggPG), dioleoyl-PG (DOPG), sulfoquinovosyldiacylglycerol (SQDG) and digalactosyl-DG (DGDG) with and without cholesterol are presented. The observed intensity decay curves are analyzed simultaneously in terms of the Brownian rotational diffusion model. The analysis thus yields the isotropic fluorescence decay, the initial anisotropy r (0), the order parameters P ₂ and P ₂ as well as the diffusion coefficient of the long molecular axis. It is shown that increasing unsaturation in the acyl chains of the PC lipids results in an increase in the rotational diffusion rates of the probes and a decrease in the order parameter P ₂. However, the value of P ₂ remains unchanged. The corresponding orientational distribution function of the probes is bimodal, with fractions lying preferentially parallel and perpendicular to the local vesicle surface. Surprisingly, the fraction of probe molecules lying with their long axes parallel to the bilayer surface increases with increasing unsaturation with a concomitant narrowing in the width of the distribution of the fraction lying perpendicular to it. As expected, cholesterol is found to increase the order parameters in all the systems and to suppress the tendency of the molecules to lie parallel to the bilayer surface. Furthermore, the rotational diffusion coefficients of the probes is found to increase in all the systems except for DLPC. Interestingly, the effects of unsaturation on the reorientational dynamics of TMA-DPH molecules in the vesicle systems are opposite to those found in the corresponding planar multibilayers (Deinum et al. 1988), whereas the same cholesterol effect is observed for the two systems. Nevertheless, the TMA-DPH molecules exhibit higher diffusion coefficients in the vesicle than in the planar multibilayer systems. In addition, a unimodal distribution of the probe molecules is found in the multibilayer systems. The differences between the two systems are ascribed to the differences in the radius of curvature and the hydration of the bilayers. Lastly we rationalize the bimodal distribution of the TMA-DPH molecules in the vesicles in terms of their observed partition between the lipid and aqueous phases.Abbreviations DPH 1,6-diphenyl-1,3,5-hexatriene - TMA-DPH 1-[4-(trimethylammonio)-phenyl]-6-phenyl-1,3,5-hexatriene - POPC palmitoyloleoylphosphatidylcholine - EggPC PC extracted from egg yolk - DOPC dioleoyl-PC - DLPC dilinoleoyl-PC - EggPG phosphatidylglycerol extracted from egg yolk - DOPG dioleoyl-PG - SQDG sulfoquinovosyldiacylglycerol - DGDG di-galactosyl-DG - HPTLC high performance thin layer chromatography     

A membranotropic region in the C-terminal domain of Hepatitis C virus protein NS4B: Interaction with membranes

We have identified a membrane-active region in the HCV NS4B protein by studying membrane rupture induced by a NS4B-derived peptide library on model membranes. This segment corresponds to one of two previously predicted amphipathic helix and define it as a new membrane association domain. We report the binding and interaction with model membranes of a peptide patterned after this segment, peptide NS4B_H2, and show that NS4B_H2 strongly partitions into phospholipid membranes, interacts with them, and is located in a shallow position in the membrane. Furthermore, changes in the primary sequence cause the disruption of the hydrophobicity along the structure and prevent the resulting peptide from interacting with the membrane. Our results suggest that the region where the NS4B_H2 is located might have an essential role in the membrane replication and/or assembly of the viral particle through the modulation of the replication complex. Our findings therefore identify an important region in the HCV NS4B protein which might be implicated in the HCV life cycle and possibly in the formation of the membranous web.     

Cell envelope fluidity modification for an effective glutamate excretion in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> 2262

1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was used to assess the cell envelope fluidity of Corynebacterium glutamicum 2262 during a temperature-triggered glutamate producing process. Because the fluorescence lifetime of TMA-DPH was shown to be constant all over the process, fluorescence anisotropy can be considered as a good index of cell envelope fluidity. When the temperature of the fed-batch culture was increased from 33 to 39°C to induce glutamate excretion, the fluorescence anisotropy values decreased from 0.212 ± 0.002 to 0.186 ± 0.002 (corresponding to an increase in the cell fluidity), while the specific glutamate production rate reached its maximal value. The increase in fluidity of the C. glutamicum cell envelope was not due to a physical effect related to the temperature elevation, but rather to an alteration of the composition of the cell envelope. Using a mutant devoid of corynomycolates, significant differences in fluorescence anisotropy values were obtained compared to the wild-type strain, suggesting that TMA-DPH is mainly anchored into the corynomycomembrane. Differences in fluorescence anisotropy were also observed when the bacteria were cultivated at 33, 36, 38, and 39°C in batch cultures, and a linear relationship was obtained between the maximum specific glutamate production rate and the measured fluidity. When using the glutamate non-producing variant of C. glutamicum 2262, the fluorescence anisotropy remained constant at 0.207 ± 0.003 whatever the applied temperature shift. This suggests that the fluidity of the Corynebacteria mycomembrane plays an important role in glutamate excretion during the temperature-triggered process.