A differential polarized phase fluorometric study of the effects of high hydrostatic pressure upon the fluidity of cellular membranes

The effects of high hydrostatic pressure (up to 2 kbar) upon the fluidity and order of the synaptic and myelin membrane fractions of goldfish brain have been studied by using steady-state and differential polarized phase fluorometry. Probe motion provided a measure of membrane order (r infinity) and probe rotational rate (R). Membrane order became progressively greater as pressure was increased up to approximately 2 kbar. This effect was similar over the temperature range 5.6-34.3 degrees C. An increase in pressure of 1 kbar had an effect on membrane order that was equivalent to a 13-19 degrees C reduction in temperature. Membrane order was essentially identical during pressurization and depressurization. At 5.6 degrees C, pressurization caused a large increase in R, and similar, though less dramatic, anomalies occurred at higher temperatures. It is suggested that this is due to the segregation of probe molecules in highly ordered membranes, which leads either to excitation transfer between 1,6-diphenyl-1,3,5-hexatriene (DPH) molecules or to changes in the rotational motion of DPH from "sticking" to "slipping".     

Structural changes in lipid bilayers and biological membranes caused hydrostatic pressure

By use of neutron diffraction, the structural parameters of oriented multilayers of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine with deuteriocarbon chains/cholesterol (molar ratio 70:30), multilamellar lipid vesicles composed of pure lipids and lipid/cholesterol mixtures, and crystalline purple membrane patches from Halobacterium halobium have been measured at pressures up to 2 kbar. Pressurization of the oriented 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/cholesterol multilayers results in an in-plane compression with the mean deuteriocarbon chain spacing of 4.44 A obtained under ambient conditions decreasing by 3-7% at 1.9 kbar. The thickness for this bilayer increases by approximately equal to 1.5 A, but the net bilayer volume decreases and the isothermal compressibility is estimated to be in the range (-0.1 to -0.6) X 10(-4)/bar at 19.0 degrees C. The d spacings for multilamellar vesicles composed of lipids in the liquid crystalline state and lipid/cholesterol mixtures increase linearly as a function of pressure, suggesting that these bilayers are also compressed in the membrane plane. For 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and 1,2-distearoyl-sn-glycero-3-phosphatidylcholine MLVs in the gel state, the d spacing decreases with pressure. For 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, the hexagonally packed chains are anisotropically compressed in the bilayer plane, resulting in a pseudohexagonal chain packing at 1.9 kbar. The bilayer compressibility is (-0.4 or -0.5) X 10(-4)/bar depending on whether the chain tilt increases with pressure or terminal methyl groups of apposing lipid monolayers approach each other.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of the NMDA receptor noncompetitive antagonist MK-801 with model and native membranes.

MK-801, a noncompetitive antagonist of the NMDA (N-methyl-D-aspartate) receptor, has protective effects against excitotoxicity and ethanol withdrawal seizures. We have determined membrane/buffer partition coefficients (Kp[mem]) of MK-801 and its rates of association with and dissociation from membranes. Kp[mem] (+/- SD) = 1137 (+/- 320) in DOPC membranes and 485 (+/- 99) in synaptoneurosomal (SNM) lipid membranes from rat cerebral cortex (unilamellar vesicles). In multilamellar vesicles, Kp[mem] was higher: 3374 (+/- 253) in DOPC and 6879 (+/- 947) in SNM. In cholesterol/DOPC membranes, Kp[mem] decreased as the cholesterol content increased. MK-801 associated with and dissociated from membranes rapidly. Addition of ethanol to SNM did not affect Kp[mem]. MK-801 decreased the cooperative unit size of DMPC membranes. The decrease was smaller than that caused by 1,4-dihydropyridine drugs, indicating a weaker interaction with the hydrocarbon core. Small angle x-ray diffraction, with multilayer autocorrelation difference function modeling, indicated that MK-801 in a cholesterol/DOPC membrane (mole ratio = 0.6) causes a perturbation at approximately 16.0 A from the bilayer center. In bilayers of cholesterol/DOPC = 0.15 (mole ratio) or pure DOPC, the perturbation caused by MK-801 was more complex. The physical chemical interactions of MK-801 with membranes in vitro are consistent with a fast onset and short duration of action in vivo.     

Polyphosphoinositide activation of cholesterol side chain cleavage with purified cytochrome P-450scc

Highly purified beef adrenal cytochrome P-450 specific for cholesterol side chain cleavage (P-450-scc) has been reconstituted with sonicated vesicles containing cholesterol and either dimyristoyl phosphatidylcholine (DMPC) or dioleoyl phosphatidylcholine (DOPC). When cholesterol was present in DMPC vesicles at 1:15 molar ratio, cardiolipin and L-alpha-phosphatidylinositol 4-monophosphate (DPI) increased side chain cleavage by at least 5-fold (0.7 min-1-3.5 min-1). In DOPC vesicles, a smaller increase was observed (2.8 min-1-5.0 min-1). Activator phospholipids increased the rate of transference of cholesterol both to and from the cytochrome when, respectively, cholesterol-free P-450scc and cholesterol-P-450scc complex are combined with either DMPC or DOPC vesicles. Transfer of cholesterol to and from cytochrome P-450 occurred with similar first order rate constants and was also independent of the concentrations of cholesterol vesicles and P-450. It is suggested that transfer in both directions is limited by the rate of insertion of P-450scc into the membrane. Phospholipid stimulatory effects for both cholesterol transfer and for activation of side chain cleavage occurred with the same ranking, even though cholesterol transfer, following reconstitution, was 5-10 times slower than the turnover of side chain cleavage. DPI increased Vmax for side chain cleavage in both DMPC and DOPC vesicles to the same rate (12 min-1) without effect on the Km for cholesterol, while cardiolipin both produced a similar increase in Vmax and decreased Km (cholesterol). This activation by DPI is attributed to more favorable incorporation of P-450scc in these membranes and is consistent with previously reported effects of acidic phospholipids on other mitochondrial proteins.     

Temperature-pressure phase diagram of a heterogeneous anionic model biomembrane system: results from a combined calorimetry, spectroscopy and microscopy study

By using Fourier transform infrared (FT-IR) spectroscopy in combination with differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), ultrasound velocimetry, Laurdan fluorescence spectroscopy, fluorescence microscopy and atomic force microscopy (AFM), the temperature and pressure dependent phase behavior of the five-component anionic model raft lipid mixture DOPC/DOPG/DPPC/DPPG/cholesterol (20:5:45:5:25 mol%) was investigated. A temperature range from 5 to 65 °C and a pressure range up to 16 kbar were covered to establish the temperature-pressure phase diagram of this heterogeneous model biomembrane system. Incorporation of 10-20 mol% PG still leads to liquid-ordered (l(o))-liquid-disordered (l(d)) phase coexistence regions over a wide range of temperatures and pressures. Compared to the corresponding neutral model raft mixture (DOPC/DPPC/Chol 25:50:25 mol%), the p,T-phase diagram is - as expected and in accordance with the Gibbs phase rule - more complex, the phase sequence as a function of temperature and pressure is largely similar, however. This anionic heterogeneous model membrane system will serve as a more realistic model biomembrane system to study protein interactions with anionic lipid bilayers displaying liquid-disordered/liquid-ordered domain coexistence over a wide range of the temperature-pressure plane, thus allowing also studies of biologically relevant systems encountered under extreme environmental conditions.     

Quantitation of lateral stress in lipid layer containing nonbilayer phase preferring lipids by frequency-domain fluorescence spectroscopy.

Frequency-resolved fluorescence measurements have been performed to quantitate the lateral stress of the lipid layer containing nonbilayer phase preferring dioleoylphosphatidylethanolamine (DOPE). On the basis of a new rotational diffusion model, the wobbling diffusion constant (Dw), the curvature-related hopping diffusion constant (DH), and the two local orientational order parameters ([P2] and [P4]) of 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl] carbonyl]-3-sn-phosphatidylcholine (DPH-PC) in fully hydrated DOPE and DOPE/dioleoylphosphatidylcholine (DOPC) mixtures were calculated from the frequency-domain anisotropy data. The values of [P2], [P4], and DH for DOPE were found to increase significantly at approximately 12 degrees C, the known lamellar liquid crystalline (L alpha) to inverted hexagonal (HII) phase transition temperature of DOPE. Similar features as well as a decline of Dw were detected in the DOPE/DOPC mixtures as the DOPE content was increased from 85% to 90% at 23 degrees C, corresponding to the known lyotropic phase transition of the DOPE/DOPC. In contrast, for DOPC (0-40 degrees C) and DOPE/DOPC (0-100% DOPE at 3 degrees C), which remained in the L alpha phase, these changes were not detected. The most probable local orientation of DPH-PC in the DOPE/DOPC mixtures shifted progressively toward the normal of the lipid/water interface as the content of DOPE increased. We concluded that the curvature-related lateral stress in the lipid layer increases with the content of the nonbilayer phase preferring lipids.     

Effects of cis and trans unsaturation on the structure of phospholipid bilayers: a high-pressure infrared spectroscopic study

In order to compare the effects of cis and trans unsaturation on the structure and packing of phospholipid bilayers, infrared spectra of aqueous dispersions of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dielaidoyl-sn-glycero-3-phosphocholine (DEPC) were measured in a diamond anvil cell at 28 degrees C as a function of pressure up to 36 kbar. The infrared spectra indicate that DEPC and DOPC undergo pressure-induced liquid-crystalline to gel phase transitions at critical pressures of 0.7 and 5.2 kbar, respectively. Below their respective critical pressures, the infrared spectra of DOPC and DEPC are essentially indistinguishable, whereas above these pressures, there are very pronounced differences in the barotropic behavior of these two lipids. Specifically, at the 5.2-kbar transition in DOPC, there are significant changes in the frequencies, intensities, and widths of bands associated with the interfacial C = O groups, the olefinic CH = CH groups, and the terminal CH3 groups, whereas the corresponding bands of DEPC are, by contrast, relatively insensitive to the pressure-induced phase transition. The unusual band shape changes in DOPC are attributed to a unique packing arrangement of the oleoyl acyl chains required to accommodate the bent geometries of adjacent cis double bonds. Moreover, above 5 kbar in DEPC, well-defined correlation field splittings of the CH2 scissoring and rocking modes are observed, with magnitudes very similar to those observed at comparable pressures in saturated lipid systems. The absence of correlation field splittings of the corresponding bands of DOPC up to 36 kbar suggests that the bent oleoyl acyl chains are closely packed with all chains oriented parallel to each other.     

Pressure effects on the lateral distribution of cholesterol in lipid bilayers: a time-resolved spectroscopy study.

The effects of hydrostatic pressure and temperature on the phase behavior and physical properties of the binary mixture palmitoyloleoylphosphatidylcholine/cholesterol, over the 0-40 molar % range of cholesterol compositions, were determined from the changes in the fluorescence lifetime distribution and anisotropy decay parameters of the natural lipid trans-parinaric acid (t-PnA). Pressurized samples were excited with a Ti-sapphire subpicosecond laser, and fluorescence decays were analyzed by the quantified maximum entropy method. Above the transition temperature (T(T) = -5 degrees C), at atmospheric pressure, two liquid-crystalline phases, alpha and beta, are formed in this system. At each temperature and cholesterol concentration below the transition pressure, the fluorescence lifetime distribution pattern of t-PnA was clearly modulated by the pressure changes. Pressure increased the fraction of the liquid-ordered beta-phase and its order parameter, but it decreased the amount of cholesterol in this phase. Palmitoyloleoylphosphatidylcholine/cholesterol phase diagrams were also determined as a function of temperature and hydrostatic pressure.     

Differential effect of pressure and temperature on the catalytic behaviour of wild-type human butyrylcholinesterase and its D70G mutant.

The combined action of temperature (10-35 degrees C) and pressure (0. 001-2 kbar) on the catalytic activity of wild-type human butyrylcholinesterase (BuChE) and its D70G mutant was investigated at pH 7.0 using butyrylthiocholine as the substrate. The residue D70, located at the mouth of the active site gorge, is an essential component of the peripheral substrate binding site of BuChE. Results showed a break in Arrhenius plots of wild-type BuChE (at Tt approximately 22 degrees C) whatever the pressure (dTt/dP = 1.6 +/- 1.5 degrees C.kbar-1), whereas no break was observed in Arrhenius plots of the D70G mutant. These results suggested a temperature-induced conformational change of the wild-type BuChE which did not occur for the D70G mutant. For the wild-type BuChE, at around a pressure of 1 kbar, an intermediate state, whose affinity for substrate was increased, appeared. This intermediate state was not seen for the mutant enzyme. The wild-type BuChE remained active up to a pressure of 2 kbar whatever the temperature, whereas the D70G mutant was found to be more sensitive to pressure inactivation (at pressures higher than 1.5 kbar the mutant enzyme lost its activity at temperatures lower than 25 degrees C). The results indicate that the residue D70 controls the conformational plasticity of the active site gorge of BuChE, and is involved in regulation of the catalytic activity as a function of temperature.     

Fluid-phase chain unsaturation controlling domain microstructure and phase in ternary lipid bilayers containing GalCer and cholesterol

We report the microstructure and phase behavior of three ternary mixtures each containing a long-chain saturated glycosphingolipid, galactosylceramide (GalCer), and cholesterol at room temperature. The unsaturation level of the fluid-phase component was varied by lipid choice, i.e., saturated 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), singly unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), or doubly unsaturated 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). GalCer was used because of its biological significance, for example, as a ligand in the sexual transmission of HIV and stimulator of natural killer T-cells. Supported lipid bilayers of the ternary mixtures were imaged by atomic force microscopy and GalCer-rich domains were characterized by area/perimeter ratios (A/P). GalCer domain phase transitions from solid (S) to liquid (L) phase were verified by domain behavior in giant unilamellar vesicles, which displayed two-dimensional microstructure similar to that of supported lipid bilayers. As cholesterol concentration was increased, we observed approximately 2.5, approximately 10, and approximately 20-fold decreases in GalCer domain A/P for bilayers in L-S phase coexistence containing DOPC, POPC, and DLPC, respectively. The transition to L-L phase coexistence occurred at approximately 10 mol % cholesterol for bilayers containing DOPC or POPC and was accompanied by maintenance of a constant A/P. L-L phase coexistence did not occur for bilayers containing DLPC. We systematically relate our results to the impact of chain unsaturation on the interaction of the fluid-phase lipid and cholesterol. Physiologically, these observations may give insight into the interplay of fatty acid chain unsaturation, sterol concentration, and lipid hydrophobic mismatch in membrane phenomena.     

Effects of steroid molecules on the dynamical structure of dioleoylphosphatidylcholine and digalactosyldiacylglycerol bilayers

The ESR spectra of cholestane spin labels (CSL) in dioleoylphosphatidylcholine (DOPC) bilayers containing 20 wt% of cholesterol, 7-dehydrocholesterol, beta-sitosterol, stigmasterol and lanosterol exhibit a marked similarity, thus indicating that these steroids induced the same effects on the lipid bilayer over the temperature range 21-55 degrees C. The incorporation of these steroids into the DOPC bilayers enhances the orientational order of the CSL molecules at every temperature studied, but only induces a pronounced slow-down in their rotational motions at temperatures above 35 degrees C. Similar results were obtained in DOPC/ergosterol multilamellar liposomes, but the changes are now less pronounced than in the other five DOPC/steroid systems. In contrast, the addition of stigmasterol to digalactosyldiacylglycerol (DGDG) bilayers appears to increase the order parameter mean value of P2, without affecting the diffusion coefficients. Furthermore, the incorporation of 7-dehydrocholesterol to DGDG bilayers causes a large enhancement in the orientational order, but has only a small effect on D perpendicular of the CSL molecules. Importantly, this latter effect appears to be independent of temperature. The marked changes in the rates of the rotational motion brought about by the addition of steroids, contrasts with the lack of a significant effect of unsaturation on the bilayer dynamics reported by us previously (Korstanje et al. (1989), Biochim. Biophys. Acta 980, 225-233, and 982, 196-204).     

Study of interaction of long-chain n-alcohols with fluid DOPC bilayers by a lateral pressure sensitive fluorescence probe

The excimer 1,2-dipyrenedecanoyl-sn-glycero-3-phosphatidylcholine (dipy10PC) fluorescence probe was used to determine effects of aliphatic alcohols (CnH2n+1OH, n = 12-18 is the even number of carbons in alkyl chain) on fluid dioleoylphosphatidylcholine (DOPC) +dioleoylphosphatidylserine (DOPS) bilayers in multilamellar vesicles at molar ratio DOPC/DOPS = 24.7. The excimer to monomer fluorescence intensity ratio increases with the increase of CnH2n+1OH/DOPC molar ratio and decreases with the CnH2n+1OH alkyl chain length n at a constant CnH2n+1OH/DOPC = 0.4 molar ratio. These effects indicate changes in the bilayer lateral pressure on the level of pyrenyl moieties location.     

Effects of temperature and pressure on the lateral organization of model membranes with functionally reconstituted multidrug transporter LmrA

To contribute to the understanding of membrane protein function upon application of pressure, we investigated the influence of hydrostatic pressure on the conformational order and phase behavior of the multidrug transporter LmrA in biomembrane systems. To this end, the membrane protein was reconstituted into various lipid bilayer systems of different chain length, conformation, phase state and heterogeneity, including raft model mixtures as well as some natural lipid extracts. In the first step, we determined the temperature stability of the protein itself and verified its reconstitution into the lipid bilayer systems using CD spectroscopic and AFM measurements, respectively. Then, to yield information on the temperature and pressure dependent conformation and phase state of the lipid bilayer systems, generalized polarization values by the Laurdan fluorescence technique were determined, which report on the conformation and phase state of the lipid bilayer system. The temperature-dependent measurements were carried out in the temperature range 5-70 °C, and the pressure dependent measurements were performed in the range 1-200 MPa. The data show that the effect of the LmrA reconstitution on the conformation and phase state of the lipid matrix depends on the fluidity and hydrophobic matching conditions of the lipid system. The effect is most pronounced for fluid DMPC and DMPC with low cholesterol levels, but minor for longer-chain fluid phospholipids such as DOPC and model raft mixtures such as DOPC/DPPC/cholesterol. The latter have the additional advantage of using lipid sorting to avoid substantial hydrophobic mismatch. Notably, the most drastic effect was observed for the neutral/glycolipid natural lipid mixture. In this case, the impact of LmrA incorporation on the increase of the conformational order of the lipid membrane was most pronounced. As a consequence, the membrane reaches a mechanical stability which makes it very insensitive to application of pressures as high as 200 MPa. The results are correlated with the functional properties of LmrA in these various lipid environments and upon application of high hydrostatic pressure and are discussed in the context of other work on pressure effects on membrane protein systems.     

Pressure-induced conformational changes in a human Bence-Jones protein (Mcg)

The effect of high static pressures on the internal structure of the immunoglobulin light chain (Bence-Jones) dimer from the patient Mcg was assessed with measurements of intrinsic protein fluorescence polarization and intensity. Depolarization of intrinsic fluorescence was observed at relatively low pressures (less than 2 kbar), with a standard volume change of -93 mL/mol. The significant conformational changes indicated by these observations were not attributable to major protein unfolding, since pressures exceeding 2 kbar were required to alter intrinsic fluorescence emission maxima and yields. Fluorescence intensity and polarization measurements were used to investigate pressure effects on the binding of bis(8-anilino-naphthalene-1-sulfonate) (bis-ANS), rhodamine 123, and bis(N-methylacridinium nitrate) (lucigenin). Below 1.5 kbar the Mcg dimer exhibited a small decrease in affinity for bis-ANS (standard volume change approximately 5.9 mL/mol). At 3 kbar the binding activity increased by greater than 250-fold (volume change -144 mL/mol) and remained 10-fold higher than its starting value after decompression. With rhodamine 123 the binding activity showed an initial linear increase but plateaued at pressures greater than 1.5 kbar (standard volume change -23 mL/mol). These pressure effects were completely reversible. Binding activity with lucigenin increased slightly at low pressures (standard volume change -5.5 mL/mol), but the protein was partially denatured at pressures greater than 2 kbar. Taken in concert with the results of parallel binding studies in crystals of the Mcg dimer, these observations support the concept of a large malleable binding region with broad specificity for aromatic compounds.(ABSTRACT TRUNCATED AT 250 WORDS)     

The structure of DNA-DOPC aggregates formed in presence of calcium and magnesium ions: a small-angle synchrotron X-ray diffraction study

The structure of aggregates formed due to DNA interaction with dioleoylphosphatidylcholine (DOPC) vesicles in presence of Ca(2+) and Mg(2+) cations was investigated using synchrotron small-angle X-ray diffraction. For DOPC/DNA=1:1 mol/base and in the range of concentration of the cation(2+) 0-76.5 mM, the diffractograms show the coexistence of two lamellar phases: L(x) phase with repeat distance d(Lx) approximately 8.26-7.39 nm identified as a phase where the DNA strands are intercalated in water layers between adjacent lipid bilayers, and L(DOPC) phase with repeat distance d(DOPC) approximately 6.45-5.65 nm identified as a phase of partially dehydrated DOPC bilayers without any divalent cations and DNA strands. The coexistence of these phases was investigated as a function of DOPC/DNA molar ratio, length of DNA fragments and temperature. If the amount of lipid increases, the fraction of partially dehydrated L(DOPC) phase is limited, depends on the portion of DNA in the sample and also on the length of DNA fragments. Thermal behaviour of DOPC+DNA+Ca(2+) aggregates was investigated in the range 20-80 degrees C. The transversal thermal expansivities of both phases were evaluated.     

Reorientational and Conformational Ordering Processes at Elevated Pressures in 1,2-Dioleoyl Phosphatidylcholine: A Raman and Infrared Spectroscopic Study

Raman and infrared spectra of fully hydrated bilayers of 1,2-dioleoyl phosphatidylcholine (DOPC) were measured at increasing hydrostatic pressures up to -37 kbar. Under ambient conditions aqueous dispersions of DOPC are in the liquid crystalline state. The application of an external hydrostatic pressure induces conformational and dynamic ordering processes in DOPC, which trigger a first-order structural phase transition at 5 kbar from a disordered liquid crystalline state to a highly ordered gel state. In the gel phase the methylene chains of each molecule are fully extended and the two all-trans chain segments on both sides of the rigid cis double bond form a bent structure. The bent oleoyl chains in each molecule, as well as in neighboring molecules are packed parallel to each other. To achieve this parallel interchain packing, the double bonds of the sn-1 and sn-2 chains of each molecule must be aligned at the same position with respect to the bilayer interface which is achieved by a rotation of the C—C bonds in the glycerol moiety in the head group. The extremely strong interchain interactions in the gel phase of DOPC are unique for this lipid with cis dimono-unsaturated acyl chains. Our experimental results suggest that in the pressure-induced gel phase of DOPC the olefinic CH bonds are rotated out of the phase of the bent oleoyl chains and that the oleoyl chains of opposing bilayers bend towards opposite directions.     

Application of high hydrostatic pressure for increasing activity and stability of enzymes

Elevated hydrostatic pressure has been used to increase catalytic activity and thermal stability of alpha-chymotrypsin (CT). For an anilide substrate, characterized by a negative value of the reaction activation volume (DeltaV( not equal)), an increase in pressure at 20 degrees C results in an exponential acceleration of the hydrolysis rate catalyzed by CT reaching a 6.5-fold increase in activity at 4700 atm (4.7 kbar). Due to a strong temperature dependence of DeltaV( not equal), the acceleration effect of high pressure becomes more pronounced at high temperatures. For example, at 50 degrees C, under a pressure of 3.6 kbar, CT shows activity which is more than 30 times higher than the activity at normal conditions (20 degrees C, 1 atm). At pressures of higher than 3.6 kbar, the enzymatic activity is decreased due to a pressure-induced denaturation.Elevated hydrostatic pressure is also efficient for increasing stability of CT against thermal denaturation. For example, at 55 degrees C, CT is almost instantaneously inactivated at atmospheric pressure, whereas under a pressure of 1.8 kbar CT retains its anilide-hydrolyzing activity during several dozen minutes. Additional stabilization can be achieved in the presence of glycerol, which is most effective for protection of CT at an intermediate concentration of 40% (v/v). There has been observed an additivity in stabilization effects of high pressure and glycerol: thermal inactivation of pressure-stabilized CT can be decelerated in a supplementary manner by addition of 40% (v/v) glycerol. The protection effect of glycerol on the catalytic activity and stability of CT becomes especially pronounced when both extreme factors of temperature and pressure reach critical values. For example, at approximately 55 degrees C and 4.7 kbar, enzymatic activity of CT in the presence of 40% (v/v) glycerol is severalfold higher than in aqueous buffer.The results of this study are discussed in terms of the hypotheses which explain the action of external and medium effects on protein structure, such as preferential hydration and osmotic pressure. (c) 1996 John Wiley & Sons, Inc.     

Interactions of cholesterol hemisuccinate with phospholipids and (Ca2+-Mg2+)-ATPase

Cholesterol hemisuccinate has been shown to equilibrate readily with liposomes and with the (Ca2+-Mg2+)-ATPase from sarcoplasmic reticulum and has been used to modify the sterol content of these membranes. Cholesterol hemisuccinate incorporates into dioleoylphosphatidylcholine (DOPC) up to a molar ratio of 3:1 sterol to DOPC. Effects on lipid order as detected by electron spin resonance and fluorescence polarization are comparable to those of cholesterol. Binding constants have been determined, and the uncharged form of the sterol binds more strongly than the anionic form. Binding to DOPC and to the lipid component of the ATPase system is comparable. From use of the fluorescence quenching properties of 1,2-bis(9,10- dibromooleoyl )phosphatidylcholine and dibromocholesterol hemisuccinate, two classes of binding sites on the ATPase have been deduced. At the lipid/protein interface, the binding constant for cholesterol hemisuccinate is considerably less than that for DOPC. At the second set of sites ( nonannular sites), binding occurs with Kd = 0.55 in molar ratio units. The effect of cholesterol hemisuccinate on the activity of the ATPase depends on the phospholipid present in the system: ATPase reconstituted with DOPC is inhibited whereas ATPase reconstituted with dimyristoleoylphosphatidylcholine is activated. We conclude that changes in membrane fluidity are not important in determining ATPase activity in these systems.     

Characterization of the pressure-induced intermediate and unfolded state of red-shifted green fluorescent protein--a static and kinetic FTIR,UV/VIS and fluorescence spectroscopy study

The green fluorescence proteins (GFP) are widely used as reporters in molecular and cell biology. For their use it in high-pressure microbiology and biotechnology studies, their structural properties, thermodynamic parameters and stability diagrams have to be known. We investigated the pressure stability of the red-shifted green fluorescent protein (rsGFP) using Fourier-transform infrared spectroscopy, fluorescence and UV/Vis spectroscopy. We found that rsGFP does not unfold up to approximately 9kbar at room temperature. Its unique three-dimensional structure is held responsible for the high-pressure stability. At higher temperatures, its secondary structure collapses below 9kbar (e.g. the denaturation pressure at 58 degrees C is 7.8kbar). The analysis of the IR data shows that the pressure-denatured state contains more disordered structures at the expense of a decrease of intramolecular beta-sheets. As indicated by the large volume change of DeltaV degrees (u) approximately -250(+/-50)mlmol(-1) at 58 degrees C, this highly cooperative transition can be interpreted as a collapse of the beta-can structure of rsGFP. For comparison, the temperature-induced unfolding of rsGFP has also been studied. At high temperature (T(m)=78 degrees C), the unfolding resulted in the formation of an aggregated state. Contrary to the pressure-induced unfolding, the temperature-induced unfolding and aggregation of GFP is irreversible. From the FT-IR data, a tentative p,T-stability diagram for the secondary structure collapse of GFP has been obtained. Furthermore, changes in fluorescence and absorptivity were found which are not correlated to the secondary structural changes. The fluorescence and UV/Vis data indicate smaller conformational changes in the chromophore region at much lower pressures ( approximately 4kbar) which are probably accompanied by the penetration of water into the beta-can structure. In order to investigate also the kinetics of this initial step, pressure-jump relaxation experiments were carried out. The partial activation volumes observed indicate that the conformational changes in the chromophore region when passing the transition state are indeed rather small, thus leading to a comparably small volume change of -20 ml mol(-1) only. The use of the chromophore absorption and fluorescence band of rsGFP in using GFP as reporter for gene expression and other microbiological studies under high pressure conditions is thus limited to pressures of about 4kbar, which still exceeds the pressure range relevant for studies in vivo in micro-organisms, including piezophilic bacteria from deep-sea environments.