Specific interaction of cytosolic and mitochondrial glyoxalase II with acidic phospholipids in form of liposomes results in the inhibition of the cytosolic enzyme only

Kinetics of cytosolic recombinant human glyoxalase II and bovine liver mitochondrial glyoxalase II were studied in the presence of liposomes made of different phospholipids (PLs). Neutral PLs such as egg phosphatidylcholine or dipalmitoylphosphatidylcholine did not affect the enzymatic activity of either enzymatic form. Liposomes made of dioleoyl phosphatidic acid or cardiolipin or phosphatidylserine also did not affect the enzymatic activity of mitochondrial glyoxalase II. Conversely, these negatively charged PLs exerted noncompetitive inhibition on cytosolic glyoxalase II only, dioleoyl phosphatidic acid and bovine brain phosphatidylserine exerting the highest and lowest inhibition, respectively. Binding studies, carried out by using a resonant mirror biosensor, revealed that liposomes made of negatively charged PLs interact specifically with both enzymatic forms of glyoxalase II, whereas interactions were not detected with neutral PLs. Once bound on glyoxalase II, negatively charged liposomes could not be removed by 3 M NaCl, suggesting that interactions between glyoxalase II and negatively charged PLs, besides ionic, may be also hydrophobic. These data suggest a possible role of negatively charged phospholipids in the regulation of level of lactoylglutathione in the cell. The data are also discussed in terms of a possible regulation of reduced glutathione supply to mitochondria.     

Conformational study of purified epoxide hydrolase from rat liver

Hepatic epoxide hydrolase (EC 3.3.2.3) was purified from phenobarbital-treated rats by ion-exchange chromatography followed by hydrophobic chromatography. The enzyme had a specific activity of 300--400 nmol min-1 mg-1 protein with benzo[a]pyrene-4,5-oxide as the substrate. Circular dichroism (CD) spectra of the purified enzyme gave two negative bands, centered at 210 nm and 222 nm, respectively. The mean residue ellipticity at 222 nm was 12,9000 deg X cm(2) X dmol(-1), which indicated the presence of about 35% alpha-helical structures. Sodium dodecyl sulfate (SDS) greatly affected the shape of the CD spectra, which were gradually shifted to the blue. This suggested a decrease in the aggregation state of the protein. Electrostatic interactions were important in the organization of the enzyme structure since the conformation was stable between pH 7.4 and pH 10. At pH-values 5.0, 6.0 and 12.0, the CD bands underwent considerable changes in both amplitude and shape. Moreover there was a good correlation between the optimal pH range of the epoxide hydrolase activity and the organization state of the protein. After membrane reconstitution with liposomes, the conformation of the enzyme was not significantly modified by the presence of dimyristoyl L-alpha-phosphatidylcholine or other phospholipids. This constancy was obtained over a wide range of molar ratios of phospholipids to protein (0--500). However, phospholipids did increase the thermal stability of the enzyme. Fluorescence measurements of diphenylhexatriene (DPH) bound to dimyristoyl L-alpha-phosphatidylcholine indicated that addition of epoxide hydrolase modified the thermal transition of the lipid phase. On the other hand, electron paramagnetic resonance (EPR) signals of the nitroxide-labelled fatty acid, 2-(14-carboxy-tetradecyl)-2-ethyl-4,4-dimethyl-3,3,-oxazolidiny-oxyl, bound to the phospholipid, indicated that the presence of the protein decreased by about 53% the correlation time of the label, suggesting that its motion had increased. In conclusion, phospholipid-epoxide hydrolase interactions enhanced the fluidity of dimyristoyl L-alpha-phosphatidylcholine liposomes without changing the secondary structure of the enzyme. Electrostatic interactions also played an important role in the conformational stability of the protein.     

Fluorescence spectroscopy studies of the association of membrane and soluble forms of chromaffin granule dopamine-beta-monooxygenase to bipolar phospholipid liposomes and micelles

The association of membrane and soluble forms of dopamine-beta-monooxygenase to liposomes and micelles made from phosphatidylcholine and lysophosphatidylcholine respectively has been studied using the fluorescence spectroscopy technique. As it was shown in our previous study these bipolar phospholipids activate the reaction catalyzed by the enzyme. Effects of pH and ionic strength on the association process were also studied, and efficiency of the association for apo- and holoenzyme was compared. The data obtained demonstrate that electrostatic attraction is involved in the association process. It was also shown that the membrane dopamine-beta-monooxygenase associated with phospholipid liposomes and micelles with higher efficiency than the soluble one did, which might be due to the involvement of the hydrophobic interactions in the association process. The results of the experiments also suggest that this process is specific and depends on the enzyme conformation, particularly on its quaternary structure. The participation of the hydrophobic peptide of the membrane dopamine-beta-monooxygenase in the formation and stabilization of the enzyme-phospholipid complex in vivo is proposed.     

Membrane fusion between liposomes composed of acidic phospholipids and neutral phospholipids induced by melittin: a differential scanning calorimetric study

Melittin-induced membrane fusion between neutral and acidic phospholipids was examined in liposome systems with a high-sensitivity differential scanning calorimeter. Membrane fusion could be detected by calorimetric measurement by observing thermograms of mixed liposomal lipids. The roles of hydrophobic and electrostatic interactions were investigated in membrane fusion induced by melittin. Melittin, a bee venom peptide, is composed of a hydrophobic region including hydrophobic amino acids and a positively charged region including basic amino acids. When phosphatidylcholine liposomes were prepared in the presence of melittin, reductions in the phase transition enthalpies were observed in the following order; dimyristoylphosphatidylcholine (DMPC) > dipalmitoylphosphatidylcholine (DPPC) > distearoylphosphatidylcholine (DSPC) > dielaidoylphosphatidylcholine (DEPC). The plase transition enthalpy of an acidic phospholipid, dipalmitoylphosphatidylserine (DPPS), was raised by melittin at low concentrations, then reduced at higher concentrations. DPPC liposomes prepared in melittin solution were fused with DPPS liposomes when the liposomal dispersions were mixed and incubated. Similar fusion was observed between dipalmitoylphosphatidylcholine and dimyristoylphosphatidic acid (DMPA) liposomes. These results indicate that a peptide including hydrophobic and basic regions can mediate membrane fusion between neutral and acidic liposomes by hydrophobic and electrostatic interactions.     

Calreticulin is a thermostable protein with distinct structural responses to different divalent cation environments

Calreticulin is a soluble calcium-binding chaperone of the endoplasmic reticulum (ER) that is also detected on the cell surface and in the cytosol. Calreticulin contains a single high affinity calcium-binding site within a globular domain and multiple low affinity sites within a C-terminal acidic region. We show that the secondary structure of calreticulin is remarkably thermostable at a given calcium concentration. Rather than corresponding to complete unfolding events, heat-induced structural transitions observed for calreticulin relate to tertiary structural changes that expose hydrophobic residues and reduce protein rigidity. The thermostability and the overall secondary structure content of calreticulin are impacted by the divalent cation environment, with the ER range of calcium concentrations enhancing stability, and calcium-depleting or high calcium environments reducing stability. Furthermore, magnesium competes with calcium for binding to calreticulin and reduces thermostability. The acidic domain of calreticulin is an important mediator of calcium-dependent changes in secondary structure content and thermostability. Together, these studies indicate interactions between the globular and acidic domains of calreticulin that are impacted by divalent cations. These interactions influence the structure and stability of calreticulin, and are likely to determine the multiple functional activities of calreticulin in different subcellular environments.     

Redox- and pH-dependent association of plastocyanin with lipid bilayers: effect on protein conformation and thermal stability

The effect of electrostatic interactions on the conformation and thermal stability of plastocyanin (Pc) was studied by infrared spectroscopy. Association of any of the two redox states of the protein with positively charged membranes at neutral pH does not significantly change the secondary structure of Pc. However, upon membrane binding, the denaturation temperature decreases, regardless of the protein redox state. The extent of destabilization depends on the proportion of positively charged lipid headgroups in the membrane, becoming greater as the surface density of basic phospholipids increases. In contrast, at pH 4.8 the membrane binding-dependent conformational change becomes redox-sensitive. While the secondary structures and thermal stabilities of free and membrane-bound oxidized Pc are similar under acidic conditions, the conformation of the reduced form of the protein drastically rearranges upon membrane association. This rearrangement does not depend on electrostatic interactions to occur, since it is also observed in the presence of uncharged lipid bilayers. The conformational transition, only observed for reduced Pc, involves the exposure of hydrophobic regions that leads to intermolecular interactions at the membrane surface. Membrane-mediated partial unfolding of reduced Pc can be reversed by readjusting the pH to neutrality, in the absence of electrostatic interactions. This redox-dependent behavior might reflect specific structural requirements for the interaction of Pc with its redox partners.     

Membrane interactions of rat intestinal alkaline phosphatase: role of polar head groups

Lipid-protein interactions with purified membranous intestinal alkaline phosphatase have been studied by using rat intestine. The enzyme was incorporated equally well into neutral lecithin and anionic liposomes, including those made from phosphatidic acid alone. It could not be solubilized with chaotropic salts nor by phospholipases C and D from either native membranes or phospholipid vesicles. Detergents effected nearly complete release of enzyme from the vesicles. Phosphatase activity was lost upon treatment with phospholipase D alone. The activity was restored with free choline, or choline containing phospholipids, but not by the addition of other phospholipids or amines. The catalytic activity was also lower when the enzyme was bound to a phosphatidylcholine vesicle containing additional phosphatidic acid. Neither phosphatidylserine nor phosphatidylinositol addition altered enzyme activity. These results show that the enzyme binds to the membrane by a primary hydrophobic interaction with membrane phospholipids without requiring the polar head group and that the enzyme activity is affected via a secondary interaction with choline. We suggest that choline protects the active site of brush border alkaline phosphatase from inhibition by endogenous membrane phosphate groups.     

Inhibition of skeletal muscle sarcoplasmic reticulum CaATPase activity by calmidazolium

Calmidazolium, a lipophilic cation and putative calmodulin-specific antagonist, inhibited potently the calcium ATPase of sarcoplasmic reticulum (SR) vesicles isolated from skeletal muscle. Based on steady-state measurements of catalytic activity over a range of MgATP, calmidazolium, and SR protein concentrations, the calculated values of the inhibition constant (KI) and binding stoichiometry were 0.06 microM and 770 nmol/mg protein, respectively. SR CaATPase inhibition apparently is not a general property of lipophilic cations since the hydrophobic anion tetraphenylboron inhibited catalysis, whereas its cationic analog, tetraphenylarsonium, did not. Enzyme inhibition by calmidazolium was noncompetitive with respect to the substrates Ca2+ and MgATP. In the presence of other SR CaATPase inhibitors, calmidazolium was competitive with respect to quercetin and noncompetitive with respect to trifluoperazine and propranolol. While calmidazolium inhibited enzyme phosphorylation by MgATP, catalysis was more sensitive to the inhibitor. Binding of calmidazolium to SR membranes produced morphological changes seen by electron microscopy as membrane thickening and loss of resolution of surface detail. Our results show that calmidazolium is a high-affinity, noncompetitive inhibitor of skeletal SR CaATPase activity, and they suggest that this inhibition is based on binding to the membrane phospholipids rather than specific antagonism of enzyme activation by calmodulin.     

Characteristics of binding of zwitterionic liposomes to water-soluble proteins

The interactions of zwitterionic phospholipids phosphatidylcholine and phosphatidylethanolamine with protein proteinase inhibitors aprotinin and Bowman-Birk soybean proteinase inhibitor have been investigated. An increase in the hydrophobicity of the liposome surface was shown to be an important factor for the formation of proteoliposomes. According to ³¹P-NMR spectra, incorporation of the proteins into the liposomes does not influence the structural organization of the surface of the liposomes. Increasing the ionic strength does not inhibit the process of proteoliposome formation. Fluorescence assay of the complexes of anthracene-labeled phospholipids with the rhodamine B-labeled protein showed that after the encapsulation into the liposomes, the protein is located inside the particles and between the bilayers. Also, the effect of phospholipids with saturated fatty acid residues on the protein-lipid interaction was studied by differential scanning calorimetry. The results indicate that water-soluble proteins efficiently interact with zwitterionic phospholipids, and the encapsulation of the proteins into the liposomes is provided by electrostatic and hydrophobic forces (in the case of aprotinin) or predominantly by hydrophobic forces (Bowman-Birk soybean proteinase inhibitor).     

Inhibition of PhoE translocation across Escherichia coli inner-membrane vesicles by synthetic signal peptides suggests an important role of acidic phospholipids in protein translocation

To obtain insight into the mechanism of precursor protein translocation across membranes, the effect of synthetic signal peptides and other relevant (poly)peptides on in vitro PhoE translocation was studied. The PhoE signal peptide, associated with inner membrane vesicles, caused a concentration-dependent inhibition of PhoE translocation, as a result of a specific interaction with the membrane. Using a PhoE signal peptide analog and PhoE signal peptide fragments, it was demonstrated that the hydrophobic part of the peptide caused the inhibitory effect, while the basic amino terminus is most likely important for an optimal interaction with the membrane. A quantitative analysis of our data and the known preferential interaction of synthetic signal peptides with acidic phospholipids in model membranes strongly suggest the involvement of negatively charged phospholipids in the inhibitory interaction of the synthetic PhoE signal peptide with the inner membrane. The important role of acidic phospholipids in protein translocation was further confirmed by the observation that other (poly)peptides, known to have both a high affinity for acidic lipids and hydrophobic interactions with model membranes, also caused strong inhibition of PhoE translocation. The implication of these results with respect to the role of signal peptides in protein translocation is indicated.     

The amyloid precursor protein interacts with neutral lipids.

The amyloid protein precursor (APP) was incorporated into liposomes or phospholipid monolayers. APP insertion into liposomes required neutral lipids, such as L-alpha-phosphatidylcholine, in the target membrane. It was prevented in vesicles containing L-alpha-phosphatidylserine. The insertion was enhanced in acidic solutions, suggesting that it is modulated by specific charge/charge interactions. Surface-active properties and behaviour of APP were characterized during insertion of the protein in monomolecular films of L-alpha-phosphatidylcholine, L-alpha-phosphatidylethanolamine or L-alpha-phosphatidylserine. The presence of the lipid film enhanced the rate of adsorption of the protein at the interface, and the increase in surface pressure was consistent with APP penetrating the lipid film. The adsorption of APP on the lipid monolayers displayed a significant head group dependency, suggesting that the changes in surface pressure produced by the protein were probably affected by electrostatic interactions with the lipid layers. Our results indicate that the penetration of the protein into the lipid monolayer is also influenced by the hydrophobic interactions between APP and the lipid. CD spectra showed that a large proportion of the alpha-helical secondary structure of APP remained preserved over the pH or ionic strength ranges used. Our findings suggest that APP/membrane interactions are mediated by the lipid composition and depend on both electrostatic and hydrophobic effects, and that the variations observed are not due to major secondary structural changes in APP. These observations may be related to the partitioning of APP into membrane microdomains.     

Effect of pH on the stability and structure of yeast hexokinase A. Acidic amino acid residues in the cleft region are critical for the opening and the closing of the structure

pH and salts have a marked effect on the stability, structure, and function of many globular proteins due to their ability to influence the electrostatic interactions. In this work, calorimetry, CD, and fluorescence studies have been carried out to understand the pH-dependent conformational changes of the two-domain protein yeast hexokinase A. In conjunction with the crystal structural data available, the present results have enabled the complete characterization and analysis of the pH-dependent conformational changes of the enzyme that have strong implications in understanding its structure-function relationship. The calorimetric profiles show a single thermal transition in the acidic pH range, whereas two independent transitions were observed in the alkaline pH range, suggesting the structural merger of the domains at the acidic pH. Comparison of the thermal transitions at pH 8.5 studied by different techniques suggests that the first transition corresponds to the smaller domain, and the second transition corresponds to the larger domain. The acid-denatured state of hexokinase A has high secondary structure content with little or no tertiary interactions and binds to the hydrophobic dye 8-anilinonaphthalene-1-sulfonic acid, suggesting that it is a molten globule-like state, whereas the alkali-denatured state is less structured than the acid-denatured state but more structured than the urea-denatured state, suggestive of a premolten globule-like state. Structural analysis using the published hexokinase B structure as well as the hexokinase A structure with the revised amino acid sequence in conjunction with the results obtained by us suggests that the ionization state of the acidic residues at the active site could regulate domain movements that are responsible for the opening and the closure of the cleft between the two domains and in turn affect the structure and function of the enzyme.     

The role of phospholipids in the modulation of enzyme activities in the chromaffin granule membrane

(1) 93% of protein of chromaffin granule membranes can be solubilized by 1.3% (w/v) sodium cholate. The solubilized material can be substantially delipidated by ammonium sulphate precipitation. After three such cycles less than 2% of the endogenous phospholipids remain. (2) The chromaffin granule membrane Mg2+-ATPase depends on the presence of phospholipids for retention of its full activity. Soybean and extracted chromaffin granule phospholipids fully reactivate the delipidated enzyme provided only one delipidation step is used. (3) Successive ammonium sulphate precipitation steps result in a delipidated, and deactivated ATPase preparation which can be only partially reactivated on re-addition of phospholipids. (4) The phospholipid specificity for reactivation of the Mg2+-ATPase is broad. Although acidic phospholipids allow higher activities than neutral phospholipids, the main requirement appears to be the hydrophobic environment provided by the phospholipid hydrocarbon chains. (5) Correlations between changes in slope in the Arrhenius plot of the Mg2+-ATPase, and phase transitions in the phospholipid used for reactivation suggest that the 'fluidity' of the hydrocarbon chains modulates the activity of the enzyme.     

Generation of fatty acids from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cardiolipin liposomes that stabilize recombinant human serum albumin

Abstract

Context: At elevated temperatures, studies have shown that serum albumin undergoes irreversible changes to its secondary structure. Anionic fatty acids and/or anionic surfactants have been shown to stabilize human serum albumin (HSA) against thermal denaturation through bridging hydrophobic domains and cationic amino acids residues of the protein.

Objective: As albumin can readily interact with a variety of liposomes, this study proposes that cardiolipin delivered via 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes can improve the thermal stability of recombinant HSA produced in Saccharomyces cerevisiae (ScrHSA) in a similar manner to anionic fatty acids.

Materials and methods: Thermal stability and structure of ScrHSA in the absence and presence of DPPC/cardiolipin liposomes was assessed with U/V circular dichroism spectropolarimetry and protein thermal stability was confirmed with differential scanning calorimetry.

Results: Although freshly prepared DPPC/cardiolipin liposomes did not improve the stability of ScrHSA, DPPC/cardiolipin liposomes incubated at room temperature for 7?d (7dRT) dramatically improved the thermal stability of the protein. Mass spectrometry analysis identified the presence of fatty acids in the 7dRT liposomes, not identified in freshly prepared liposomes, to which the improved stability was attributed.

Discussion and conclusion: The generation of fatty acids is attributed to either the chemical hydrolysis or oxidative cleavage of the unsaturated acyl chains of cardiolipin. By modulating the lipid composition through the introduction of lipids with higher acyl chain unsaturation, it may be possible to generate the stabilizing fatty acids in a more rapid manner.     

Binding of Synapsin I to Synaptic Vesicles: Clues from the Study of its Interactions with Liposomes

Abstract

Synapsin I is a major brain phosphoprotein which interacts with synaptic vesicles and actin in a phosphorylation-dependent fashion. The binding of synapsin I to synaptic vesicles involves interactions with the phospholipid and protein components of the vesicle membrane. The highly hydrophobic NH₂-terminal head region of the protein binds with high-affinity to acidic phospholipids and penetrates the hydrophobic core of the membrane, whereas the basic COOH-terminal tail region does not significantly contribute to this binding. The interaction with phospholipids increases the amount of α-helix in the secondary structure of synapsin I, but does not markedly affect the microenvironment of tryptophan and cysteine residues present in the head region. The results suggest that synapsin I binds to synaptic vesicle phospholipids through amphiphilic and positively charged domains present in its NH2-terminal region and that such an interaction contributes to the high-affinity binding of synapsin I to synaptic vesicles.     

Bile salt hydrolase, the member of Ntn-hydrolase family: differential modes of structural and functional transitions during denaturation

Conformational transitions and functional stability of the bile salt hydrolase (BSH; cholylglycine EC: 3.5.1.24) from Bifidobacterium longum (BlBSH) cloned and expressed in E. coli were studied under thermal, chemical and pH-mediated denaturation conditions using fluorescence and CD spectroscopy. Thermal and Gdn-HCl-mediated denaturation of BlBSH is a multistep process of inactivation and unfolding. The inactivation and unfolding of the enzyme was found to be irreversible. Enzyme activity seems sensitive to even minor conformational changes at the active site. Thermal denaturation as such did not result in any insoluble protein aggregates. However, on treating with 0.25 - 1 M Gdn-HCl the enzyme showed increasing aggregation at temperatures of 40 - 55 degrees C indicating more complex structural changes taking place in the presence of chemical denaturants. The enzyme secondary structure was still intact at acidic pH (pH 1 - 3). The perturbation in the tertiary structure at the acidic pH was detected through freshly formed solvent exposed hydrophobic patches on the enzyme. These changes could be due to the formation of an acid-induced molten globule-like state.     

Ultracentrifugation studies of the location of the site involved in the interaction of pig heart lactate dehydrogenase with acidic phospholipids at low pH. A comparison with the muscle form of the enzyme

Lactate dehydrogenase (LDH) from the pig heart interacts with liposomes made of acidic phospholipids most effectively at low pH, close to the isoelectric point of the protein (pH = 5.5). This binding is not observed at neutral pH or high ionic strength. LDH-liposome complex formation requires an absence of nicotinamide adenine dinucleotides and adenine nucleotides in the interaction environment. Their presence limits the interaction of LDH with liposomes in a concentration-dependent manner. This phenomenon is not observed for pig skeletal muscle LDH. The heart LDH-liposome complexes formed in the absence of nicotinamide adenine dinucleotides and adenine nucleotides are stable after the addition of these substances even in millimolar concentrations. The LDH substrates and studied nucleotides that inhibit the interaction of pig heart LDH with acidic liposomes can be ordered according to their effectiveness as follows: NADH > NAD > ATP = ADP > AMP > pyruvate. The phosphorylated form of NAD (NADP), nonadenine nucleotides (GTP, CTP, UTP) and lactate are ineffective. Chemically cross-linked pig heart LDH, with a tetrameric structure stable at low pH, behaves analogously to the unmodified enzyme, which excludes the participation of the interfacing parts of subunits in the interaction with acidic phospholipids. The presented results indicate that in lowered pH conditions, the NADH-cofactor binding site of pig heart LDH is strongly involved in the interaction of the enzyme with acidic phospholipids. The contribution of the ATP/ADP binding site to this process can also be considered. In the case of pig skeletal muscle LDH, neither the cofactor binding site nor the subunit interfacing areas seem to be involved in the interaction.     

Interaction of mitochondrial aspartate aminotransferase with negatively charged lecithin liposomes.

Several kinds of hydrophilic proteins were examined to determine their interaction with artificial liposomes. Mitochondrial aspartate aminotransferase (m-GOT) [EC 2.6.1.1], as well as cytochrome c, was found to interact strongly with negatively charged liposomes. In each case, an appreciable amount of the protein bound to liposomes remained unreleased after raising the salt concentration in the medium. The m-GOT tightly bound to the liposomes was also found to become latent in its enzymatic activity, and could be reversibly activated by solubilization of the liposomes with detergent. This is also the case for cytochrome c, which ceases to be reducible by external reductant, such as dithionite. Furthermore, the tightly bound m-GOT was not susceptible to the proteolytic action of trypsin, or that of Nagarse. From these observations it can be inferred that these basic proteins interact with acidic liposomes not only electrostatically but also hydrophobically. This kind of hydrophobic interaction was not observed in the combination of positively charged liposomes and acidic proteins, including s-GOT. Mitochondrial GOT was shown to be bound to isolated intact mitochondrial, but the bound enzyme was fully active, in contrast to the case of acidic liposomes. The hydrophobic interaction of water-soluble protein with liposomes is discussed in connection with the penetration of matrix enzyme through mitochondrial membranes.     

Low pH-induced conformational changes in vesicular stomatitis virus glycoprotein involve dramatic structure reorganization

Membrane fusion is the key step in the entry of enveloped animal viruses into their host cells. Fusion of vesicular stomatitis virus with membranes occurs at acidic pH and is mediated by its envelope glycoprotein, the G protein. To study the structural transitions induced by acidic pH on G protein, we have extracted the protein from purified virus by incubation with nonionic detergent. At pH 6.0, purified G protein was able to mediate fusion of either phospholipid vesicles or Vero cells in culture. Intrinsic fluorescence studies revealed that changes in the environment of Trp residues occurred as pH decreases. In the absence of lipidic membranes, acidification led to G protein aggregation, whereas protein-protein interactions were substituted by protein-lipid interactions in the presence of liposomes. 1,1'-Bis(4-aniline-5-naphthalene sulfonate) (bis-ANS) binding was utilized to probe the degree of exposure of hydrophobic regions of G protein during acidification. Bis-ANS binding was maximal at pH 6.2, suggesting that a hydrophobic segment is exposed to the medium at this pH. At pH 6.0, a dramatic decrease in bis-ANS binding was observed, probably due to loss of tridimensional structure during the conformational rearrangement. This hypothesis was confirmed by circular dichroism analysis at different pH values, which showed a great decrease in alpha-helix content at pH values close to 6.0, suggesting that a reorganization of G protein secondary structure occurs during the fusion reaction. Our results indicate that G protein undergoes dramatic structural changes at acidic pH and acquires a conformational state able to interact with the target membrane.     

Novel organization and properties of annexin 2-membrane complexes

Annexin 2 belongs to the annexin family of proteins that bind to phospholipid membranes in a Ca(2+)-dependent manner. Here we show that, under mild acidic conditions, annexin 2 binds to and aggregates membranes containing anionic phospholipids, a fact that questions the mechanism of its interaction with membranes via Ca(2+) bridges only. The H(+) sensitivity of annexin 2-mediated aggregation is modulated by lipid composition (i.e. cholesterol content). Cryo-electron microscopy of aggregated liposomes revealed that both the monomeric and the tetrameric forms of the protein form bridges between the liposomes at acidic pH. Monomeric annexin 2 induced two different organizations of the membrane junctions. The first resembled that obtained at pH 7 in the presence of Ca(2+). For the tetramer, the arrangement was different. These bridges seemed more flexible than the Ca(2+)-mediated junctions allowing the invagination of membranes. Time-resolved fluorescence analysis at mild acidic pH and the measurement of Stokes radius revealed that the protein undergoes conformational changes similar to those induced by Ca(2+). Labeling with the lipophilic probe 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine indicated that the protein has access to the hydrophobic part of the membrane at both acidic pH in the absence of Ca(2+) and at neutral pH in the presence of Ca(2+). Models for the membrane interactions of annexin 2 at neutral pH in the presence of Ca(2+) and at acidic pH are discussed.