Peptide fragment studies on the folding elements of dihydrofolate reductase from Escherichia coli

One of the necessary conditions for a protein to be foldable is the presence of a complete set of “folding elements” (FEs) that are short, contiguous peptide segments distributed over an amino acid sequence. The FE‐assembly model of protein folding has been proposed, in which the FEs play a role in guiding structure formation through FE–FE interactions early in folding. However, two major issues remain to be clarified regarding the roles of the FEs in determining protein foldability. Are the FEs AFUs that can form nativelike structures in isolation? Is the presence of only the FEs without mutual connections a sufficient condition for a protein to be foldable? Here, we address these questions using peptide fragments corresponding to the FEs of DHFR from Escherichia coli. We show by CD measurement that the FE peptides are unfolded under the native conditions, and some of them have the propensities toward non‐native helices. MD simulations also show the non‐native helical propensities of the peptides, and the helix contents estimated from the simulations are well correlated with those estimated from the CD in TFE. Thus, the FEs of DHFR are not AFUs, suggesting the importance of the FEs in nonlocal interactions. We also show that equimolar mixtures of the FE peptides do not induce any structural formation. Therefore, mutual connections between the FEs, which should strengthen the nonlocal FE–FE interactions, are also one of the necessary conditions for a protein to be foldable. Proteins 2006. © 2005 Wiley‐Liss, Inc.     

Probing the interactions between the folding elements early in the folding of Escherichia coli dihydrofolate reductase by systematic sequence perturbation analysis

One of the necessary conditions for a protein to be foldable is the presence of a complete set of folding elements (FEs) that are short contiguous peptide segments distributed over an amino acid sequence. Previous studies indicated the FE assembly model of protein folding, in which the FEs interact with each other and coalesce to form an intermediate(s) early in the folding reaction. This suggests that a clue to the understanding of the determinants of protein foldability can be found by investigating how the FEs interact with each other early in the folding and thereby elucidating roles of the FEs in protein folding. To reveal the formation process of FE-FE interactions, we studied the early folding events of Escherichia coli dihydrofolate reductase (DHFR) utilizing systematic sequence perturbation analysis. Here, systematic single amino acid substitutions were introduced inside of the FEs (W30X in FE2, V40X in FE3, N59X in FE4, and I155X in FE10; X refers to various amino acid residues), and their kinetic refolding reactions were measured by stopped-flow circular dichroism and fluorescence. We show that the interactions around Trp30 and Ile155 are formed in the burst phase intermediate, while those around Val40 and Asn59 are formed in the transition state of the subsequent folding phase (tau5-phase) and in much later processes, respectively. These and previous results suggest that FE2 and FE10, and also FE1 and FE7, involved in the loop subdomain of DHFR, interact with each other within a millisecond time range, while the stable FE3-FE4 interactions are formed in the later processes. This may highlight the important roles of the FEs mainly inside of the loop subdomain in formation of the burst phase intermediate having a hydrophobic cluster and native-like overall topology and in acquisition of the foldability of DHFR.     

Phase behaviour of mixtures of lipid X with phosphatidylcholine and phosphatidylethanolamine

The effect of increasing concentrations of lipid X (2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine 1-phosphate) on the phase behaviour of EPC (egg phosphatidylcholine) and EPE (egg phosphatidylethanolamine) is studied at a pH greater than or equal to 7 where lipid X carries one to two negative charges. Small amounts of lipid X (molar ratio approximately 0.01) induce continuous swelling of EPC and EPE bilayers and consequently the formation of large unilamellar vesicles in excess water. In many respects, the effect of lipid X on EPC and EPE bilayers is similar to that of phosphatidic acid. However, lipid X/EPC mixtures form micelles in excess lipid X whereas mixtures of phosphatidic acid/EPC vesiculate at all ratios. The same is true for lipid X/EPE mixtures. Small unilamellar vesicles of an average diameter of 40 nm form spontaneously upon dispersion of a dry lipid X/EPE film (molar ratio = 10). Unsonicated dispersions of lipid X/EPC (molar ratio = 1) are subjected to pH-jump treatment which involves raising of the pH to 11-12 and subsequent lowering of the pH to between 7.5 and 8.5. Such a treatment has little effect on the vesicle size and size distribution as compared to a control dispersion at pH 8.2. The mean size is determined to be 92 +/- 60 nm. Electron micrographs of freeze-fractured samples of lipid X/EPC (molar ratio = 1) reveal the presence of mainly micelles at pH 12. Upon lowering the pH to neutrality these micelles become unstable and aggregate/fuse rapidly to unilamellar vesicles (average diameter 95 +/- 40 nm). Sonication of equimolar mixtures of lipid X and EPC at pH 7 yields small unilamellar vesicles of a diameter of 20-25 nm as well as mixed micelles of a size between 15 and 17 nm. This behaviour is again different from that of mixed EPC/phosphatidic acid dispersions which form small unilamellar vesicles. The presence of lipid X in such mixtures does not prevent the aggregation/fusion to larger vesicles during freezing of the dispersion. As with pure EPC bilayers, stabilization is, however, achieved in the presence of 10% sucrose. This indicates that the covalently bonded glucosamine group of lipid X cannot substitute water of hydration in neighbouring EPC molecules.     

Reactive oxygen species formation and cell death in catalase-deficient tobacco leaf disks exposed to cadmium

The physiological responses of tobacco (Nicotiana tabacum L.) to oxidative stress induced by cadmium were examined with respect to reactive oxygen species (ROS) formation, antioxidant enzymes activities, and cell death appearance in wild-type SR1 and catalase-deficient CAT1AS plants. Leaf disks treated with 100 or 500 µM CdCl₂ increased Evans blue staining and leakage of electrolytes in SR1 or CAT1AS plants, more pronouncedly in the transgenic cultivar, but without evidence of lipid peroxidation in any of the cultivars compared to controls. Cadmium significantly reduced the NADPH oxidase-dependent O ₂ ^? formation in a dose dependent manner in SR1 very strongly at 500 µM (to 5% of the activity in the nontreated SR1 leaf disks). In CAT1AS, the NADPH oxidase activity was constitutively reduced at 50% with respect to that of SR1, but the magnitude of the decay was less prominent in this cultivar, reaching an average of 64% of the C at 21 h, for both Cd concentrations. Hydrogen peroxide formation was only slightly increased in SR1 or CAT1AS leaf disks at 21 h of exposure compared to the respective controls. Cd increased superoxide dismutase activity more than six times at 21 h in CAT1AS, but not in SR1 and reduced catalase activity by 59% at 21 h of treatment only in SR1 plants. Despite that catalase expression was constitutively lower in CATAS1 compared to SR1 nontreated leaf disks, 500 µM CdCl₂ almost doubled it only in CAT1AS at 21 h. The mechanisms underlying Cd-induced cell death were possibly not related exclusively to ROS formation or detoxification in tobacco SR1 or CAT1AS plants.     

Micelle-vesicle transition of egg phosphatidylcholine and octyl glucoside

The dissolution and formation of egg phosphatidylcholine (PC) vesicles by the detergent octyl glucoside were examined systematically by using resonance energy transfer between fluorescent lipid probes, turbidity, and gel filtration chromatography. Resonance energy transfer was exquisitely sensitive to the intermolecular distance when the lipids were in the lamellar phase and to the transitions leading to mixed micelles. Turbidity measurements provided information about the aggregation of lipid and detergent. Several reversible discrete transitions between states of the PC-octyl glucoside system were observed by both methods during dissolution and vesicle formation. These states could be described as a series of equilibrium structures that took the forms of vesicles, open lamellar sheets, and mixed micelles. As detergent was added to an aqueous suspension of vesicles, the octyl glucoside partitioned into the vesicles with a partition coefficient of 63. This was accompanied by leakage of small molecules and vesicle swelling until the mole fraction of detergent in the vesicles was just under 50% (detergent:lipid ratio of 1:1). Near this point, a transition was observed by an increase in turbidity and release of large molecules like inulin, consistent with the opening of vesicles. Both a turbidity maximum and a sharp increase in fluorescence were observed at a detergent to lipid mole ratio of 2.1:1. This was interpreted as the lower boundary of a region where both lamellar sheets and micelles are at equilibrium. At a detergent:lipid ratio of 3.0:1, another sharp change in resonance energy transfer and clarification of the suspension were observed, demarcating the upper boundary of this two-phase region. This latter transition is commonly referred to as solubilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aggregation behavior of lipid IVA in aqueous solutions at physiological pH. 1: Simple buffer solutions.

We have investigated the aggregation behaviour of lipid IVA (a bioactive precursor of lipid A and the lipid anchor of lipopolysaccharide) in aqueous solutions in the physiological pH range using dynamic light scattering, nuclear magnetic resonance, fluorescence, surface pressure, electron microscopy and force field simulation studies. The sonication of lipid IVA in PBS, Tris and Hepes produces vesicles which are stable in the concentration range of 10(-3) - 10(-7) M, possibly even at lower concentrations. The vesicle size is not sensitive to the nature of the buffer, only to the pH and to some extent to the ionic strength. The long time stability of the small unilamellar vesicles as well as the structureless 1H-NMR spectra might be attributed to a rigid surface structure. This structure is also supported by the simulation studies. We have tentatively proposed a coexistence of micelles and/or other aggregates with the bilayered vesicles at higher lipid concentrations in order to explain some of the experimental observations.     

Characterization of hatching-associated changes in the sea urchin fertilization envelope

The embryo of the sea urchin Strongylocentrotus purpuratus hatches from the fertilization envelope (FE) via synthesis and secretion of a hatching enzyme and by ciliary activity. Although the basic characteristics of the hatching enzyme are known, little is understood about changes in the FE during hatching. We have studied the biochemical changes in FEs during hatching. Polyacrylamide gel analysis revealed an increasingly complex polypeptide spectrum of the extractable fraction of FEs isolated during development. Immunoblotting of these polypeptides (using antiserum against the soluble polypeptides extracted from FEs isolated at 30 minutes postinsemination) revealed a decrease in the soluble FE components during hatching. Immunochemical analysis of hatching medium showed a strong correlation between the soluble FE components released and the hatching interval. Immunoblotting of hatching media indicated the presence of soluble FE polypeptides of similar and lower molecular weights than those obtained for extracts of FEs. These results imply that the hatching-associated changes in the FE of S purpuratus occur via proteolysis of FE components, which are derived from the paracrystalline protein fraction, a subset of cortical granule proteins.     

Reconstituted phosphatidylserine synthase from Escherichia coli is activated by anionic phospholipids and micelle-forming amphiphiles.

The activity of phosphatidylserine (PS) synthase (CDP-1, 2-diacyl-sn-glycerol: l-serine O-phosphatidyltransferase, EC 2.7.8. 8) from Escherichia coli was studied after reconstitution with lipid vesicles of various compositions. PS synthase exhibited practically no activity in the absence of a detergent and with the substrate CDP-diacylglycerol (CDP-DAG) present only in the lipid vesicles. Inclusion of octylglucoside (OG) in the assay mixture increased the activity 20- to 1000-fold, the degree of activation depending on the lipid composition of the vesicles. Inclusion of additional CDP-DAG in the assay mixture increased the activity 5- to 25-fold. When the fraction of phosphatidylglycerol (PG) was increased from 15 to 100 mol% in the vesicles the activity increased 10-fold using the assay mixture containing OG. The highest activities were exhibited with the anionic lipids synthesized by E. coli, namely PG, diphosphatidylglycerol (DPG), and phosphatidic acid, while phosphatidylinositol gave a lower activity. Cryotransmission electron microscopy showed that transformation of the vesicles to micelles brings about an activation of the enzyme that is proportional to the degree of micellization. Thus, the activity of PS synthase is modulated by the lipid aggregate structure and by the fraction and type of anionic phospholipid in the aggregates. The increase in the activity caused by PG and DPG is physiologically relevant; it may be part of a regulatory mechanism that keeps the balance between phosphatidylethanolamine, and the sum of PG and DPG, nearly constant in wild-type E. coli cells.     

Functional roles of the major chloroplast lipids in the violaxanthin cycle

Monogalactosyldiacylglyceride (MGDG) and digalactosyldiacylglyceride (DGDG) are the major membrane lipids of chloroplasts. The question of the specialized functions of these unique lipids has received limited attention. One function is to support violaxanthin de-epoxidase (VDE) activity, an enzyme of the violaxanthin cycle. To understand better the properties of this system, the effects of galactolipids and phosphatidylcholines on VDE activity were examined by two independent methods. The results show that the micelle-forming lipid (MGDG) and bilayer forming lipids (DGDG and phosphatidylcholines) support VDE activity differently. MGDG supported rapid and complete de-epoxidation starting at a threshold lipid concentration (10 μM) coincident with complete solubilization of violaxanthin. In contrast, DGDG supported slow but nevertheless complete to nearly complete de-epoxidation at a lower lipid concentration (6.7 μM) that did not completely solubilize violaxanthin. Phosphotidylcholines showed similar effects as DGDG except that de-epoxidation was incomplete. Since VDE requires solubilized violaxanthin, aggregated violaxanthin in DGDG at low concentration must become solubilized as de-epoxidation proceeds. High lipid concentrations had lower activity possibly due to formation of multilayered structures (liposomes) that restrict accessibility of violaxanthin to VDE. MGDG micelles do not present such restrictions. The results indicate VDE operates throughout the lipid phase of the single bilayer thylakoid membrane and is not limited to putative MGDG micelle domains. Additionally, the results also explain the differential partitioning of violaxanthin between the envelope and thylakoid as due to the relative solubilities of violaxanthin and zeaxanthin in MGDG, DGDG and phospholipids. The violaxanthin cycle is hypothesized to be a linked system of the thylakoid and envelope for signal transduction of light stress.     

A comparison of fertilization envelope development in three species of Strongylocentrotus (S. franciscanus, S. droebachiensis, and S. purpuratus)

The ultrastructure of fertilization envelope (FE) development and the polypeptide spectra of Strongylocentrotus franciscanus and S. droebachiensis envelopes were compared to S. purpuratus. In S. franciscanus, the FE reached its maximum thickness of 67 nm by 3 minutes postinsemination (PI), and final structuralization was observed by 40 minutes PI. The fully formed FE did not have microvillar impressions (casts) and was symmetrical, with outer double laminar elements surrounding an amorphous central region. Isolated S. franciscanus FEs were soluble in reducing and denaturing solvents and the same set of 33 polypeptides ranging from 18.5 to 260 kD was detected in FEs isolated from 10 to 180 minutes PI. The S. droebachiensis FE retained microvillar casts, assumed its definitive form by 3 minutes PI, and was 70 nm thick between microvillar impressions. Isolated S. droebachiensis FEs were partially soluble in reducing and denaturing solvents, and the polypeptide spectra of FEs isolated between 10 and 60 minutes PI were identical and showed 14 polypeptides from 18.5 to 265 kD. Antisera against extracted FEs and the FE extract from S. purpuratus were immunologically cross-reactive (using an enzyme-linked immunosorbent assay) with S. franciscanus and S. droebachiensis FE preparations; immunoblots identified 13 and 5 cross-reactive polypeptides, respectively. Most of the cross-reactive polypeptides were of slightly different molecular weight. Based on comparative ultrastructural, solubility, and electrophoretic data, we suggest that S. droebachiensis FE development is most like that observed in S. purpuratus.     

Interactions of the antimicrobial peptide Ac-FRWWHR-NH(2) with model membrane systems and bacterial cells.

The acetylated and amidated hexapeptide FRWWHR (combi-2), previously identified by combinatorial chemistry methods, shows strong antimicrobial activity. The binding of the peptide to 1-palmitoyl-2-oleoyl-sn-glycero-3-[(phospho-rac-(1-glycerol)] (POPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles was studied using fluorescence spectroscopy and isothermal titration calorimetry (ITC). Differential scanning calorimetry (DSC) with dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) multilamellar vesicles was performed to determine changes in the lipid phase behaviour upon binding the peptide. Two-dimensional proton nuclear magnetic resonance (NMR) spectroscopy, to solve the bound peptide structure, was performed in the presence of dodecylphosphatidylcholine (DPC) and sodium dodecyl sulphate (SDS) micelles. The fluorescence, ITC and DSC studies indicate that the peptide interacts preferentially with lipid vesicles containing negatively charged head groups. Conformational information determined using NMR indicate that the combi-2 peptide adopts a coiled amphipathic conformation when bound to SDS and DPC micelles. Leakage assays indicate that the peptide is not very efficient at causing leakage from calcein-filled large unilamellar vesicles comprised of POPG/POPC (1 : 1). The rapid passage of either the fluorescent-tagged peptides combi-2 or the previously studied peptide Ac-RRWWRF-NH(2) (combi-1) into Escherichia coli and Staphylococcus aureus suggests that instead of membrane disruption, the main bactericidal site of action of these peptides might be located inside bacteria.     

Mixed lipid aggregates containing gangliosides impose different 2H-NMR dynamical parameters on water environment depending on their lipid composition

Water dynamics in samples of ceramide tetrasaccharide (G_g4Cer) vesicles and G_M1 ganglioside micelles at 300:1 water/lipid mole ratio were studied by using deuterium nuclear magnetic resonance (²H-NMR). G_M1 imposes a different restriction on water dynamics that is insensitive to temperatures either above or below its phase transition temperature or below the freezing point of water. The calculated correlation times are in the range of 10^?10 s, typical of water molecules near to the polar groups. Pure G_M1 micelles have two distinct water microenvironments dynamically characterized. Their dynamic parameters remain constant with temperature ranging from ?18 to 32°C, but the amount of strongly associated water is modified. By contrast, a mixture of single soluble carbohydrates corresponding to G_M1 polar head group does not preserve the dynamic parameters of water hydration when the temperature is varied. Incorporation of cholesterol or lysophosphatidylcholine into G_M1 micelles substantially increases the mobility of water molecules compared with that found in pure G_M1 micelles. The overall results indicate that both the supramolecular organization and the local surface quality (lipid–lipid interaction) strongly influence the interfacial water mobility and the extent of hydration layers in glycosphingolipid aggregates.     

Fluorescence study on the interaction of a multiple antigenic peptide from hepatitis A virus with lipid vesicles

The interaction of the multiple antigenic peptide MAP4VP3 with lipid membranes has been studied by spectroscopic techniques. MAP4VP3 is a multimeric peptide that corresponds to four units of the sequence 110-121 of the capsid protein VP3 of hepatitis A virus. In order to evaluate the electrostatic and hydrophobic components on the lipid-peptide interaction, small unilamelar vesicles of different compositions, including zwitterionic dipalmitoylphosphatidylcholine (DPPC), anionic dipalmitoylphosphatidylcholine/phatidylinositol (DPPC:PI 9:1), and cationic dipalmitoylphosphatidylcholine/stearylamine (DPPC:SA 9.5:0.5), were used as membrane models. Intrinsic tryptophan fluorescence changes and energy transfer experiments show that MAP4VP3 binds to all three types of vesicles with the same stoichiometry, indicating that the electrostatic component of the interaction is not important for binding of this anionic peptide. Steady-state polarization experiments with vesicles labeled with 1,6-diphenyl-1,3,5-hexatriene or with 1-anilino-8-naphtalene sulphonic acid indicate that MAP4VP3 induces a change in the packing of the bilayers, with a decrease in the fluidity of the lipids and an increase in the temperature of phase transition in all the vesicles. The percentage of lipid exposed to the bulk aqueous phase is around 60% in intact vesicles, and it does not change upon binding of MAP4VP3 to DPPC vesicles, indicating that the peptide does not alter the permeability of the membrane. An increase in the amount of lipid exposed to the aqueous phase in cationic vesicles indicates either lipid flip-flop or disruption of the vesicles. Binding to DPPC vesicles occurs without leakage of entrapped carboxyfluorescein, even at high mol fractions of peptide. However, a time-dependent leakage is seen with cationic DPPC/SA and anionic DPPC/PI vesicles, indicating that the peptide induces membrane destabilization and not lipid flip-flop. Resonance energy transfer experiments show that MAP4VP3 leakage from cationic vesicles is due to membrane fusion, whereas leakage from anionic vesicles is not accompanied by lipid mixing. Results show that MAP4VP3 interacts strongly with the lipid components of the membrane, and although binding is not of electrostatic nature, the bound form of the peptide has different activity depending on the membrane net charge; thus, it is membrane disruptive in cationic and anionic vesicles, whereas no destabilizing effect is seen in DPPC vesicles.     

Reconstituted phosphatidylserine synthase from Escherichia coli is activated by anionic phospholipids and micelle-forming amphiphiles

The activity of phosphatidylserine (PS) synthase (CDP-1,2-diacyl-sn-glycerol: l-serine O-phosphatidyltransferase, EC 2.7.8.8) from Escherichia coli was studied after reconstitution with lipid vesicles of various compositions. PS synthase exhibited practically no activity in the absence of a detergent and with the substrate CDP-diacylglycerol (CDP-DAG) present only in the lipid vesicles. Inclusion of octylglucoside (OG) in the assay mixture increased the activity 20- to 1000-fold, the degree of activation depending on the lipid composition of the vesicles. Inclusion of additional CDP-DAG in the assay mixture increased the activity 5- to 25-fold. When the fraction of phosphatidylglycerol (PG) was increased from 15 to 100 mol% in the vesicles the activity increased 10-fold using the assay mixture containing OG. The highest activities were exhibited with the anionic lipids synthesized by E. coli, namely PG, diphosphatidylglycerol (DPG), and phosphatidic acid, while phosphatidylinositol gave a lower activity. Cryotransmission electron microscopy showed that transformation of the vesicles to micelles brings about an activation of the enzyme that is proportional to the degree of micellization. Thus, the activity of PS synthase is modulated by the lipid aggregate structure and by the fraction and type of anionic phospholipid in the aggregates. The increase in the activity caused by PG and DPG is physiologically relevant; it may be part of a regulatory mechanism that keeps the balance between phosphatidylethanolamine, and the sum of PG and DPG, nearly constant in wild-type E. coli cells.     

Peptide hormone-membrane interactions: the aggregational and conformational state of lipo-gastrin derivatives and their receptor binding affinity.

The (2RS)-1,2-dipalmitoyl-3-mercaptoglycerol/-, (2RS)-1,2-dimyristoyl-3-mercaptoglycerol/-, and (2RS)-1-myristoyl-2-palmitoyl-3-mercaptoglycerol/maleoyl-bet a-alanyl- [Nle15]-human-gastrin-(2-17) adducts were prepared as lipo-gastrin derivatives of explicitly primary amphiphilic properties. As representative of this class of lipo-gastrins, the dimyristoyl derivative has been thoroughly characterized in its aggregational state since, among the three compounds, theoretically it should exhibit the lowest degree of lipid character. It aggregates in aqueous solution to form monodispersed unilamellar spherical vesicles with dislocation of the peptide moiety at the bilayer surface in predominantly unordered structure. The liposomes are remarkably stable toward solubilization with trifluoroethanol and toward vesicle to micelle transition with neutral and negatively charged surfactants even above their critical micellar concentrations. Asymmetric fusion with the detergent micelles induces polydispersion of the liposomes in terms of shape and size without affecting in significant manner the mode of display of the gastrin portions at the bilayer surface. Only the positively charged hexadecyltrimethylammonium hydroxide provokes the collapse of the vesicles into mixed micelles with concomitant altered dislocation of the gastrin-peptide in the new aggregational state. Despite the lipid properties of the gastrin derivatives, i.e., formation of liposomes, they retain remarkable receptor affinities (IC50 = 1.5 x 10(-9) M for myristoyl-palmitoyl-gastrin, IC50 = 2.0 x 10(-9) M for di-myristoyl-gastrin and IC50 = 3.1 x 10(-9) M for di-palmitoyl-gastrin vs IC50 = 2.8 x 10(-10) M for Nle15-gastrin). Since the displacement of radiolabeled Nle15-gastrin from rat pancreatic acinar cell line membrane preparations by both the parent gastrin hormone and the three lipo-gastrins occurs in parallel manner, the data support a mechanism of receptor occupancy via accumulation of the gastrins at the membrane surface and their two-dimensional diffusion to the target receptor. Thereby the differentiated decrease of affinity in function of fatty acid chain length has to be attributed to the energetically more or less favored transfer of the monomers from the donor vesicles to the acceptor membranes. Moreover, according to this model migration of the lipo-gastrins with their interdigitating di-fatty-acyl moieties should be delayed, again in lipid structure-dependent manner, in comparison to the parent gastrin molecule, which is free to float in the membrane interfacial phase.     

The effects of lipids on the structure of the eukaryotic cytolysin equinatoxin II: a synchrotron radiation circular dichroism spectroscopic study

Synchrotron radiation circular dichroism (SRCD) spectroscopy studies of the eukaryotic pore-forming protein equinatoxin II (EqtII) were carried out in solution and in the presence of micelles or small unilamellar vesicles (SUV) of different lipid composition. The SRCD structural data was correlated with calcein leakage from SUV and with partitioning of EqtII to liposomes, and micelles, according to haemolysis assays. The structure of EqtII in water and dodecylphosphocholine micelles as determined by SRCD was similar to the values calculated from crystal and solution structures of the protein, and no changes were observed with the addition of sphingomyelin (SM). SM is required to trigger pore formation in biological and model membranes, but our results suggest that SM alone is not sufficient to trigger dissociation of the N-terminal helix and further structural rearrangements required to produce a pore. Significant changes in conformation of EqtII were detected with unsaturated phospholipid (DOPC) vesicles when SM was added, but not with saturated phospholipids (DMPC), which suggests that not only is membrane curvature important, but also the fluidity of the bilayer. The SRCD data indicated that the EqtII structure in the presence of DOPC:SM SUV represents the 'bound' state and the 'free' state is represented by spectra for DOPC or DOPC:Chol vesicles, which correlates with the high lytic activity for SUV of DOPC:SM. The SRCD results provide insight into the lipid requirements for structural rearrangements associated with EqtII toxicity and lysis.     

Characteristics of spectrin-induced leakage of extruded, phosphatidylserine vesicles

At neutral pH spectrin induces modest leakage of trapped calcein from reverse-phase or extruded, but not sonicated, vesicles composed of phosphatidylserine, but not phosphatidylcholine. The extent of leakage from extruded vesicles is not or is only slightly affected by magnesium ions at a physiological concentration or calcium ions at a greater than physiological concentration, respectively. In addition to accounting for several previously discrepant observations on the lytic effects of spectrin, these findings indicate that some proteins like spectrin may destabilize vesicles with low curvature more readily than vesicles of high curvature, in contrast to certain amphiphilic peptides. 60% less leakage is induced from phosphatidylserine vesicles by heat-denatured than by native spectrin. In contrast, both trypsin- and subtilisin-treated spectrins, if sufficiently digested, induce several-fold more leakage than undigested spectrin. Since spectrin prepared either by 1 M Tris dissociation of Triton-extracted cytoskeletons or by low ionic strength extraction of ghosts released the same amounts of calcein from vesicles of various compositions, these effects are unlikely to reflect artifacts of spectrin preparation. Furthermore, spectrin is unlikely to promote leakage in vivo, since vesicles composed of phosphatidylserine, cholesterol and/or phosphatidylethanolamine, which constitute the lipid composition of the inner monolayer of the red cell membrane, did not leak on addition of spectrin, whereas vesicles composed of phosphatidylserine and phosphatidylcholine, did leak in the presence of spectrin.     

Physical-chemical behavior of dietary and biliary lipids during intestinal digestion and absorption. 1. Phase behavior and aggregation states of model lipid systems patterned after aqueous duodenal contents of healthy adult human beings

We developed equilibrium phase diagrams corresponding to aqueous lipid compositions of upper small intestinal contents during lipid digestion and absorption in adult human beings. Ternary lipid systems were composed of a physiological mixture of bile salts (BS), mixed intestinal lipids (MIL), principally partially ionized fatty (oleic) acid (FA) plus racemic monooleylglycerol (MG), and cholesterol (Ch), all at fixed aqueous-electrolyte concentrations, pH, temperature, and pressure. The condensed phase diagram for typical physiological conditions (1 g/dL total lipids, FA:MG molar ratio of 5:1, pH 6.5, 0.15 M Na+ at 37 degrees C) was similar to that of a dilute model bile [BS/lecithin (PL)/Ch] system [Carey, M. C., & Small, D. M. (1978) J. Clin. Invest. 61, 998-1026]. We identified two one-phase zones composed of mixed micelles and lamellar liquid crystals, respectively, and two two-phase zones, one composed of Ch monohydrate crystals and Ch-saturated micelles and the other of physiologic relevance composed of Ch- and MIL-saturated mixed micelles and unilamellar vesicles. A single large three-phase zone in the system was composed of Ch-saturated micelles, Ch monohydrate crystals, and liquid crystals. Micellar phase boundaries for otherwise typical physiological conditions were expanded by increases in total lipid concentration (0.25-5 g/dL), pH (5.5-7.5), and FA:MG molar ratio (5-20:1), resulting in a reduction of the size of the physiological two-phase zone. Mean particle hydrodynamic radii (Rh), measured by quasielastic light scattering (QLS), demonstrated an abrupt increase from micellar (less than 40 A) to micelle plus vesicle sizes (400-700 A) as this two-phase zone was entered. With relative lipid compositions within this zone, unilamellar vesicles formed spontaneously following coprecipitation, and their sizes changed markedly as functions of time, reaching equilibrium values only after 4 days. Further, vesicle Rh values were influenced appreciably by MIL:mixed bile salt (MBS) ratio, pH, total lipid concentration, and FA:MG ratio, but not by Ch content. In comparison, micellar systems equilibrated rapidly, and their Rh values only slightly influenced by physical-chemical variables of physiological importance. In contrast to the BS-PL-Ch system [Mazer, N. A., & Carey, M. C. (1983) Biochemistry 22, 426-442], no divergence in micellar sizes occurred as the micellar phase boundary was approached. The ionization state of FA at simulated "intestinal" pH values (5.5-7.5) in the micellar and physiologic two-phase zones was principally that of 1:1 sodium hydrogen dioleate, an insoluble swelling "acid soap" compound. By phase separation and analysis, tie-lines for the constituent phase in the two-phase zone demonstrated that the mixed micelles were saturated with MIL and Ch and the coexisting vesicles were saturated with MBS, but not with Ch.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hardening of the sea urchin fertilization envelope by peroxidase-catalyzed phenolic coupling of tyrosines

Within minutes after its elevation from the egg surface, the sea urchin fertilization envelope (FE) becomes "hardened" by a reaction that renders it resistant to agents that solubilize, denature or degrade most proteins. Peroxidase activity is released into the surrounding seawater from Stronglyocentrotus purpuratus eggs during fertilization. Evidence from several sources indicate that the catalytic action of the peroxidase is responsible for hardening the FE through the phenolic coupling of tyrosyl residues of the FE proteins. First, the peroxidase is localized within the hardened FE and within the crystalline FE precursor material released from egg cortical granules during the fertilization reaction. Second, a direct correlation is established between the effectiveness of compounds in inhibiting the cortical granule peroxidase (CGP) and their effectiveness in inhibiting hardening of the FE. Third, the CGP catalyzes the cross-linking of tyrosines in solution, a reaction known to be catalyzed by horseradish peroxidase (HRP). Fourth, acid hydrolysates of hardened FEs contain cross-linked tyrosines that are identified by comparing their chromatographic ultraviolet absorption and fluorescent characteristics to those known for cross-linked tyrosines formed by HRP. Finally, when eggs are fertilized in the presence of 125I, the CGP heavily labels proteins of the FE and of the crystalline FE precursor material released with the enzyme from the cortical granules. The iodide label reflects the localization of the CGP and may reflect the sites of peroxidase-generated tyrosyl phenyl radicals involved in the tyrosine coupling reaction. Maximal iodide labeling occurs during the first 5 min period following fertilization, corresponding to the period of FE hardening.     

The effects of lipids on the structure of the eukaryotic cytolysin equinatoxin II: A synchrotron radiation circular dichroism spectroscopic study

Synchrotron radiation circular dichroism (SRCD) spectroscopy studies of the eukaryotic pore-forming protein equinatoxin II (EqtII) were carried out in solution and in the presence of micelles or small unilamellar vesicles (SUV) of different lipid composition. The SRCD structural data was correlated with calcein leakage from SUV and with partitioning of EqtII to liposomes, and micelles, according to haemolysis assays. The structure of EqtII in water and dodecylphosphocholine micelles as determined by SRCD was similar to the values calculated from crystal and solution structures of the protein, and no changes were observed with the addition of sphingomyelin (SM). SM is required to trigger pore formation in biological and model membranes, but our results suggest that SM alone is not sufficient to trigger dissociation of the N-terminal helix and further structural rearrangements required to produce a pore. Significant changes in conformation of EqtII were detected with unsaturated phospholipid (DOPC) vesicles when SM was added, but not with saturated phospholipids (DMPC), which suggests that not only is membrane curvature important, but also the fluidity of the bilayer. The SRCD data indicated that the EqtII structure in the presence of DOPC:SM SUV represents the ‘bound’ state and the ‘free’ state is represented by spectra for DOPC or DOPC:Chol vesicles, which correlates with the high lytic activity for SUV of DOPC:SM. The SRCD results provide insight into the lipid requirements for structural rearrangements associated with EqtII toxicity and lysis.