Spin-label ESR studies on the interaction of bovine spinal cord myelin basic protein with dimyristoylphosphatidylglycerol dispersions

Electron spin resonance (ESR) spectroscopy and chemical binding assays were used to study the interaction of bovine spinal cord myelin basic protein (MBP) with dimyristoylphosphatidylglycerol (DMPG) membranes. Increasing binding of MBP to DMPG bilayers resulted in an increasing motional restriction of PG spin-labeled at the C-5 atom position in the acyl chain, up to a maximum degree of association of 1 MBP molecule per 36 lipid molecules. ESR spectra of PG spin-labels labeled at other positions in the sn-2 chain showed a similar motional restriction, while still preserving the chain flexibility gradient characteristic of fluid lipid bilayers. In addition, labels at the C-12 and C-14 atom positions gave two-component spectra, suggesting a partial hydrophobic penetration of the MBP into the bilayer. Spectral subtractions were used to quantitate the membrane penetration in terms of the stoichiometry of the lipid-protein complexes. Approximately 50% of the spin-labeled lipid chains were directly affected at saturation protein binding. The salt and pH dependence of the ESR spectra and of the protein binding demonstrated that electrostatic interaction of the basic residues of the MBP with the PG headgroups is necessary for an effective association of the MBP with phospholipid bilayers. Binding of the protein, and concomitant perturbation of the lipid chain mobility, was reduced as the ionic strength increased, until at salt concentrations above 1 M NaCl the protein was no longer bound. The binding and ESR spectral perturbation also decreased as the protein charge was reduced by pH titration to above the pI of the protein at approximately pH 10.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selectivity of interaction of phospholipids with bovine spinal cord myelin basic protein studied by spin-label electron spin resonance

The selectivity of interaction between bovine spinal cord myelin basic protein (MBP) and eight different spin-labeled lipid species in complexes with dimyristoylphosphatidylglycerol (DMPG) and between spin-labeled phosphatidylglycerol and spin-labeled phosphatidylcholine in complexes of MBP with various mixtures of DMPG and dimyristoylphosphatidylcholine (DMPC) has been studied by electron spin resonance (ESR) spectroscopy. In DMPC/DMPG mixtures, the protein binding gradually decreased with increasing mole fraction of DMPC in a nonlinear fashion. The lipid-protein binding assays indicated a preferential binding of the protein to phosphatidylglycerol relative to phosphatidylcholine without complete phase separation of the two lipids. The outer hyperfine splittings (2Amax) of both phosphatidylglycerol and phosphatidylcholine labeled at C-5 of the sn-2 chain (5-PGSL and 5-PCSL, respectively) were monitored in the lipid-protein complexes as a function of the mole fraction of DMPC. The increases in the value of Amax induced on binding of the protein were larger for 5-PGSL than for 5-PCSL, up to 0.25 mole fraction of DMPC. Beyond this mole fraction the spectral perturbations induced by the protein were similar for both lipid labels. The ESR spectra of phosphatidylglycerol and phosphatidylcholine labeled at C-12 of the sn-2 chain were two component in nature, indicating indicating a direct interaction of the protein with the lipid chains, at mole fractions of DMPC up to 0.25. Quantitation of the motionally restricted spin-label population by spectral subtraction again indicated a preferential interaction of the protein with phosphatidylglycerol relative to phosphatidylcholine. Up to DMPC mode fractions of 0.25, the microenvironment of the protein was enriched in DMPG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid-protein and protein-protein interactions in double recombinants of myelin proteolipid apoprotein and myelin basic protein with dimyristoylphosphatidylglycerol.

The integral proteolipid apoprotein (PLP) from bovine spinal cord has been reconstituted in dimyristoylphosphatidylglycerol (DMPG) bilayers, and the mutual interactions on binding the peripheral myelin basic protein (MBP) have been studied. Quantitation of protein and lipid contents in the MBP-PLP-DMPG double recombinants at different PLP:DMPG ratios led to the conclusion that MBP binds only to the DMPG lipid headgroups and is hindered from interaction with the first shell of lipids surrounding the PLP. No specific PLP-MBP association could be detected. Electron spin resonance (ESR) spectra of phosphatidylglycerol spin-labeled at position n = 5 in the sn-2 chain showed that complexation of MBP with the PLP-DMPG recombinants leads to a decrease in lipid chain mobility to an extent which correlates with the degree of MBP binding. At low DMPG:PLP ratios, the perturbations of lipid mobility by both proteins are mutually enhanced. In single recombinants of PLP with DMPG, the ESR spectra of phosphatidylglycerol spin-labeled at position n = 14 in the sn-2 chain indicated that approximately 10 lipids/protein are motionally restricted by direct contact with the intramembranous surface of the protein. This number is in agreement with earlier results for reconstitutions of PLP in dimyristoylphosphatidylcholine (DMPC) [Brophy, P. J., Horváth, L. I., & Marsh, D. (1984) Biochemistry 23, 860-865] and is consistent with a hexameric arrangement of the PLP molecules in DMPG bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fragments from actin binding protein (ABP-280; filamin) insert into reconstituted lipid layers.

Previous computer analyses suggested two possible lipid binding sites, residues 49-71 and 131-155, of the primary amino acid sequence on ABP-280 (filamin), which could facilitate membrane attachment/insertion. We expressed these regions as fusion proteins with schistosomal GST and investigated their interaction with mixtures of zwitterionic (dimyristoyl-l-alpha-phosphatidylcholine, DMPC) and anionic (dimyristoyl-l-alpha-phosphatidylglycerol, DMPG) phospholipids in reconstituted lipid bilayers by differential scanning calorimetry (DSC). Using vesicles of mixed DMPC/DMPG with increasing fusion protein concentrations, we established in calorimetric assays a decrease of the main chain transition enthalpy, DeltaH, and a shift in chain melting temperature. This is indicative of the insertion of these fragments into the hydrophobic region of lipid membranes. We confirmed these findings by the film balance technique using lipid monolayers (DMPG). The binding judged from both methods was of moderate affinity.     

Spin-label ESR of bacteriophage M13 coat protein in mixed lipid bilayers. Characterization of molecular selectivity of charged phospholipids for the bacteriophage M13 coat protein in lipid bilayers

Bacteriophage M13 major coat protein has been incorporated at different lipid/protein ratios in lipid bilayers consisting of various ratios of dimyristoylphosphatidylcholine (DMPC) to dimyristoylphosphatidylglycerol (DMPG). Spin-label ESR experiments were performed with phospholipids labeled at the C-14 position of the sn-2 chain. For M13 coat protein recombinants with DMPC alone, the relative association constants were determined for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels and found to be 1.0, 1.0, and 2.1 relative to the background DMPC, respectively. The number of association sites for each phospholipid on the protein was found to be 4 per protein monomer. The intrinsic off-rates for lipid exchange at the intramembranous surface of the protein in DMPC alone at 30 degrees C were found to be 5 X 10(6), 6 X 10(6), and 2 X 10(6) s-1 for the phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid spin-labels, respectively. Adding DMPG to the DMPC lipid system increased the exchange rates of the lipids on and off the protein. By gel filtration chromatography, it is found that protein aggregation is reduced after addition of DMPG to the lipid system. This is in agreement with measurements of tryptophan fluorescence, which show a decrease in quenching efficiency after introduction of DMPG in the lipid system. The results are interpreted in terms of a model relating the ESR data to the size of the protein-lipid aggregates.     

Assistance of maltose binding protein to the in vivo folding of the disulfide-rich C-terminal fragment from Plasmodium falciparum merozoite surface protein 1 expressed in Escherichia coli

The C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 (F19) is a leading candidate for the development of a malaria vaccine. Successful vaccination trials on primates, immunochemistry, and structural studies have shown the importance of its native conformation for its protective role against infection. F19 is a disulfide-rich protein, and the correct pairing of its 12 half-cystines is required for the native state of the protein. F19 has been produced in the Escherichia coli periplasm, which has an oxidative environment favorable for the formation of disulfide bonds. F19 was either expressed as a fusion with the maltose binding protein (MBP) or directly addressed to the periplasm by fusing it with the MBP signal peptide. Direct expression of F19 in the periplasm led to a misfolded protein with a heterogeneous distribution of disulfide bridges. On the contrary, when produced as a fusion protein with E. coli MBP, the F19 moiety was natively folded. Indeed, after proteolysis of the fusion protein, the resulting F19 possesses the structural characteristics and the immunochemical reactivity of the analogous fragment produced either in baculovirus-infected insect cells or in yeast. These results demonstrate that the positive effect of MBP in assisting the folding of passenger proteins extends to the correct formation of disulfide bridges in vivo. Although proteins or protein fragments fused to MBP have been frequently expressed with success, our comparative study evidences for the first time the helping property of MBP in the oxidative folding of a disulfide-rich protein.     

Coupling of antibody-binding fragments to solid-phase supports: site-directed binding of F(ab')2 fragments.

A method to covalently bind antibody fragments, via their carboxyl termini to solid supports, is presented. The strategy involves: (1) reversibly blocking all the accessible carboxyl groups on the antibody molecule with phenylhydrazine, (2) exposing the carboxyl termini of the fragment by enzymatic digestion with pepsin and (3) subsequently coupling the fragment to an appropriate support. Experiments with an anti-bovine serum albumin monoclonal antibody and C-14 phenylhydrazine revealed that the blocking step was nearly completely reversible with a dilute solution of FeCl3. Radioiodinated blocked F(ab')2 fragments were then coupled to an amino-functionalized Sepharose 4B column, and characterized as to their coupling capacity (mass of protein coupled/ml of bead), and antigen-binding activity. The coupling capacity of the blocked fragments was found to be 12%, half the coupling efficiency of unmodified radioiodinated F(ab')2. The antigen-binding capacity (mol antigen bound per mol antibody coupled) for the blocked F(ab')2, on the other hand, was found to be 1.9, which was approx. 3.5-times greater than for the unmodified F(ab')2. Comparisons with other conventional coupling techniques were also made. These preliminary studies suggest that this technique can provide one with the means to obtain more uniform and active populations of immobilized antibody fragments.     

Modulation of cytochrome C coupling to anionic lipid monolayers by a change of the phase state: a combined neutron and infrared reflection study

The effect of monolayer domain formation on the electrostatic coupling of cytochrome c from the subphase to a monolayer at the air/water interface was studied using a combination of neutron reflection (NR) and infrared reflection absorption spectroscopy (IRRAS) techniques. The monolayers consisted of a binary mixture of the zwitterionic phosphatidylcholine and the anionic phosphatidylglycerol. For a monolayer of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylglycerol (DMPG, 30 mol%), which exhibits a non-ideal mixing of the two lipid components, we observed a significantly higher protein coupling to the liquid-condensed phase compared to the liquid-expanded state. In contrast, this higher protein binding was not observed when the two lipids had identical chain lengths (nearly ideal mixing). Similarly, for an equimolar mixture of DPPC and DMPG, we did not observe significant differences in the protein binding for the two phase states. The results strongly suggest that the domain formation in a condensed monolayer under non-ideal lipid mixing conditions is crucial for the cytochrome c binding strength. Furthermore, this study demonstrates the significant advantages of gathering information on protein-monolayer coupling by the combined use of a dedicated IRRAS set-up with the NR technique.     

Secondary structure of charge isomers of myelin basic protein before and after phosphorylation

Human myelin basic protein (MBP) was fractionated into several of its charge isomers (components). Of these, the secondary structures of four isomers before and after phosphorylation have been studied by circular dichroism (CD). None of the four showed any alpha-helical structure. All of the components showed varying amounts of beta-structure, random structure, and turns. Component 1 (C-1), the most cationic of the components, showed 13%; component 2 (C-2) had 19%; C-3, 17%; and C-4, 24% of beta-structure. Each of the four components was phosphorylated with protein kinase C, from human brain. The extent of phosphorylation varied considerably from 2.8 +/- 0.6 mol of PO4/mol of protein in C-1 to 5.2 +/- 0.8 mol of PO4/mol of protein in C-4. The effect of phosphorylation on the secondary structure was to induce beta-structure in all the components. The largest change in beta-structure was in C-1 and the least in C-4. The surprising result is that although the components were phosphorylated to different extents, the amount of beta-structure in all four components increased to a final proportion of 35-40%. Treatment of phosphorylated C-1 with acid phosphatase removed 50% of the total radioactivity. Although the remainder represented approximately 1 mol of PO4/mol of protein, the proportion of beta-structure was unaltered. We concluded that a single phosphorylation site identified as residues 5-13 represented a critical size for stabilization of beta-structure of MBP in solution and that phosphorylation at the other sites had little influence on secondary structure.     

Intramolecular organization of Class I H-2 MHC antigens; localization of the alloantigenic determinants and the beta 2 m binding site to different regions of the H-2 Kb glycoprotein

The intramolecular organization of the membrane integrated Class I major histocompatibility complex (MHC) molecule H-2Kb (Kb) was analyzed. After the removal of the two carbohydrate moieties by glycosidase enzymes, proteolytic digestion of the Kb molecule yielded: 1) several fragments with the beta 2 microglobulin (beta 2 m) subunit still bound and 2) one fragment carrying alloantigenic activity but lacking the beta 2 m. Isolation of the beta 2 m binding fragments showed them to be derived from the C-2 domain by partial N-terminal sequence analysis. One fragment extended to the C-terminus and the other fragment had lost the transmembrane region. Such studies conclusively show that the beta 2 m subunit is bound in the third domain, i.e., C-2, of the Kb 44,000 m.w. heavy chain. The alloantigenic fragment also isolated from the proteolytic digest consists of the first 180 residues of the 44,000 m.w. heavy chain, i.e., domains N and C-1, and carried alloantigenic determinants detected by several monoclonal antibodies as well as alloantisera. The present studies indicate that the external region of the Class I molecules has two functional regions. The first 180 residues bear the recognition elements for the immune system, and the next 90 residues (180-270) are involved in binding to beta 2 m.     

Interaction of glycosylated human myelin basic protein with lipid bilayers

Myelin basic protein (MBP), isolated from normal human myelin, was glycosylated with UDP-N-acetyl-D-galactosamine and a glycosyltransferase isolated from porcine submaxillary glands. MBP containing 0.85 mol of N-acetyl-D-galactosamine per mole of protein was oxidized at carbon 6 by galactose oxidase and complexed with a spin-label, Tempoamine, in order to study its interactions with lipids. When the spin-labeled MBP was reacted with lipid vesicles consisting of DSPG, DPPG, and DMPG, most of the spin-label was motionally restricted in the gel phase, with a correlation time greater than 10(-8)s. The motion increased with increasing temperature and was sensitive to the lipid phase transition. Interaction with the gel phase of DPPA caused much less motional restriction of the probe. However, melting of the lipid allowed increased interaction and motional restriction of the probe, which was only partially reversed on cooling back to the gel phase. The motional restriction of the probe in these lipids is attributed to its penetration partway into the lipid bilayer in both the gel and liquid-crystalline phases. The fact that the probe bound to the protein can penetrate partway into the bilayer suggests that other hydrophobic side chains and residues of the protein can similarly penetrate into the bilayer. Additional evidence for penetration was provided by digestion of the lipid-bound protein with endoproteinase Lys-C. When nonglycosylated and glycosylated MBP in solution was treated with Lys-C, extensive digestion occurred. A single radioactive peptide which eluted at 25 min was identified as residues 92-105.(ABSTRACT TRUNCATED AT 250 WORDS)     

ADP-Ribosylation of Human Myelin Basic Protein

Abstract: When isolated myelin membranes were ADP-ribosylated by [³²P]NAD⁺ either in the absence of toxin (by the membrane ADP-ribosyltransferase) or in the presence of cholera toxin, the same proteins were ADP-ribosylated in both cases and myelin basic protein (MBP) was the major radioactive product. Therefore, cholera toxin was considered a good model for ADP-ribosylation of myelin proteins. Although purified human MBP migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 20 kDa, the microheterogeneity that is masked under these conditions can be clearly demonstrated on alkaline-urea gels at pH 10.6. At this pH, MBP is resolved into several components that differ one from the other by a single charge (charge isomers). These charge isomers can be resolved on CM52 columns at pH 10.6, and several can be ADP-ribosylated. Component 1 (C-1), the most cationic charge isomer, incorporated 1.79 mol of ADP-ribose/mol of protein. C-2 and C-3 (which differ from C-1 by the loss of one and two positive charges, respectively) incorporated slightly less at 1.67 and 1.63 mol of ADP-ribose/mol of protein, respectively, whereas C-8, the least cationic, incorporated less than 0.11 mol/mol of protein. In the presence of neutral hydroxylamine, the ADP-ribosyl bond was shown to have a half-life of about 80 min, suggesting an N-glycosidic linkage between ADP-ribose and an arginyl residue of the protein. As MBP contains several components that are ADP-ribosylated to different specific activities, the use of MBP, ADP-ribosylated in the natural membrane, to identify the sites involved would yield a mixture of peptides difficult to resolve. Therefore, to identify the sites ADP-ribosylated, an endoproteinase Lys-C digest of C-1 ADP-ribosylated by cholera toxin was prepared. Two radioactive peptides were isolated by reversed-phase HPLC. Amino acid and sequence analyses identified the radioactive peptides as residues 5–13 and 54–58 of the human sequence (sp. act., 0.89 and 0.62 nmol of ADP-ribose/nmol of peptide, respectively). The ADP-ribosylated residues were identified as Arg⁹ and Arg⁵⁴ by automated and manual Edman sequencing. Taken together with our previous observation that MBP binds GTP at a single site, these data suggest that MBP functions as part of a signal transduction system in myelin.     

Binding of RPE65 fragments to lipid monolayers and identification of its partners by glutathione S-transferase pull-down assays

RPE65 is the major component of the retinal pigment epithelium (RPE) microsomal membrane, and it plays a critical role in the binding of retinoids involved in the visual cycle. To understand how RPE65 binds to membranes, we have expressed and purified soluble fragments of human RPE65 fused to glutathione S-transferase (GST). The interaction between two fragments of RPE65 (F1 and F2 which include residues 1-125 and 126-250, respectively) and lipid monolayers has been studied by surface pressure, ellipsometry, and surface rheology measurements. Surface pressure and ellipsometry clearly showed a rapid adsorption of F2 to lipid monolayers whereas the kinetics of binding of F1 was much slower. Furthermore, the data suggest that the F2 fragment inserts into the lipid monolayer. Surface rheology showed a clear increase in monolayer rigidity only in the presence of F2, thereby demonstrating high intermolecular interactions of this fragment. This observation is further supported by the GST pull-down assays which demonstrated that F2 cosediments with full-length RPE65, suggesting that RPE65 has the propensity to form clusters or oligomers. The structure homology modeling of RPE65 based on a related family member, apocarotene 15',15'-oxygenase, further suggests that a hydrophobic patch located in the F2 region might be responsible for membrane binding. The present work shows that F2 interacts much stronger with lipid monolayers than does F1, which suggests that the region of RPE65 located between residues 126-250 should be very important for its membrane binding. Moreover, given that these fragments are not acylated, these data also suggest that an effective binding of RPE65 to membranes can be achieved without palmitoylation. Furthermore, GST pull-down assays also indicated that F2 interacts with 11-cis-retinol dehydrogenase, which supports previous data suggesting that it could act as a partner of RPE65.     

Formation of domains of cationic or anionic lipids in binary lipid mixtures increases the electrostatic coupling strength of water-soluble proteins to supported bilayers

The electrostatic binding strength of water-soluble proteins having either an excess positive (cytochrome c) or negative (beta-lactoglobulin) electric charge to oppositely charged supported planar bilayers (SPBs) was studied as a function of the bilayer phase state (fluid or gel phase) by IR-ATR spectroscopy. The bilayer consisted of mixtures of zwitterionic DEPC with either cationic DMTAP or anionic DMPG. We observed drastic differences in the binding strength of both proteins for the two bilayer phase states, with the gel phase exhibiting a higher binding strength than the fluid phase, under conditions where the two lipid components had different hydrophobic chain lengths resulting in a nonideal mixing behavior. In addition, for beta-lactoglobulin we observed a strong binding to a gel phase SPB comprising DEPC/DMTAP, while raising the temperature of the SPB above the chain melting transition temperature of the mixture resulted in a complete unbinding of the protein. In contrast, for DMPC/DMTAP having the same cationic charge content but no hydrophobic chain mismatch, no phase-dependent coupling strength of the protein to the SPB was observed. Our results suggest that the formation of charge-enriched domains by partial demixing of the bilayer lipids at the transition to the gel state is crucial for modulation of the protein binding strength to the SPB, while the intrinsic charge of the solid support surface is of minor importance.     

Spin-label electron spin resonance study of bacteriophage M13 coat protein incorporation into mixed lipid bilayers

The major coat protein of bacteriophage M13 was incorporated in mixed dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (80/20 w/w) vesicles probed with different spin-labeled phospholipids, labeled on the C-14 atom of the sn-2 chain. The specificity for a series of phospholipids was determined from a motionally restricted component seen in the electron spin resonance (ESR) spectra of vesicles with the coat protein incorporated. At 30 degrees C and pH 8, the fraction of motionally restricted phosphatidic acid spin-label is 0.36, 0.52, and 0.72 for lipid/protein ratios of 18, 14, and 9 mol/mol, respectively. The ESR spectra, analyzed by digital subtraction, resulted in a phospholipid preference following the pattern cardiolipin = phosphatidic acid greater than stearic acid = phosphatidylserine = phosphatidylglycerol greater than phosphatidylcholine = phosphatidylethanolamine. The specificities found are related to the composition of the target Escherichia coli cytoplasmic membrane.     

In vitro maximum binding of antigenic peptides to murine MHC class II molecules does not always take place at the acidic pH of the in vivo endosomal compartment.

Presentation of Ag to the T cell requires binding of specific peptide fragments of the Ag to MHC II molecules. The ability of a peptide to bind to MHC class II appears to be pH dependent. Recent reports indicate that the binding of peptide to MHC class II molecules takes place primarily within an endosomal compartment of the cell at around pH 5. In this study, we have explored the in vitro pH dependence of peptide binding to different haplotypes of murine MHC class II molecules. The binding of peptides to MHC II was analyzed and quantitated by silica gel TLC, using radiolabeled peptides. The MBP peptide fragments, MBP(1-14)A4 and MBP(88-101)Y88, bound maximally at pH 8 to IAk and IAs, respectively. The binding of PLP peptide fragment, PLP(138-151)Y138, to IAs was maximal at around neutral pH. The maximum binding of an OVA peptide fragment, OVA(323-340)Y340, to IAd, was found to occur at pH 6. Results presented in this report thus suggest that the in vitro maximum binding of peptide is pH dependent and does not always occur at pH 5. The optimum pH range for maximum binding may depend on the nature and net charge of the peptide and its interaction with MHC class II molecules.     

Lipid binding-induced conformational change in human apolipoprotein E. Evidence for two lipid-bound states on spherical particles

Apolipoprotein (apo) E contains two structural domains, a 22-kDa (amino acids 1-191) N-terminal domain and a 10-kDa (amino acids 223-299) C-terminal domain. To better understand apoE-lipid interactions on lipoprotein surfaces, we determined the thermodynamic parameters for binding of apoE4 and its 22- and 10-kDa fragments to triolein-egg phosphatidylcholine emulsions using a centrifugation assay and titration calorimetry. In both large (120 nm) and small (35 nm) emulsion particles, the binding affinities decreased in the order 10-kDa fragment approximately 34-kDa intact apoE4 > 22-kDa fragment, whereas the maximal binding capacity of intact apoE4 was much larger than those of the 22- and 10-kDa fragments. These results suggest that at maximal binding, the binding behavior of intact apoE4 is different from that of each fragment and that the N-terminal domain of intact apoE4 does not contact lipid. Isothermal titration calorimetry measurements showed that apoE binding to emulsions was an exothermic process. Binding to large particles is enthalpically driven, and binding to small particles is entropically driven. At a low surface concentration of protein, the binding enthalpy of intact apoE4 (-69 kcal/mol) was approximately equal to the sum of the enthalpies for the 22- and 10-kDa fragments, indicating that both the 22- and 10-kDa fragments interact with lipids. In a saturated condition, however, the binding enthalpy of intact apoE4 (-39 kcal/mol) was less exothermic and rather similar to that of each fragment, supporting the hypothesis that only the C-terminal domain of intact apoE4 binds to lipid. We conclude that the N-terminal four-helix bundle can adopt either open or closed conformations, depending upon the surface concentration of emulsion-bound apoE.     

1,2-Dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) monolayers: influence of temperature, pH, ionic strength and binding of alkaline earth cations

Ion binding and lipid ionization of the acidic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) in monolayers was studied by measuring the lateral pressure Pi as a function of the molecular area A at the air/water interface at different temperatures. The pH of the subphase (pH 2 and 7) and the ionic strength (NaCl) was varied. In addition, different divalent cations (1mM MgCl2, CaCl2 and SrCl2, pH 7) were added. DMPG is partly protonated on pure water at pH 7. An increase in the NaCl concentration in the subphase leads to film expansion. This effect is caused by an ionization of the headgroup of DMPG, i.e. a shift of the apparent pK. More condensed films are obtained on pure water at pH 2, due to the reduction of electrostatic repulsion by headgroup protonation and the possibility for the formation of a hydrogen bonding network. The divalent cations Mg2+, Ca2+ and Sr2+ interact differently with a DMPG monolayer in pure water at pH 7. In the presence of 1mM CaCl2 a condensation of the DMPG film is induced, whereas an expansion of the monolayer is observed in the presence of Mg2+ and Sr2+. Two counteracting effects are operative: (a) ionization of the headgroup due to electrostatic screening leads to film expansion and (b) binding of the divalent cations to the lipid headgroups leads to condensation. The latter effect is more pronounced in the case of Ca2+, whereas the binding of Mg2+ and Sr2+ to DMPG is weaker. Site-specific cation binding has to be assumed in addition to electrostatic effects.     

The major ribosome binding site of Escherichia coli ribosomal protein S1 is located in its N-terminal segment

We have cleaved protein S1, which is the largest and the most elongated protein of the Escherichia coli ribosome, using cyanogen bromide and isolated two fragments that retain the functional domains of the intact molecule. The fragments (denoted S1-F2a and S1-F2b) showed molecular weights of 24,000 and 22,500 by dodecyl sulphate/polyacrylamide gel electrophoresis. Fragment F2a is shown to be the N-terminal segment containing about 32% of the peptide chain length of S1. Fragment F2b is derived from another (probably C-terminal) region of S1.Fragment F2a binds to 30 S ribosomal subunits with a strength and specificity comparable to the binding of intact S1. It also binds to matrix-bound poly(U) but the binding is salt-sensitive, unlike the binding of intact S1. Fragment F2b binds only very weakly to poly(U) and does not bind to 30 S subunits. These results are discussed with respect to the ribosome binding domain(s) of protein S1 and the possible interdependence of the multiple functional domains in this large protein.     

Aqueous dispersions of DMPG in low salt contain leaky vesicles

Aqueous dispersions of dimyristoyl phosphatidylglycerol (DMPG), at low ionic strength, display uncommon thermal behavior. Models for such behavior need to assign a form to the lipid aggregate. Although most studies accept the presence of lipid vesicles in the lipid gel and fluid phases, this is still controversial. With electron spin resonance (ESR) spectra of spin labels incorporated into DMPG aggregates, quantification of [(14)C]sucrose entrapped by the aggregates, and viscosity measurements, we demonstrate the existence of leaky vesicles in dispersions of DMPG at low ionic strength, in both gel and fluid phases of the lipid. As a control system, the ubiquitous lipid dimyristoyl phosphatidylcholine (DMPC) was used. For DMPG in the gel phase, spin labeling only indicated the presence of lipid bilayers, strongly suggesting that DMPG molecules are organized as vesicles and not micelles or bilayer fragments (bicelles), as the latter has a non-bilayer structure at the edges. Quantification of [(14)C]sucrose entrapping by DMPG aggregates revealed the presence of highly leaky vesicles. Due to the short hydrocarbon chains ((14)C atoms), DMPC vesicles were also found to be partially permeable to sucrose, but not as much as DMPG vesicles. Viscosity measurements, with the calculation of the intrinsic viscosity of the lipid aggregate, showed that DMPG vesicles are rather similar in the gel and fluid phases, and quite different from aggregates observed along the gel-fluid transition. Taken together, our data strongly supports that DMPG forms leaky vesicles at both gel and fluid phases.