Ionic interactions in nanofiltration of beta casein peptides

Nanofiltration (NF) membrane technology shows interesting potentials for separating organic components on the basis of solute charge and size in the range of 300-1000 g mol-1. Separation properties of two inorganic NF membranes were studied with a set of 10 small peptides (molecular mass range: 300-900 g mol-1; 3 < pI < 10) contained in a well-characterized tryptic beta casein hydrolysate. Peptides transmission strongly depended on ionic interactions in the system. Physicochemical conditions such as ionic strength and especially pH were crucial to the separation, because the membrane and peptides showed amphoteric properties. Thus, the three categories of peptides (acid, basic, neutral) were separated according to their pI because of presumed concentration gradients of charged peptides at the membrane: positive for basic peptides and negative for acid peptides. At optimum pH 8 this led to high transmissions of basic peptides (even over 100%), intermediate transmissions for neutral peptides, and low transmissions for acid peptides. The addition of multicharged cationic and anionic species in the hydrolysate induced a markedly enhanced selectivity when the polyelectrolyte was a membrane coion and a complete reversion of selectivity when it was a membrane counterion. Copyright 1998 John Wiley & Sons, Inc.     

Two-dimensional gel electrophoresis of membrane proteins using anionic and cationic detergents. Application to the study of mitochondrial F0-F1-ATPase

Polyacrylamide gel electrophoresis in the presence of a cationic detergent, tetradecyltrimethylammonium bromide (TDAB) has been compared to electrophoresis in the presence of an anionic detergent, sodium dodecyl sulfate (SDS). Although, in both systems, the peptides generally migrated as a function of their molecular weight, the TDAB electrophoresis permitted us to obtain a much better resolution of several peptides of the mitochondrial F0-F1-ATPase, especially for the alpha and beta subunits and for the oligomycin sensitivity conferring protein (OSCP). The differences between the two electrophoretic profiles have been used to devise a new technique of two-dimensional electrophoresis using successively anionic and cationic detergents. This method could be very useful in the case of membrane proteins, which are generally soluble only in the presence of powerful ionic detergents. It has been particularly successful in resolving the small peptides of the F0-F1-ATPase which were difficult to differentiate by other techniques in one- or two-dimensional polyacrylamide gel electrophoresis.     

Selective separation of cationic peptides from a tryptic hydrolysate of beta-lactoglobulin by electrofiltration

Electrofiltration (EF) was used to selectively separate cationic (basic) peptides contained in a tryptic beta-lactoglobulin (beta-LG) hydrolysate, with particular emphasis on the isolation of basic sequence beta-LG 142-148, which is a potential antihypertensive peptide. Both the influence of feed solution pH and operating parameters (transmembrane pressure, feed velocity) were assessed to find optimum conditions enabling the fractionation between peptides during EF. The cathode (-) was inserted in the permeate side to increase the separation of basic peptides contained in the tryptic beta-LG hydrolysate as compared to conventional NF. The highest separation factor between basic and neutral peptides was obtained at pH 9 using G-10 membrane with a molecular weight cut-off (MWCO) of 2,500 g/mol, at 5 V with the lowest transmembrane pressure (0.344 MPa) and feed velocity (0.047 m/s). The transmission behavior of the peptides during EF was better explained when taking into account the positive/negative charge ratio. Because of its 3+/1- charge ratio, beta-LG 142-148 had the highest transmission during EF. Consequently, its relative concentration was raised from 3.5% in the initial tryptic beta-LG hydrolysate up to 38% in the permeate. The electric field seemed more effective when the convective/shearing forces were minimized.     

Four gel systems for electrophoretic fractionation of membrane proteins using ionic detergents

Membrane proteins were fractionated electrophoretically in polyacrylamide gels containing either anionic or cationic detergent at either pH 2.4 or 8.3. In all four systems, polypeptides migrated as monomers bearing the charge of the detergent ion and semi-logarithmic molecular weight–mobility relationships pertained. Electrophoresis of erythrocyte membrane proteins in these systems yielded very similar protein staining patterns but revealed significant differences in sialoglycoprotein migration.     

Molecular mechanism of opioid receptor selection

Preferred conformations, orientations, and accumulations of 26 opioid peptides on lipid membranes were estimated and compared with pharmacologic and selective binding data taken from the literature. Interaction with mu-receptors was governed by the net positive charge effective at the message domain of the agonist peptides z(eff) as the Boltzmann term ez(eff) that determines relative accumulation on anionic biologic membranes. Selection for delta-receptors was reduced by z(eff) and correlated with e-z(eff). Selection for kappa-receptors was governed by the peptide amphiphilic moment A. A pronounced scalar magnitude A and almost perpendicular orientation of the N-terminal message domain as an alpha-helix were favorable for kappa-site selection. Potencies as kappa-agonists and binding affinities correlated with A X ez(eff). The classical site selectivity caused by the receptor requirements for a complementary fit of the agonist to the discriminator site is thus crucially supplemented by a selection mechanism based on peptide membrane interactions (membrane requirements). In the model presented here, the delta-site is exposed to the aqueous compartment surrounding the target cell at a distance comparable to or greater than the Debye-Hückel length and is in a cationic vicinity. The mu-site is exposed to the anionic fixed-charge compartment of the membrane in aqueous surroundings. The kappa-site is buried in a more hydrophobic membrane compartment close to the fixed-charge compartment. The relative accumulation of the opioid message domains in these compartments is determined by the address domains and constitutes a major part of the site selection mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessing changes in Cd phytoavailability to tomato in amended calcareous soils

An efficient production method of heme-iron-enriched peptide was developed based on enzymatic hydrolysis. Hemoglobin hydrolysis, carried out stepwise with commercially available exopeptidase and endopeptidase, resulted in an increased degree of hydrolysis (DH). Exopeptidase-catalyzed protein hydrolysis formed low molecular weight peptides and amino acids. Different process parameters including dialysis and ultra- and diafiltration were evaluated. Heme/peptide ratio increased as molecular weight cut-off (MWCO) of the dialysis membrane increased. When the hydrolysate was dialyzed against sodium phosphate buffer, a higher heme/ peptide ratio was obtained. The heme/peptide ratio of the hydrolysate reached up to 25.4% when the dialysis was carried out with a membrane of 12-14 kDa MWCO. Also, the ratio was improved by the use of ultrafiltration and diafiltration on the pilot-scale.     

Membrane association, electrostatic sequestration, and cytotoxicity of Gly-Leu-rich peptide orthologs with differing functions

The skins of closely related frog species produce Gly-Leu-rich peptide orthologs that have very similar sequences, hydrophobicities, and amphipathicities but differ markedly in their net charge and membrane-damaging properties. Cationic Gly-Leu-rich peptides are hemolytic and very potent against microorganisms. Peptides with no net charge have only hemolytic activity. We have used ancestral protein reconstruction and peptide analogue design to examine the roles of electrostatic and hydrophobic interactions in the biological activity and mode of action of functionally divergent Gly-Leu-rich peptides. The structure and interaction of the peptides with anionic and zwitterionic model membranes were investigated by circular dichroism with 2-dimyristoyl-sn-glycero-3-phosphatidylcholine or 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol vesicles and surface plasmon resonance with immobilized bilayers. The results, combined with antimicrobial assays, the kinetics of bacterial killing, and membrane permeabilization assays, reveal that Gly, Val, Thr, and Ile can all be accommodated in an amphipathic alpha helix when the helix is in a membrane environment. Binding to anionic and zwitterionic membranes fitted to a 2-stage interaction model (adsorption to the membrane followed by membrane insertion). The first step is governed by hydrophobic interactions between the nonpolar surface of the peptide helix and the membranes. The strong binding of Gly-Leu-rich cationic peptides to anionic membranes is due to the second binding step and involves short-range Coulombic interactions that prolong the residence time of the membrane-inserted peptide. The data demonstrate that evolution has positively selected charge-altering nucleotide substitutions to generate an orthologous cationic variant of neutral hemolytic peptides that bind to and permeate bacterial cell membranes.     

An anionic antimicrobial peptide from toad Bombina maxima

Amphibian skin is a rich resource of antimicrobial peptides like maximins and maximins H from toad Bombina maxima. A novel cDNA clone encoding a precursor protein that comprises maximin 3 and a novel peptide, named maximin H5, was isolated from a skin cDNA library of B. maxima. The predicted primary structure of maximin H5 is ILGPVLGLVSDTLDDVLGIL-NH2. Containing three aspartate residues and no basic amino acid residues, maximin H5 is characterized by an anionic property. Different from cationic maximin H peptides, only Gram-positive strain Staphylococcus aureus was sensitive to maximin H5, while the other bacterial and fungal strains tested were resistant to it. The presence of metal ions, like Zn2+ and Mg2+, did not increase its antimicrobial potency. Maximin H5 represents the first example of potential anionic antimicrobial peptides from amphibians. The results provide the first evidence that, together with cationic antimicrobial peptides, anionic antimicrobial peptides may also exist naturally as part of the innate defense system.     

The calmodulin antagonist W-7 inhibits the epithelial Na+/H+ exchanger via modulating membrane surface potential

NHE3 is regulated via alterations in membrane surface charge. This is achieved through altered binding of cationic regions in the cytosolic-terminus of the exchanger with the inner leaflet of the plasma membrane. Calmodulin antagonists, including W-7, regulate surface potential and inhibit NHE3 activity. Utilizing fluorescent protein conjugated membrane probes we show that binding of cationic, but not hydrophobic peptides, to the plasma membrane is prevented by W-7. An interaction between cationic regions in the regulatory, cytosolic domain of NHE3 to anionic phospholipids in either reconstituted liposomes or the plasma membrane in cell culture is similarly prevented by W-7, at a concentration that inhibits the exchanger. We propose therefore that W-7 inhibits NHE3 activity, at least in part, by altering the association of cationic segments within the carboxy-terminus of the exchanger with anionic phospholipids in the plasma membrane.     

Effect of lipid composition on the topography of membrane-associated hydrophobic helices: stabilization of transmembrane topography by anionic lipids

To investigate the effect of lipid structure upon the membrane topography of hydrophobic helices, the behavior of hydrophobic peptides was studied in model membrane vesicles. To define topography, fluorescence and fluorescence quenching methods were used to determine the location of a Trp at the center of the hydrophobic sequence. For peptides with cationic residues flanking the hydrophobic sequence, the stability of the transmembrane (TM) configuration (relative to a membrane-bound non-TM state) increased as a function of lipid composition on the order: 1:1 (mol:mol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine ∼ 6:4 POPC:cholesterol < POPC ∼ dioleoylphosphatidylcholine (DOPC) < 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt (DOPG) ≤ 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] sodium salt (DOPS), indicating that the anionic lipids DOPG and DOPS most strongly stabilized the TM configuration. TM stabilization was near maximal at 20-30 mol% anionic lipid, which are physiologically relevant values. TM stabilization by anionic lipid was observed for hydrophobic sequences with a diverse set of sequences (including polyAla), diverse lengths (from 12 to 22 residues), and various cationic flanking residues (H, R, or K), but not when the flanking residues were uncharged. TM stabilization by anionic lipid was also dependent on the number of cationic residues flanking the hydrophobic sequence, but was still significant with only one cationic residue flanking each end of the peptide. These observations are consistent with TM-stabilizing effects being electrostatic in origin. However, Trp located more deeply in DOPS vesicles relative to DOPG vesicles, and peptides in DOPS vesicles showed increased helix formation relative to DOPG and all other lipid compositions. These observations fit a model in which DOPS anchors flanking residues near the membrane surface more strongly than does DOPG and/or increases the stability of the TM state to a greater degree than DOPG. We conclude that anionic lipids can have significant and headgroup structure-specific effects upon membrane protein topography.     

Why and how are peptide-lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes

Animals as well as plants defend themselves against invading pathogenic microorganisms utilizing cationic antimicrobial peptides, which rapidly kill various microbes without exerting toxicity against the host. Physicochemical peptide-lipid interactions provide attractive mechanisms for innate immunity. Many of these peptides form cationic amphipathic secondary structures, typically alpha-helices and beta-sheets, which can selectively interact with anionic bacterial membranes by the aid of electrostatic interactions. Rapid, peptide-induced membrane permeabilization is an effective mechanism of antimicrobial action. This review article summarizes interactions with lipid bilayers of magainins (alpha-helix) and tachyplesins (beta-sheet) discovered in frog skin and horseshoe crab hemolymph, respectively, as archetypes, emphasizing that the mode of interaction is strongly dependent on the physicochemical properties not only of the peptide, but also of the target membrane.     

Membrane interaction and perturbation mechanisms induced by two cationic cell penetrating peptides with distinct charge distribution

Independently from the cell penetrating peptide uptake mechanism (endocytic or not), the interaction of the peptide with the lipid bilayer remains a common issue that needs further investigation. The cell penetrating or antimicrobial properties of exogenous peptides require probably different preliminary interactions with the plasma membrane. Herein, we have employed (31)P NMR, differential scanning calorimetry and CD to study the membrane interaction and perturbation mechanisms of two basic peptides with similar length but distinct charge distribution, penetratin (non-amphipathic) and RL16, a secondary amphipathic peptide. The peptide effects on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dipalmitoleoyl phosphatidylethanolamine (DiPoPE) were investigated. We have found that, even though both peptides are cationic, their interaction with zwitterionic versus anionic lipids is markedly distinct. Penetratin greatly affects the temperature, enthalpy and cooperativity of DMPG main phase transition but does not affect those of DMPC while RL16 presents opposite effects. Additionally, it was found that penetratin induces a negative curvature whereas RL16 induces a positive one, since a decrease in the fluid lamellar to inverted hexagonal phase transition temperature of DiPoPE (T(H)) was observed for penetratin and an increase for RL16. Contrary to penetratin, (31)P NMR of samples containing DMPC MLVs and RL16 shows an isotropic signal indicative of the formation of small vesicles, concomitant with a great decrease in sample turbidity both below and at the phase transition temperature. Opposite effects were also observed on DMPG where both peptides provoke strong aggregation and precipitation. Both CPPs adopt helical structures when contacting with anionic lipids, and possess a dual behavior by either presenting their cationic or hydrophobic domains towards the phospholipid face, depending on the lipid nature (anionic vs zwitterionic, respectively). Surprisingly, the increase of electrostatic interactions at the water membrane interface prevents the insertion of RL16 hydrophobic region in the bilayer, but is essential for the interaction of penetratin. Modulation of amphipathic profiles and charge distribution of CPPs can alter the balance of hydrophobic and electrostatic membrane interaction leading to translocation or and membrane permeabilisation. Penetratin has a relative pure CPP behavior whereas RL16 presents mixed CPP/AMP properties. A better understanding of those processes is essential to unveil their cell translocation mechanism.     

Membrane order perturbation in the presence of antimicrobial peptides by H solid-state NMR spectroscopy

The ²H solid-state NMR spectra of deuterated fatty acyl chains provide direct access to the order of the hydrophobic membrane interior. From the deuterium order parameter profiles of perdeuterated fatty acyl chains the membrane hydrophobic thickness can be calculated. Here we show data obtained from POPC, POPE and mixed POPE/POPG bilayers, representative of bacterial membranes, in the presence of cholesterol or ergosterol and antimicrobial peptaibols. Whereas sterols have a strong ordering effect also on these membranes, the peptides exhibit neutral or disordering effects. By comparing with data from the literature it becomes obvious that cationic amphipathic peptides that probably reside within the interface of phospholipid membranes tend to strongly disorder the packing of the fatty acyl chains, an effect that has been correlated to antimicrobial and DNA transfection activities. In contrast transmembrane sequences or hydrophobic peptides that probably partition deeply into the membrane tend to have only modest disordering activities. The ²H solid-state NMR approach has also been used to monitor the lateral separation of domains rich in anionic phospholipids in the presence of cationic peptides and has thereby provided important insights into their mechanisms of action.     

Selective cytotoxicity following Arg-to-Lys substitution in tritrpticin adopting a unique amphipathic turn structure

In antimicrobial peptides, the cationic property due to basic amino acids has been widely recognized as an important factor to promote electrostatic interaction with negatively charged phospholipids. However, little is known about the differences between two basic residues, Arg and Lys, in membrane binding affinity. Tritrpticin is an Arg- or Trp-rich antimicrobial peptide with a broad spectrum of antibacterial and antifungal activity. To investigate the structural and functional differences between Arg and Lys residues, here we designed and synthesized Arg-containing peptides, tritrpticin and SYM11, and their counterpart Lys-substituted peptides, TRK and SYM11KK, respectively. Although there were no remarkable conformational differences between Arg-containing and Lys-substituted peptides, TRK and SYM11KK exhibited almost two-fold enhanced antibacterial activity but significantly reduced hemolytic activity as compared to tritrpticin and SYM11, respectively. Furthermore, Arg-containing peptides showed strong binding affinity to both zwitterionic and anionic liposomes, whereas Lys-substituted peptides interacted weakly with zwitterionic liposomes but strongly with anionic liposomes. These results suggest that the primary amine of Lys interacts less electrostatically with zwitterionic phospholipids than the guanidinium group of Arg. Our results obtained in this study may be helpful in the design of drugs that target negatively charged phospholipids.     

Structural determinants of antimicrobial and antiplasmodial activity and selectivity in histidine-rich amphipathic cationic peptides

Designed histidine-rich amphipathic cationic peptides, such as LAH4, have enhanced membrane disruption and antibiotic properties when the peptide adopts an alignment parallel to the membrane surface. Although this was previously achieved by lowering the pH, here we have designed a new generation of histidine-rich peptides that adopt a surface alignment at neutral pH. In vitro, this new generation of peptides are powerful antibiotics in terms of the concentrations required for antibiotic activity; the spectrum of target bacteria, fungi, and parasites; and the speed with which they kill. Further modifications to the peptides, including the addition of more hydrophobic residues at the N terminus, the inclusion of a helix-breaking proline residue or using D-amino acids as building blocks, modulated the biophysical properties of the peptides and led to substantial changes in toxicity to human and parasite cells but had only a minimal effect on the antibacterial and antifungal activity. Using a range of biophysical methods, in particular solid-state NMR, we show that the peptides are highly efficient at disrupting the anionic lipid component of model membranes. However, we also show that effective pore formation in such model membranes may be related to, but is not essential for, high antimicrobial activity by cationic amphipathic helical peptides. The information in this study comprises a new layer of detail in the understanding of the action of cationic helical antimicrobial peptides and shows that rational design is capable of producing potentially therapeutic membrane active peptides with properties tailored to their function.     

Membrane-active peptides and the clustering of anionic lipids

There is some overlap in the biological activities of cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs). We compared nine AMPs, seven CPPs, and a fusion peptide with regard to their ability to cluster anionic lipids in a mixture mimicking the cytoplasmic membrane of Gram-negative bacteria, as measured by differential scanning calorimetry. We also studied their bacteriostatic effect on several bacterial strains, and examined their conformational changes upon membrane binding using circular dichroism. A remarkable correlation was found between the net positive charge of the peptides and their capacity to induce anionic lipid clustering, which was independent of their secondary structure. Among the peptides studied, six AMPs and four CPPs were found to have strong anionic lipid clustering activity. These peptides also had bacteriostatic activity against several strains (particularly Gram-negative Escherichia coli) that are sensitive to lipid clustering agents. AMPs and CPPs that did not cluster anionic lipids were not toxic to E. coli. As shown previously for several types of AMPs, anionic lipid clustering likely contributes to the mechanism of antibacterial action of highly cationic CPPs. The same mechanism could explain the escape of CPPs from intracellular endosomes that are enriched with anionic lipids.     

Isolation and characterization of a cDNA encoding rat cationic trypsinogen

A cDNA encoding rat cationic trypsinogen has been isolated by immunoscreening from a rat pancreas cDNA library. The protein encoded by this cDNA is highly basic and contains all of the structural features observed in trypsinogens. The amino acid sequence of rat cationic trypsinogen is 75% and 77% homologous to the two anionic rat trypsinogens. The homology of rat cationic trypsinogen to these anionic trypsinogens is lower than its homology to other mammalian cationic trypsinogens, suggesting that anionic and cationic trypsins probably diverged prior to the divergence of rodents and ungulates. The most unusual feature of this trypsinogen is the presence of an activation peptide containing five aspartic acid residues, in contrast to all other reported trypsinogen activation peptides which contain four acidic amino acid residues. Comparisons of cationic and anionic trypsins reveal that the majority of the charge changes occur in the C-terminal portion of the protein, which forms the substrate binding site. Several regions of conserved charge differences between cationic and anionic trypsins have been identified in this region, which may influence the rate of hydrolysis of protein substrates.     

Effects of arginine density on the membrane-bound structure of a cationic antimicrobial peptide from solid-state NMR

Solid-state NMR spectroscopy is used to determine the membrane-bound topological structure of a cationic β-hairpin antimicrobial peptide in which the number of Arg residues has been halved. The parent peptide, PG-1, was previously found to form transmembrane β-barrels in anionic membranes where the Arg residues complex with the lipid phosphate groups to cause toroidal pore defects in the membrane. In comparison, the charge-attenuated and less active mutant studied here forms β-sheets that lie on the surface of the zwitterionic membrane and only partially insert into the anionic membrane. The mutant also exhibits much looser contact with the lipid headgroups. These results indicate that transmembrane insertion and tight Arg-phosphate association are two important elements for strong antimicrobial activities of this class of peptides. Comparison with other β-hairpin antimicrobial peptides studied so far further suggests a relative potency scale for the various mechanisms of action for the β-sheet family of antimicrobial peptides. The transmembrane insertion-toroidal pore mechanism is the most potent in disrupting the lipid bilayer, followed by the large-amplitude in-plane motional mechanism. The carpet model, where peptides aggregate on the membrane surface to cause lateral expansion and eventual micellization of the membrane, is a weaker mechanism of action.     

Surface charges associated with fenestrated brain capillaries. I. In vitro labeling of anionic sites

Ferritin derivatives with different pI values and the basic dye ruthenium red have been used as cationic probes to localize anionic sites associated with fenestrated brain capillaries. Cationic ferritin was found in the endothelial basement membrane and the basement membrane of the perivascular cellular linings in amounts far exceeding those observed with anionic derivatives, the degree being greater for the more cationized ferritin molecules. Labeling of the luminal endothelial front with cationic ferritin was only achieved when a serum- or albumin-free medium was applied. Furthermore, the striated collagen fibers were coated with cationic ferritin molecules in a highly ordered fashion. Ruthenium red localized to the same sites. The findings suggest the existence of a perivascular charge filter around fenestrated capillaries of the brain. Some physiological roles of this filter are discussed, as related to its possible function in regulating homeostasis of cerebrospinal fluid.