Comprehensive analysis of all triple helical repeats in beta-spectrins reveals patterns of selective evolutionary conservation

The spectrin superfamily (spectrin, alpha-actinin, utrophin and dystrophin) has in common a triple helical repeating unit of ~106 amino acid residues. In spectrin, alpha and beta chains contain multiple copies of this repeat. beta-spectrin chains contain the majority of binding activities in spectrin and are essential for animal life. Canonical beta-spectrins have 17 repeats; beta-heavy spectrins have 30. Here, the repeats of five human beta-spectrins, plus beta-spectrins from several other vertebrates and invertebrates, have been analysed. Repeats 1, 2, 14 and 17 in canonical beta are highly conserved between invertebrates and vertebrates, and repeat 8 in some isoforms. This is consistent with conservation of critical functions, since repeats 1, 2 and 17 bind alpha-spectrin. Repeats 1 of beta-spectrins are not always detected by SMART or Pfam tools. A profile hidden Markov model of beta-spectrin repeat 1 detects alpha-actinins, but not utrophin or dystrophin. Novel examples of repeat 1 were detected in the spectraplakins MACF1, BPAG1 and plectin close to the actin-binding domain. Ankyrin binds to the C-terminal portion of repeat 14; the high conservation of this entire repeat may point to additional, undiscovered ligand-binding activities. This analysis indicates that the basic triple helical repeat pattern was adapted early in the evolution of the spectrin superfamily to encompass essential binding activities, which characterise individual repeats in proteins extant today.     

Phospholipid binding by proteins of the spectrin family: a comparative study

Erythroid and neuronal spectrin (fodrin) are both known to interact strongly with the aminophospholipids that occur in the inner leaflet of plasma membranes. In erythroid spectrin the positions of the binding sites within the constituent (alphaI and betaI) polypeptide chains have been defined, and also the importance of the lipid interaction in regulating the properties of the membrane. Here we report the locations of the corresponding binding sites in the alphaII and betaII chains that make up the fodrin molecule. Of the 10 lipid-binding repeats in the erythroid spectrin chains 5 are conserved in fodrin; one cluster of 3 consecutive structural repeating units in alphaI erythroid spectrin (repeats 8-10) is displaced by one repeat in alphaII fodrin (repeats 9-11). Fodrin also contains one binding site at the N-terminus of the alphaII chain, not present in the erythroid protein. The regions of the two spectrins containing equivalent lipid-binding sites show a much higher degree of sequence identity than corresponding repeats that do not share this property. The evolutionary conservation of the distribution of a large proportion of strong lipid-binding sites in the polypeptide chains of these two proteins of disparate character argues for a specific function of fodrin-phospholipid interactions in the neuron.     

Is spectrin a calmodulin-binding protein?

The binding of calmodulin to spectrin from human erythrocytes has been studied by affinity chromatography on sepharose-calmodulin column. The alpha and beta spectrin chains, dissociated in 6-7 M urea, both bound to the sepharose-calmodulin column, but with different affinities. Both chains were eluted together by EGTA. Binding sites for calmodulin are, therefore, present in both alpha and beta chains. However, intact purified spectrin dimers did not bind to the sepharose-calmodulin column, which renders a physiological role of calmodulin-binding to spectrin rather unlikely.     

Molecular epitopes of the ankyrin-spectrin interaction

Isoforms of ankyrin and its binding partner spectrin are responsible for a number of interactions in a variety of human cells. Conflicting evidence, however, had identified two different, non-overlapping human erythroid ankyrin subdomains, Zu5 and 272, as the minimum binding region for beta-spectrin. Complementary studies on the ankyrin-binding domain of spectrin have been somewhat more conclusive yet have not presented binding in terms of well-phased, integral numbers of spectrin repeats. Thus, the objective of this study was to clearly define and characterize the minimal ankyrin-spectrin binding epitopes. Circular dichroism (CD) wavelength spectra of the aforementioned ankyrin subdomains show that these fragments are 30-60% unstructured. In contrast, human erythroid beta-spectrin repeats 13, 14, 15, and 16 (prepared in all combinations of two adjacent repeats) demonstrated proper folding and stability as determined by CD and tryptophan wavelength and heat denaturation scans. Native polyacrylamide gel electrophoresis (PAGE) gel shifts as well as affinity pull-down assays implicated Zu5 and beta-spectrin repeats 14-15 as the minimum binding epitopes. These results were confirmed by analytical ultracentrifugation to sedimentation equilibrium by which a 1:1 complex was obtained if and only if Zu5 was mixed with beta-spectrin constructs containing repeats 14 and 15 in tandem. Surface plasmon resonance yielded a K D of 15.2 nM for binding of beta-spectrin fragments to the ankyrin subdomain Zu5, accounting for all of the binding observed between the intact molecules. Collectively, these results show the 14th and 15th beta-spectrin repeats comprise the minimal, phased region of beta-spectrin, which binds ankyrin at the Zu5 subdomain with high affinity.     

Interaction of botulinum and tetanus toxins with the lipid bilayer surface.

The interaction of botulinum neurotoxins serotypes A, B and E (from Clostridium botulinum) and of tetanus neurotoxin (from Clostridium tetani) with the surface of liposomes made of different lipid compositions was studied by photolabelling with a radioiodinated photoactive phosphatidylethanolamine analogue [125I-dipalmitoyl (3,4-azidosalicylamido)phosphatidylethanolamine]. When the vesicles were made of negatively charged lipids (asolectin), each of these neurotoxic proteins was radioiodinated, thus providing evidence for their attachment to the membrane surface. The presence of gangliosides on liposome membranes enhanced fixation of the neurotoxic proteins to the lipid vesicle surface. Both the heavy and light chains of the clostridial neurotoxins were involved in the attachment to the lipid bilayer surface. Each of the toxins tested here attached poorly to liposomes made of zwitterionic lipids (egg phosphatidylcholine), even when polysialogangliosides were present. The data suggest that the binding of botulinum and tetanus neurotoxins to their target neuronal cells involves negatively charged lipids and polysialogangliosides on the cell membrane.     

Ankyrin binds to the 15th repetitive unit of erythroid and nonerythroid beta-spectrin

Ankyrin mediates the attachment of spectrin to transmembrane integral proteins in both erythroid and nonerythroid cells by binding to the beta-subunit of spectrin. Previous studies using enzymatic digestion, 2-nitro-5-thiocyanobenzoic acid cleavage, and rotary shadowing techniques have placed the spectrin-ankyrin binding site in the COOH-terminal third of beta-spectrin, but the precise site is not known. We have used a glutathione S-transferase prokaryotic expression system to prepare recombinant erythroid and nonerythroid beta-spectrin from cDNA encoding approximately the carboxy-terminal half of these proteins. Recombinant spectrin competed on an equimolar basis with 125I-labeled native spectrin for binding to erythrocyte membrane vesicles (IOVs), and also bound ankyrin in vitro as measured by sedimentation velocity experiments. Although full length beta-spectrin could inhibit all spectrin binding to IOVs, recombinant beta-spectrin encompassing the complete ankyrin binding domain but lacking the amino-terminal half of the molecule failed to inhibit about 25% of the binding capacity of the IOVs, suggesting that the ankyrin-independent spectrin membrane binding site must lie in the amino-terminal half of beta-spectrin. A nested set of shortened recombinants was generated by nuclease digestion of beta-spectrin cDNAs from ankyrin binding constructs. These defined the ankyrin binding domain as encompassing the 15th repeat unit in both erythroid and nonerythroid beta-spectrin, amino acid residues 1,768-1,898 in erythroid beta-spectrin. The ankyrin binding repeat unit is atypical in that it lacks the conserved tryptophan at position 45 (1,811) within the repeat and contains a nonhomologous 43 residue segment in the terminal third of the repeat. It also appears that the first 30 residues of this repeat, which are highly conserved between the erythroid and nonerythroid beta-spectrins, are critical for ankyrin binding activity. We hypothesize that ankyrin binds directly to the nonhomologous segment in the 15th repeat unit of both erythroid and nonerythroid beta-spectrin, but that this sequence must be presented in the context of a properly folded spectrin "repeat unit" structure. Future studies will identify which residues within the repeat unit are essential for activity, and which residues determine the specificity of various spectrins for different forms of ankyrin.     

A beta-spectrin isoform from Drosophila (beta H) is similar in size to vertebrate dystrophin

Spectrins are a major component of the membrane skeleton in many cell types where they are thought to contribute to cell form and membrane organization. Diversity among spectrin isoforms, especially their beta subunits, is associated with diversity in cell shape and membrane architecture. Here we describe a spectrin isoform from Drosophila that consists of a conventional alpha spectrin subunit complexed with a novel high molecular weight beta subunit (430 kD) that we term beta H. The native alpha beta H molecule binds actin filaments with high affinity and has a typical spectrin morphology except that it is longer than most other spectrin isoforms and includes two knoblike structures that are attributed to a unique domain of the beta H subunit. Beta H is encoded by a different gene than the previously described Drosophila beta-spectrin subunit but shows sequence similarity to beta-spectrin as well as vertebrate dystrophin, a component of the membrane skeleton in muscle. By size and sequence similarity, dystrophin is more similar to this newly described beta-spectrin isoform (beta H) than to other members of the spectrin gene family such as alpha-spectrin and alpha- actinin.     

Ankyrin-independent membrane protein-binding sites for brain and erythrocyte spectrin

Brain spectrin reassociates in in vitro binding assays with protein(s) in highly extracted brain membranes quantitatively depleted of ankyrin and spectrin. These newly described membrane sites for spectrin are biologically significant and involve a protein since (a) binding occurs optimally at physiological pH (6.7-6.9) and salt concentrations (50 mM), (b) binding is abolished by digestion of membranes with alpha-chymotrypsin, (c) Scatchard analysis is consistent with a binding capacity of at least 50 pmol/mg total membrane protein, and highest affinity of 3 nM. The major ankyrin-independent binding activity of brain spectrin is localized to the beta subunit of spectrin. Brain membranes also contain high affinity binding sites for erythrocyte spectrin, but a 3-4 fold lower capacity than for brain spectrin. Some spectrin-binding sites associate preferentially with brain spectrin, some with erythrocyte spectrin, and some associate with both types of spectrin. Erythrocyte spectrin contains distinct binding domains for ankyrin and brain membrane protein sites, since the Mr = 72,000 spectrin-binding fragment of ankyrin does not compete for binding of spectrin to brain membranes. Spectrin binds to a small number of ankyrin-independent sites in erythrocyte membranes present in about 10,000-15,000 copies/cell or 10% of the number of sites for ankyrin. Brain spectrin binds to these sites better than erythrocyte spectrin suggesting that erythrocytes have residual binding sites for nonerythroid spectrin. Ankyrin-independent-binding proteins that selectively bind to certain isoforms of spectrin provide a potentially important flexibility in cellular localization and time of synthesis of proteins involved in spectrin-membrane interactions. This flexibility has implications for assembly of the membrane skeleton and targeting of spectrin isoforms to specialized regions of cells.     

Regulation of calpactin I phospholipid binding by calpactin I light-chain binding and phosphorylation by p60v-src.

Calpactins I and II are proteins that bind Ca2+, phospholipids, actin and spectrin; they are also major substrates of oncogene and growth-factor-receptor tyrosine kinases. Since calpactins have been proposed to provide a link between membrane lipids and the cytoskeleton, we examined in detail the interactions between purified calpactin I and phospholipid liposomes. We focused on the Ca2+-dependence, the effects of phosphorylation of calpactin I by p60v-src (the protein kinase coded for by the Rous-sarcoma-virus oncogene), and the effects of the binding of calpactin I light chain to calpactin I heavy chain. Binding of the light chain to the heavy chain increased the affinity of calpactin I for phosphatidylserine (PS) liposomes. The opposite effect was observed for phosphorylation by p60v-src; phosphorylation decreased the affinity of calpactin I for PS liposomes. These two opposite effects appeared to be independent, since phosphorylation did not prevent light-chain binding to the heavy chain. Calpactin I was found, by the use of three different techniques, to bind to phospholipid liposomes at less than 10(-8) M free Ca2+. This result is in contrast with those of previous studies, which indicated that greater than 10(-6) M free Ca2+ was required. Our findings suggest that calpactin I may be bound to phospholipids in vivo at Ca2+ concentrations of about 1.5 x 10(-7) M, typical of resting unstimulated cells, and that this interaction may be modulated by light-chain binding and phosphorylation by p60v-src.     

Biogenesis of the avian erythroid membrane skeleton: receptor-mediated assembly and stabilization of ankyrin (goblin) and spectrin

Ankyrin is an extrinsic membrane protein in human erythrocytes that links the alpha beta-spectrin-based extrinsic membrane skeleton to the membrane by binding simultaneously to the beta-spectrin subunit and to the transmembrane anion transporter. To analyse the temporal and spatial regulation of assembly of this membrane skeleton, we investigated the kinetics of synthesis and assembly of ankyrin ( goblin ) with respect to those of spectrin in chicken embryo erythroid cells. Electrophoretic analysis of Triton X-100 soluble and cytoskeletal fractions show that at steady state both ankyrin and spectrin are detected exclusively in the cytoskeleton. In contrast, continuous labeling of erythroid cells with [35S]methionine, and immunoprecipitation of ankyrin and alpha- and beta-spectrin, reveals that newly synthesized ankyrin and spectrin are partitioned into both the cytoskeletal and Triton X-100 soluble fractions. The soluble pools of ankyrin and beta-spectrin reach a plateau of labeling within 1 h, whereas the soluble pool of alpha-spectrin is substantially larger and reaches a plateau more slowly, reflecting an approximately 3:1 ratio of synthesis of alpha- to beta-spectrin. Ankyrin and beta-spectrin enter the cytoskeletal fraction within 10 min of labeling, and the amount assembled into the cytoskeletal fraction exceeds the amount present in their respective soluble pools within 1 h of labeling. Although alpha-spectrin enters the cytoskeletal fraction with similar kinetics to beta-spectrin and ankyrin, and in amounts equimolar to beta-spectrin, the amount of cytoskeletal alpha-spectrin does not exceed the amount of soluble alpha-spectrin even after 3 h of labeling. Pulse-chase labeling experiments reveal that ankyrin and alpha- and beta-spectrin assembled into the cytoskeleton exhibit no detectable turnover, whereas the Triton X-100 soluble polypeptides are rapidly catabolized, suggesting that stable assembly of the three polypeptides is dependent upon their association with their respective membrane receptor(s). The existence in the detergent-soluble compartment of newly synthesized ankyrin and alpha- and beta-spectrin that are catabolized, rather than assembled, suggests that ankyrin and spectrin are synthesized in excess of available respective membrane binding sites, and that the assembly of these polypeptides, while rapid, is not tightly coupled to their synthesis. We hypothesize that the availability of the high affinity receptor(s) localized on the membrane mediates posttranslationally the extent of assembly of the three cytoskeletal proteins in the correct stoichiometry, their stability, and their spatial localization.     

Interaction analysis of a ladder-shaped polycyclic ether and model transmembrane peptides in lipid bilayers by using Förster resonance energy transfer and polarized attenuated total reflection infrared spectroscopy

Ladder-shaped polycyclic ethers (LSPs) are predicted to interact with membrane proteins; however, the underlying mechanism has not been satisfactorily elucidated. It has been hypothesized that LSPs possess non-specific affinity to α-helical segments of transmembrane proteins. To verify this hypothesis, we constructed a model LSP interaction system in a lipid bilayer. We prepared 5 types of α-helical peptides and reconstituted them in liposomes. The reconstitution and orientation of these peptides in the liposomes were examined using polarized attenuated total reflection infrared (ATR-IR) spectroscopy and gel filtration. The results revealed that 4 peptides were retained in liposomes, and 3 of them formed stable transmembrane structures. The interaction between the LSP and the peptides was investigated using Förster resonance energy transfer (FRET). In the lipid bilayer, the LSP strongly recognized the peptides that possessed aligned hydrogen donating groups with leucine caps. We propose that this leucine-capped 16-amino acid sequence is a potential LPS binding motif.     

A Quantitative Approach to Membrane Binding of Human Ubiquitous Mitochondrial Creatine Kinase Using Surface Plasmon Resonance

We have evaluated surface plasmon resonance with avidin-biotin immobilized liposomes tocharacterize membrane binding of ubiquitous mitochondrial creatine kinase (uMtCK). Whilethe sarcomeric sMtCK isoform is well known to bind to negatively charged phospholipids,especially cardiolipin, this report provides the first experimental evidence on the membraneinteraction of an uMtCK isoform. Qualitative measurements showed that liposomes containing16% (w/w) cardiolipin bind octameric as well as dimeric human uMtCK and also cytochromec, but not bovine serum albumin. Quantitative parameters could be derived only for themembrane interaction of octameric human uMtCK using an improved analytical approach.Association and dissociation kinetics of octameric uMtCK fit well to a model for heterogeneousinteraction suggesting two independent binding sites. Rate constants of the two sites differedby one order of magnitude, while their affinity constants were both about 80–100 nM. Thedata obtained demonstrate that surface plasmon resonance with immobilized liposomes is asuitable approach to characterize the binding of peripheral proteins to a lipid bilayer and thatthis method yields consistent quantitative binding parameters.     

Lipid-binding hSH3 domains in immune cell adapter proteins

SH3 domains represent versatile scaffolds within eukaryotic cells by targeting proline-rich sequences within intracellular proteins. More recently, binding of SH3 domains to unusual peptide motifs, folded proteins or lipids has been reported. Here we show that the newly defined hSH3 domains of immune cell adapter proteins bind lipid membranes with distinct affinities. The interaction of the hSH3 domains of adhesion and degranulation promoting adapter protein (ADAP) and PRAM-1 (Promyelocytic-Retinoic acid receptor alpha target gene encoding an Adaptor Molecule-1), with phosphatidylcholine-containing liposomes is observed upon incorporation of phosphatidylserine (PS) or phosphoinositides (PIs) into the membrane bilayer. Mechanistically we show that stable association of the N-terminal, amphipathic helix with the beta-sheet scaffold favours lipid binding and that the interaction with PI(4,5)P(2)-containing liposomes is consistent with a single-site, non-cooperative binding mechanism. Functional investigations indicate that deletion of both amphipathic helices of the hSH3 domains reduces the ability of ADAP to enhance adhesion and migration in stimulated T cells.     

Targeting of antibody conjugated,phosphatidylserine-containing liposomes to vascular cell adhesion molecule 1 for controlled thrombogenesis

Phosphatidylserine (PS) membrane exposure plays an important role in blood coagulation, and the development of a liposome formulation containing PS may be of potential therapeutic utility if they can be designed to achieve tumor selective thrombosis. The objective of this study was to develop proof-of-principle data for a thrombogenic PS liposome targeted to vascular cell adhesion molecule 1 (VCAM-1) via the attachment of an anti-VCAM-1 monoclonal antibody (Ab). We have evaluated binding of the anti-VCAM-1 Ab-conjugated PS liposomes to VCAM-1 using two in vitro models, as well as assessing the ability of these liposomes to catalyze blood coagulation reactions. Binding of the Ab-conjugated PS liposomes containing 2 or 14 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) 2000] (DSPE-PEG(2000)) to interleukin 1alpha stimulated human umbilical vein endothelial cells was 8- and 16-fold higher than those without conjugated Ab, respectively, based on the percentage relative increase in cell associated lipid for these liposomes. Binding to VCAM-1-coated ELISA plates produced similar results. The VCAM-1-bound Ab-conjugated PS liposomes were capable of catalyzing blood coagulation reactions upon the exposure of the thrombogenic PS membrane surface. This control of PS surface exposure was achieved using exchangeable PEG-derivatized phosphatidylethanolamines (PE-PEG), with 97% of clotting activity recovered after PE-PEG exchanged out. Our results demonstrate the potential for considering further development of procoagulant liposomes that selectively target thrombogenesis in tumor vasculature.     

Galectin-3 interacts with membrane lipids and penetrates the lipid bilayer

The precise mechanism by which galectin-3 and other cytosolic proteins that lack signal peptides are secreted is yet to be elucidated. In the present analyses, we determined that galectin-3, a beta-galactoside binding protein, can interact directly with membrane lipids in solid phase binding assays. More interestingly, we determined by spectrophotometric methods that it can spontaneously penetrate the lipid bilayer of liposomes in either direction. These findings suggest that galectin-3 on its own has the capacity to traverse the lipid bilayer. Whereas the situation is rather simplified in liposomes, the interaction of galectin-3 with the plasma membrane may involve cholesterol-rich membrane domains where galectin-3 can be concentrated and form multimers or interact covalently with other proteins.     

Association of brain ankyrin with brain membranes and isolation of active proteolytic fragments of membrane-associated ankyrin-binding protein(s)

An assay has been developed to measure association of brain ankyrin with protein site(s) in brain membranes that are independent of spectrin and tubulin, behave as integral membrane proteins, and appear to be similar in several respects to the erythrocyte anion channel. Brain membranes were depleted of ankyrin, spectrin, and other peripheral membrane proteins by a brief incubation in 0.1 M sodium hydroxide. Binding of ankyrin to these membranes fulfilled experimentally testable criteria for a specific protein-protein association. Binding was optimal at physiological values for ionic strength and pH, was of high affinity (Kd = 20-60 nM), and the capacity of 25 pmol/mg of brain membrane protein is in the same range as the number of spectrin tetramers (30 pmol/mg). The membrane-binding site(s) for brain ankyrin are likely to be related in some way to the cytoplasmic domain of the erythrocyte anion channel since binding was inhibited by the anion channel domain and by erythrocyte ankyrin. The binding site(s) for brain ankyrin were released from the membrane by limited proteolysis as active water-soluble fragments capable of inhibiting binding of ankyrin to membranes. Ankyrin-binding fragments of Mr = 40,000 and 68,000 were selectively bound to an erythrocyte ankyrin affinity column. The fragment of Mr = 40,000 is close to the size of the cytoplasmic domain of the erythrocyte anion channel. It is likely based on these results that membrane attachment proteins for ankyrin are present in brain and other tissues and that these membrane proteins have domains homologous at least in conformation to the ankyrin-binding site of the erythrocyte anion channel.     

ATP-dependent Mechanism Protects Spectrin against Glycation in Human Erythrocytes

Human erythrocytes are continuously exposed to glucose, which reacts with the amino terminus of the β-chain of hemoglobin (Hb) to form glycated Hb, HbA1c, levels of which increase with the age of the circulating cell. In contrast to extensive insights into glycation of hemoglobin, little is known about glycation of erythrocyte membrane proteins. In the present study, we explored the conditions under which glucose and ribose can glycate spectrin, both on the intact membrane and in solution and the functional consequences of spectrin glycation. Although purified spectrin could be readily glycated, membrane-associated spectrin could be glycated only after ATP depletion and consequent translocation of phosphatidylserine (PS) from the inner to the outer lipid monolayer. Glycation of membrane-associated spectrin led to a marked decrease in membrane deformability. We further observed that only PS-binding spectrin repeats are glycated. We infer that the absence of glycation in situ is the consequence of the interaction of the target lysine and arginine residues with PS and thus is inaccessible for glycation. The reduced membrane deformability after glycation in the absence of ATP is likely the result of the inability of the glycated spectrin repeats to undergo the obligatory unfolding as a consequence of interhelix cross-links. We thus postulate that through the use of an ATP-driven phospholipid translocase (flippase), erythrocytes have evolved a protective mechanism against spectrin glycation and thus maintain their optimal membrane function during their long circulatory life span.     

A combinatorial code for the interaction of alpha-synuclein with membranes

Considerable genetic and pathological evidence has implicated the small, soluble protein alpha-synuclein in the pathogenesis of familial and sporadic forms of Parkinsons disease (PD). However, the precise role of alpha-synuclein in the disease process as well as its normal function remain poorly understood. We recently found that an interaction with lipid rafts is crucial for the normal, pre-synaptic localization of alpha-synuclein. To understand how alpha-synuclein interacts with lipid rafts, we have now developed an in vitro binding assay to rafts purified from native membranes. Recapitulating the specificity observed in vivo, recombinant wild type but not PD-associated A30P mutant alpha-synuclein binds to lipid rafts isolated from cultured cells and purified synaptic vesicles. Proteolytic digestion of the rafts does not disrupt the binding of alpha-synuclein, indicating an interaction with lipid rather than protein components of these membranes. We have also found that alpha-synuclein binds directly to artificial membranes whose lipid composition mimics that of lipid rafts. The binding of alpha-synuclein to these raft-like liposomes requires acidic phospholipids, with a preference for phosphatidylserine (PS). Interestingly, a variety of synthetic PS with defined acyl chains do not support binding when used individually. Rather, the interaction with alpha-synuclein requires a combination of PS with oleic (18:1) and polyunsaturated (either 20:4 or 22:6) fatty acyl chains, suggesting a role for phase separation within the membrane. Furthermore, alpha-synuclein binds with higher affinity to artificial membranes with the PS head group on the polyunsaturated fatty acyl chain rather than on the oleoyl side chain, indicating a stringent combinatorial code for the interaction of alpha-synuclein with membranes.     

Porphyrin-membrane interactions: binding or partition?

Porphyrins are photodynamic drugs employed in an experimental tumor-treatment modality in which cell membranes are one of the primary drug-action sites. To gain insight into the nature of the interaction of these drugs with those primary sites we have studied the affinity of porphyrins to the lipid moieties of biological membranes, at the molecular level. The association of porphyrins to large unilamellar liposomes, modeling the lipid regions of biological membranes was studied (at equilibrium) for deuteroporphyrin IX and protoporphyrin IX, at neutral pH and 37 degrees C, taking into account porphyrin aggregation. Two thermodynamic approaches were investigated: (i) Simple partition equilibria between the external aqueous phase and the lipid bilayer, for drug monomers and dimers. (ii) Binding equilibria of drug monomers and dimers to the lipid bilayer. Using two types of experimental design and processing the data according to the expectations of both approaches, three different models for the binding (differing in the participation assigned to the dimer) were considered. Our major findings are: (a) The data clearly do not fit with the expectations for simple partition equilibria, nor with binding models assuming direct participation of the dimers. (b) The data fit well with a binding process, in which the membrane binds the porphyrin monomers only, with the dimers participating indirectly through the aqueous dimerization equilibrium. (c) At 37 degrees C and neutral pH, for liposomes composed of phosphatidylcholine/cholesterol at molar ratios of 3:2, we found for both investigated species a binding constant of 2.3 x 10(4) M-1. (d) For each species the binding constant is independent of the initial and final states of drug aggregation in the aqueous phase.     

Enforcing the positive charge of N-termini enhances membrane interaction and antitumor activity of bovine seminal ribonuclease

Binding to cell membrane, followed by translocation into the cytosol and RNA degradation, is a necessary requirement to convert a ribonuclease into a cytotoxin for malignant tumor cells. In this paper, we investigate the membrane binding attitude of bovine seminal ribonuclease (BS-RNase) and its variant G38K-BS-RNase, bearing an enforced cluster of positive charges at the N-termini surface. By using a combination of biophysical techniques, including CD, SPR and ESR, we find for the two proteins a common, two-step mechanism of interaction with synthetic liposomes, an initial binding to the bilayer surface, driven by electrostatic interactions, followed by a shallow penetration in the lipid core. Protein binding effectively perturbs lipid packing and dynamics. Remarkably, the higher G38K-BS-RNase membrane interacting capability well correlates with its increased cytotoxicity for tumor cells. Overall, these studies shed light on the mechanism of membrane binding and perturbation, proving definitely the importance of electrostatic interactions in the cytotoxic activity of BS-RNase, and provide a rational basis to design proteins with anticancer potential.