Membrane binding motif of the P-type cardiotoxin

Carditoxins (CTXs) from cobra snake venoms, the basic 60-62 residue all-beta sheet polypeptides, are known to bind to and impair the function of cell membranes. To assess the membrane induced conformation and orientation of CTXs, the interaction of the P-type cardiotoxin II from Naja oxiana snake venom (CTII) with perdeuterated dodecylphosphocholine (DPC) was studied using ( 1 )H-NMR spectroscopy and diffusion measurements. Under conditions where the toxin formed a well-defined complex with DPC, the spatial structure of CTII with respect to the presence of tightly bound water molecules in loop II, was calculated using the torsion angle dynamics program DYANA. The structure was found to be similar, except for subtle changes in the tips of all three loops, to the previously described "major" form of CTII in aqueous solution illustrated by the "trans" configuration of the Val7-Pro8 peptide bond. No "minor" form with the "cis" configuration of the above bond was found in the micelle-bound state. The broadening of the CTII backbone proton signals by 5, 16-doxylstearate relaxation probes, together with modeling based on the spatial structure of CTII, indicated a periphery mode of binding of the toxin molecule to the micelle and revealed its micelle interacting domain. The latter includes a hydrophobic region of CTII within the extremities of loops I and III (residues 5-11, 46-50), the basement of loop II (residues 24-29,31-37) and the belt of polar residues encircling these loops (lysines 4,5,12,23,50, serines 11,46, histidine 31, arginine 36). It is suggested that this structural motif and the mode of binding can be realized during interaction of CTXs with lipid and biological membranes.     

Naja naja oxiana Cobra Venom Cytotoxins CTI and CTII Disrupt Mitochondrial Membrane Integrity: Implications for Basic Three-Fingered Cytotoxins

Cobra venom cytotoxins are basic three-fingered, amphipathic, non-enzymatic proteins that constitute a major fraction of cobra venom. While cytotoxins cause mitochondrial dysfunction in different cell types, the mechanisms by which cytotoxins bind to mitochondria remain unknown. We analyzed the abilities of CTI and CTII, S-type and P-type cytotoxins from Naja naja oxiana respectively, to associate with isolated mitochondrial fractions or with model membranes that simulate the mitochondrial lipid environment by using a myriad of biophysical techniques. Phosphorus-31 nuclear magnetic resonance (³¹P-NMR) spectroscopy data suggest that both cytotoxins bind to isolated mitochondrial fractions and promote the formation of aberrant non-bilayer structures. We then hypothesized that CTI and CTII bind to cardiolipin (CL) to disrupt mitochondrial membranes. Collectively, ³¹P-NMR, electron paramagnetic resonance (EPR), proton NMR (¹H-NMR), deuterium NMR (²H-NMR) spectroscopy, differential scanning calorimetry, and erythrosine phosphorescence assays suggest that CTI and CTII bind to CL to generate non-bilayer structures and promote the permeabilization, dehydration and fusion of large unilamellar phosphatidylcholine (PC) liposomes enriched with CL. On the other hand, CTII but not CTI caused biophysical alterations of large unilamellar PC liposomes enriched with phosphatidylserine (PS). Mechanistically, single molecule docking simulations identified putative CL, PS and PC binding sites in CTI and CTII. While the predicted binding sites for PS and PC share a high number of interactive amino acid residues in CTI and CTII, the CL biding sites in CTII and CTI are more divergent as it contains additional interactive amino acid residues. Overall, our data suggest that cytotoxins physically associate with mitochondrial membranes by binding to CL to disrupt mitochondrial structural integrity.     

Interaction of alpha-crystallin with lens plasma membranes. Affinity for MP26

The binding of the major water-soluble lens protein alpha-crystallin to the lens plasma membrane has been investigated by reassociating purified alpha-crystallin with alpha-crystallin-depleted membranes and with phospholipid vesicles in which the lens membrane protein MP26 had been reconstituted. alpha-Crystallin reassociates at high affinity (Kd = 13 X 10(-8)M) with alkali-washed lens plasma membranes but not with lens plasma membranes treated with guanidine/HCl, nor with phospholipid vesicles or erythrocyte membranes. Binding to lens plasma membranes is dependent on salt, temperature and pH and occurs in a saturable manner. Reconstitution of MP26 into phospholipid vesicles and subsequent analysis of alpha-crystallin binding suggests the involvement of this transmembrane protein. Binding ist not influenced by pretreatment of membranes with proteases, suggesting that the 4-kDa cytoplasmic fragment of MP26 is not necessary for alpha-crystallin binding. Labeling experiments using (trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine as a probe for intrinsic membrane proteins further showed that alpha-crystallin contains hydrophobic regions on its surface which might enable this protein to make contact with the lipid bilayer. Newly synthesized alpha-crystallin, in lens culture, is not associated with the plasma membrane, suggesting that the assembly of alpha-crystallin aggregates does not take place in a membrane-bound mode.     

Lipid phase separations induced by the association of cholera toxin to phospholipid membranes containing ganglioside GM1

The interactions of cholera toxin and their isolated binding and active subunits with phospholipid bilayers containing the toxin receptor ganglioside GM1 have been studied by using high-sensitivity differential scanning calorimetry and steady-state and time-resolved fluorescence and phosphorescence spectroscopy. The results of this investigation indicate that cholera toxin associates with phospholipid bilayers containing ganglioside GM1, independent of the physical state of the membrane. In the absence of Ca2+, calorimetric scans of intact cholera toxin bound to dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles containing ganglioside GM1 result in a broadening of the lipid phase transition peak and a slight decrease (less than 5%) in the transition enthalpy. In the presence of Ca2+ concentrations sufficient to cause ganglioside phase separation, the association of the intact toxin to the membrane results in a significant decrease of enthalpy change for the lipid transition, indicating that under these conditions the toxin molecule perturbs the hydrophobic core of the bilayer. Calorimetric scans using isolated binding subunits lacking the hydrophobic toxic subunit did not exhibit a decrease in the phospholipid transition enthalpy even in the presence of Ca2+, indicating that the binding subunits per se do not perturb the hydrophobic core of the bilayer. On the other hand, the hydrophobic A1 subunit by itself was able to reduce the phospholipid transition enthalpy when reconstituted into DPPC vesicles. These calorimetric observations were confirmed by fluorescence experiments using pyrene phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

The interaction of human apolipoprotein C-I with sub-micellar phospholipid.

Mature human apolipoprotein C-I (apoC-I), comprising 57 amino acids, is the smallest member of the plasma apolipoprotein family. Amphipathic helical regions within apoC-I, common to this class of proteins, are mediators of lipid binding, a process that underlies the functional properties of apoC-I, including the capacity to activate the plasma enzyme LCAT, to disrupt apoE mediated receptor interactions and to inhibit cholesterol ester transfer protein. To examine apoC-I/phospholipid interactions, we have developed an expression system in Escherichia coli to obtain purified apoC-I with yields of approximately 4-5 mg per L of culture. The purified product has properties similar to plasma-derived apoC-I including self-association in the lipid-free state and induced alpha-helical content in the presence of egg-yolk phosphatidylcholine and dimyristoylglycerophosphocholine vesicles. We chose the short-chain phospholipid, dihexanoylglycerophosphocholine (Hex2Gro-PCho), to examine the interaction of apoC-I with submicellar phospholipid. Circular dichroism spectroscopy and cross-linking experiments show that apoC-I acquires helical content and remains self-associated at submicellar concentrations of Hex2Gro-PCho (4 mM). Sedimentation equilibrium studies of apoC-I at submicellar levels of Hex2Gro-PCho and analysis of the effects of apoC-I on the 1H NMR spectrum of Hex2Gro-PCho indicate micelle induction by apoC-I, and establish the capacity of apoC-I to assemble individual phospholipid molecules.     

Membrane protein insertion regulated by bringing electrostatic and hydrophobic interactions into play. A case study with the translocation domain of diphtheria toxin

The study of the membrane insertion of the translocation domain of diphtheria toxin deepens our insight into the interactions between proteins and membranes. During cell intoxication, this domain undergoes a change from a soluble and folded state at alkaline pH to a functional membrane-inserted state at acid pH. We found that hydrophobic and electrostatic interactions occur in a sequential manner between the domain and the membrane during the insertion. The first step involves hydrophobic interactions by the C-terminal region. This is because of the pH-induced formation of a molten globule specialized for binding to the membrane. Accumulation of this molten globule follows a precise molecular mechanism adapted to the toxin function. The second step, as the pH decreases, leads to the functional inserted state. It arises from the changes in the balance of electrostatic attractions and repulsions between the N-terminal part and the membrane. Our study shows how the structural changes and the interaction with membranes of the translocation domain are finely tuned by pH changes to take advantage of the cellular uptake system.     

A new look at the hemolytic effect of local anesthetics, considering their real membrane/water partitioning at pH 7.4

The interaction of local anesthetics (LA) with biological and phospholipid bilayers was investigated regarding the contribution of their structure and physicochemical properties to membrane partition and to erythrocyte solubilization. We measured the partition into phospholipid vesicles—at pH 5.0 and 10.5—and the biphasic hemolytic effect on rat erythrocytes of: benzocaine, chloroprocaine, procaine, tetracaine, bupivacaine, mepivacaine, lidocaine, prilocaine, and dibucaine. At pH 7.4, the binding of uncharged and charged LA to the membranes was considered, since it results in an ionization constant (pK_app) different from that observed for the anesthetic in the aqueous phase (pK_w). Even though it occurred at a pH at which there is a predominance of the charged species, hemolysis was greatly influenced by the uncharged species, revealing that the disrupting effect of LA on these membranes is mainly a consequence of hydrophobic interactions. The correlation between the hemolytic activity and the LA potency shows that hemolytic experiments could be used for the prediction of activity in the development of new LA molecules.     

Two forms of cytotoxin II (cardiotoxin) from Naja naja oxiana in aqueous solution: spatial structures with tightly bound water molecules.

1H-NMR spectroscopy data, such as NOE intraprotein and (bound water)/protein contacts, 3J coupling constants and deuterium exchange rates were used to determine the in-solution spatial structure of cytotoxin II from Naja naja oxiana snake venom (CTII). Exploiting information from two 1H-NMR spectral components, shown to be due to cis/trans isomerization of the Val7-Pro8 peptide bond, spatial structures of CTII minor and major forms (1 : 6) were calculated using the torsion angle dynamics algorithm of the DYANA program and then energy refined using the FANTOM program. Each form, major and minor, is represented by 20 resulting conformers, demonstrating mean backbone rmsd values of 0.51 and 0.71 A, respectively. Two forms of CTII preserve the structural skeleton as three large loops, including two beta-sheets with bend regions, and demonstrate structural differences at loop I, where cis/trans isomerization occurs. The CTII side-chain distribution constitutes hydrophilic and hydrophobic belts around the protein, alternating in the trend of the three main loops. Because of the Omega-shaped backbone, formed in participation with two bound water molecules, the tip of loop II bridges the tips of loops I and III. This ensures the continuity of the largest hydrophobic belt, formed with the residues of these tips. Comparison revealed pronounced differences in the spatial organization of the tips of the three main loops between CTII and previous structures of homologous cytotoxins (cardiotoxins) in solution.     

Modulation of the lipid binding properties of the N-terminal domain of human apolipoprotein E3.

Apolipoprotein E (apoE) plays a critical role in plasma lipid homeostasis through its function as a ligand for the low-density lipoprotein (LDL) receptor family. Receptor recognition is mediated by residues 130-150 in the independently folded, 22-kDa N-terminal (NT) domain. This elongated globular four-helix bundle undergoes a conformational change upon interaction with an appropriate lipid surface. Unlike other apolipoproteins, apoE3 NT failed to fully protect human LDL from aggregation induced by treatment with phospholipase C. Likewise, in dimyristoylglycerophosphocholine (Myr2Gro-PCho) vesicle transformation assays, 100 microg apoE3 NT induced only 15% reduction in vesicle (250 microg) light scattering intensity after 30 min. ApoE3 NT interaction with modified lipoprotein particles or Myr2Gro-PCho vesicles was concentration-dependent whereas the vesicle transformation reaction was unaffected by buffer ionic strength. In studies with the anionic phospholipid dimyristoylglycerophosphoglycerol, apoE3 NT-mediated vesicle transformation rates were enhanced > 10-fold compared with Myr2Gro-PCho and activity decreased with increasing buffer ionic strength. Solution pH had a dramatic effect on the kinetics of apoE3 NT-mediated Myr2Gro-PCho vesicle transformation with increased rates observed as a function of decreasing pH. Fluorescence studies with a single tryptophan containing apoE3 NT mutant (L155W) revealed increased solvent exposure of the protein interior at pH values below 4.0. Similarly, fluorescent dye binding experiments with 8-anilino-1-naphthalene sulfonate revealed increased exposure of apoE3 NT hydrophobic interior as a function of decreasing pH. These studies indicate that apoE3 NT lipid binding activity is modulated by lipid surface properties and protein tertiary structure.     

Computer modeling of the membrane interaction of FYVE domains

FYVE domains are membrane targeting domains that are found in proteins involved in endosomal trafficking and signal transduction pathways. Most FYVE domains bind specifically to phosphatidylinositol 3-phosphate (PI(3)P), a lipid that resides mainly in endosomal membranes. Though the specific interactions between FYVE domains and the headgroup of PI(3)P have been well characterized, principally through structural studies, the available experimental structures suggest several different models for FYVE/membrane association. Thus, the manner in which FYVE domains adsorb to the membrane surface remains to be elucidated. Towards this end, recent experiments have shown that FYVE domains bind PI(3)P in the context of phospholipid bilayers and that hydrophobic residues on a conserved loop are able to penetrate the membrane interface in a PI(3)P-dependent manner.Here, the finite difference Poisson-Boltzmann (FDPB) method has been used to calculate the energetic interactions of FYVE domains with phospholipid membranes. Based on the computational analysis, it is found that (1) recruitment to membranes is facilitated by non-specific electrostatic interactions that occur between basic residues on the domains and acidic phospholipids in the membrane, (2) the energetic analysis can quantitatively differentiate among the modes of membrane association proposed by the experimentally determined structures, (3) FDPB calculations predict energetically feasible models for the membrane-associated states of FYVE domains, (4) these models are consistent with the observation that conserved hydrophobic residues insert into the membrane interface, and (5) the calculations provide a molecular model for the hydrophobic partitioning: binding of PI(3)P significantly neutralizes positive potential in the region of the hydrophobic residues, which acts as an "electrostatic switch" by reducing the energetic barrier for membrane penetration. Finally, the computational results are extended to FYVE domains of unknown structure through the construction of high quality homology models for human FYVE sequences.     

Interaction of the Water-Soluble Protein Aprotinin with Liposomes: Gel-Filtration,Turbidity Studies,and 31P NMR Studies

Abstract

The interactions of a water-soluble nonmembrane protein aprotinin with multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) from soybean phospholipids were studied using Sephadex G-75 gel chromatography combined with different methods of the analysis of the eluate fractions (fluorescence, light-scattering, turbidity; ³¹P NMR spectroscopy). The composition of the liposomes mainly containing soybean phosphatidylcholine (PC) was varied by the addition of phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lyso-phosphatidylcholine (lyso-PC). To evaluate the lipid-protein interactions, the amount of aprotinin in the MLV–aprotinin complexes was determined. Lipid–protein interactions were found to strongly depend on the liposome composition, medium pH and ionic strength. These dependencies point to the electrostatic nature of the aprotinin-lipid interactions. ³¹P NMR spectroscopy of the MLV–aprotinin complexes indicated that aprotinin influences the phospholipid structure in MLV at pH 3.0. In the case of PC:PE:PI and PC:PE:PI:lyso-PC vesicles, aprotinin induced liposome aggregation and a lamellar-to-isotropic phase transition of the phospholipids.     

Interfacial folding and membrane insertion of a designed helical peptide

Nonconstitutive membrane-active proteins, such as diphtheria toxin, must refold on membrane interfaces in the course of membrane penetration. A useful step in deciphering this process is to understand quantitatively the energetics of interface-mediated insertion of model transmembrane helices. A difficulty is that peptides that are sufficiently hydrophobic to span a lipid bilayer have a strong tendency to aggregate in the aqueous phase. To learn how to control the aqueous and membrane behavior of model peptides, we designed a 31-residue peptide (TMX-3) whose properties are described here. TMX-3 has two important structural features: a proline residue in the hydrophobic core that discourages the formation of highly helical aggregates in solution and two histidine residues that allow control of membrane and solution interactions by means of pH changes. The partitioning of TMX-3 into membranes followed complex kinetics, induced helicity, and shifted the histidine pK(a) from 6.8 to approximately 6. Topology measurements disclosed two general modes of TMX-3 binding: interfacial (IF) at low peptide concentrations and partial transmembrane (TM) insertion at higher concentrations. Both modes were reversible and, consequently, suitable for thermodynamic analysis. The free energies of IF partitioning of TMX-3 with deprotonated (pH 7.6) and protonated histidines (pH 4.5) were estimated by fluorescence titration to be -6.7 and -5.0 kcal/mol, respectively. These results show that histidine titration is likely to be important in the pH-dependent refolding of toxins on membrane interfaces and that the most favored state of TMX-3 under any conditions is the IF folded state, which emphasizes the importance of such states in the spontaneous refolding and insertion of diphtheria and other membrane toxins.     

Mapping the phospholipid-binding surface and translocation determinants of the C2 domain from cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) plays a key role in the generation of arachidonic acid, a precursor of potent inflammatory mediators. Intact cPLA2 is known to translocate in a calcium-dependent manner from the cytosol to the nuclear envelope and endoplasmic reticulum. We show here that the C2 domain of cPLA2 alone is sufficient for this calcium-dependent translocation in living cells. We have identified sets of exposed hydrophobic residues in loops known as calcium-binding region (CBR) 1 and CBR3, which surround the C2 domain calcium-binding sites, whose mutation dramatically decreased phospholipid binding in vitro without significantly affecting calcium binding. Mutation of a residue that binds calcium ions (D43N) also eliminated phospholipid binding. The same mutations that prevent phospholipid binding of the isolated C2 domain in vitro abolished the calcium-dependent translocation of cPLA2 to internal membranes in vivo, suggesting that the membrane targeting is driven largely by direct interactions with the phospholipid bilayer. Using fluorescence quenching by spin-labeled phospholipids for a series of mutants containing a single tryptophan residue at various positions in the cPLA2 C2 domain, we show that two of the calcium-binding loops, CBR1 and CBR3, penetrate in a calcium-dependent manner into the hydrophobic core of the phospholipid bilayer, establishing an anchor for docking the domain onto the membrane.     

Phosphorylation reverses the membrane association of peptides that correspond to the basic domains of MARCKS and neuromodulin.

Several groups have observed that phosphorylation causes the MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) protein to move off cell membranes and phospholipid vesicles. Our working hypothesis is that significant membrane binding of MARCKS requires both hydrophobic insertion of the N-terminal myristate into the bilayer and electrostatic association of the single cluster of basic residues in the protein with acidic lipids and that phosphorylation reverses this electrostatic association. Membrane binding measurements with myristoylated peptides and phospholipid vesicles show this hydrophobic moiety could, at best, barely attach proteins to plasma membranes. We report here membrane binding measurements with basic peptides that correspond to the phosphorylation domains of MARCKS and neuromodulin. Binding of these peptides increases sigmoidally with the percent acidic lipid in the phospholipid vesicle and can be described by a Gouy-Chapman/mass action theory that explains how electrostatics and reduction of dimensionality produce apparent cooperativity. The electrostatic affinity of the MARCKS peptide for membranes containing 10% acidic phospholipids (10(4) M-1 = chi/[P], where chi is the mole ratio of peptide bound to the outer monolayer of the vesicles and [P] is the concentration of peptide in the aqueous phase) is the same as the hydrophobic affinity of the myristate moiety for bilayer membranes. Phosphorylation decreases the affinity of the MARCKS peptide for membranes containing 15% acidic lipid about 1000-fold and produces a rapid (t1/2 < 30 s) dissociation of the peptide from phospholipid vesicles.     

The conserved undecapeptide shared by thiol-activated cytolysins is involved in membrane binding.

Thiol-activated cytolysins share a conserved hydrophobic, Trp-rich undecapeptide that is suggested to be involved in membrane binding and intercalation. The neutralizing monoclonal antibody PLY-5 recognizes all members of this toxin family and peptide mapping assigned its epitope to the undecapeptide motif. This antibody inhibited binding of the toxins to host cell membranes and the epitope was no longer available for binding when a preformed toxin/membrane complex was tested. These results confirm the model of cytolysin binding suggested by structural data.     

Interaction of the water-soluble protein aprotinin with liposomes: gel-filtration, turbidity studies, and 31P NMR studies

The interactions of a water-soluble nonmembrane protein aprotinin with multilamellar vesicles (MLV) and small unilamellar vesicles (SUV) from soybean phospholipids were studied using Sephadex G-75 gel chromatography combined with different methods of the analysis of the eluate fractions (fluorescence, light-scattering, turbidity; 31P NMR spectroscopy). The composition of the liposomes mainly containing soybean phosphatidylcholine (PC) was varied by the addition of phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lyso-phosphatidylcholine (lyso-PC). To evaluate the lipid-protein interactions, the amount of aprotinin in the MLV-aprotinin complexes was determined. Lipid-protein interactions were found to strongly depend on the liposome composition, medium pH and ionic strength. These dependencies point to the electrostatic nature of the aprotinin-lipid interactions. 31P NMR spectroscopy of the MLV-aprotinin complexes indicated that aprotinin influences the phospholipid structure in MLV at pH 3.0. In the case of PC:PE:PI and PC:PE:PI:lyso-PC vesicles, aprotinin induced liposome aggregation and a lamellar-to-isotropic phase transition of the phospholipids.     

Fatty acid transfer from intestinal fatty acid binding protein to membranes: electrostatic and hydrophobic interactions

Intestinal fatty acid binding protein (IFABP) is thought to participate in the intracellular transport of fatty acids (FAs). Fatty acid transfer from IFABP to phospholipid membranes is proposed to occur during protein-membrane collisional interactions. In this study, we analyzed the participation of electrostatic and hydrophobic interactions in the collisional mechanism of FA transfer from IFABP to membranes. Using a fluorescence resonance energy transfer assay, we examined the rate and mechanism of transfer of anthroyloxy-fatty acid analogs a) from IFABP to phospholipid membranes of different composition; b) from chemically modified IFABPs, in which the acetylation of surface lysine residues eliminated positive surface charges; and c) as a function of ionic strength. The results show clearly that negative charges on the membrane surface and positive charges on the protein surface are important for establishing the "collisional complex", during which fatty acid transfer occurs. In addition, changes in the hydrophobicity of the protein surface, as well as the hydrophobic volume of the acceptor vesicles, also influenced the rate of fatty acid transfer. Thus, ionic interactions between IFABP and membranes appear to play a primary role in the process of fatty acid transfer to membranes, and hydrophobic interactions can also modulate the rates of ligand transfer.     

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A² treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment.     

The interaction of trichosanthin with supported phospholipid membranes studied by surface plasmon resonance

Trichosanthin (TCS) is a toxic protein isolated from a Chinese herbal medicine, the root tuber of Trichosanthes kirilowii Maximowicz of the Curcurbitaceae family. It is now used in China to terminate early and mid-trimester pregnancies. The ribosome inactivating property is thought to be account for its toxicity; it can inactivate the eukaryotic ribosome through its RNA N-glycosidase activity. The interactions of TCS with biological membrane is thought to be essential for its physiological effect, for it must get across the membrane before it can enter the cytoplasm and exert its RIP function. In the present work, the interaction of TCS with supported phospholipid monolayers is studied by surface plasmon resonance. The results show that electrostatic forces dominate the interaction between TCS and negatively charged phospholipid containing membranes under acid condition and that both the pH value and the ionic strength can influence its binding. It is proposed that, besides electrostatic forces, hydrophobic interaction may also be involved in the binding process.     

Interaction of diphtheria toxin with model membranes

Low pH is believed to trigger membrane penetration by diphtheria toxin in vivo. The effect of pH upon the binding of the toxin to unilamellar model membrane vesicles was determined by using a fluorescence quenching assay. A series of studies were undertaken to determine the effect of lipid composition upon the binding of lipids to the toxin. The binding of toxin to various small unilamellar vesicles of zwitterionic or anionic lipids was similar in extent and was accompanied by deep penetration of the toxin into the fatty acyl chains, in agreement with previous studies. However, the transition pH, which is the pH at and below which toxin binding becomes significant, depended upon the fraction of anionic lipids, being highest with model membranes composed totally of anionic lipids (pH 5.8) and lowest with membranes composed of zwitterionic lipids (pH 5.2). Except for vesicle charge, the transition pH was independent of the nature of the lipid polar groups used. High ionic strength, which had no effect on the transition pH with zwitterionic vesicles, was found to shift the transition pH with totally anionic vesicles to pH 5.2. This suggests that both direct protein-lipid electrostatic interactions and the ionic double layer, which gives rise to a low local pH around anionic vesicles, contribute to the shift in the transition pH. The effect of lipid composition upon the kinetics and strength of binding was also examined. At low pH, binding was rapid and tight. Binding to vesicles containing 20 wt % anionic phosphatidylglycerol was faster and tighter than binding to vesicles of zwitterionic phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)