Interaction of trypsin with multilamellar vesicles of soybean lipids

The pH dependence of complex formation of trypsin with multilamellar vesicles (MLV) of soybean lipids has been investigated. The lipids were characterized by the same phospholipid composition, but the content of other lipids differed. Decrease of pH or introduction of negatively charged components into the lipid samples increased trypsin content in the protein-lipid complexes. This suggests electrostatic interaction between the protein and soybean lipids. The dependence of trypsin activity in the complexes with MLV on their concentration and on the presence of an ionic detergent was studied. Trypsin-MLV interaction did not result in complete inactivation of the protein molecules. Moreover, the effects of dilution and addition of ionic detergent on trypsin activity were additive. Using a fluorescence technique, complex formation with MLV was found to stabilize trypsin molecules, preventing their autolysis.     

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.     

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.     

Mass spectrometry-based footprinting of protein–protein interactions

We present a high-resolution mass spectrometric (MS) footprinting method enabling identification of contact amino acids in protein–protein complexes. The method is based on comparing surface topologies of a free protein versus its complex with the binding partner using differential accessibility of small chemical group selective modifying reagents. Subsequent MS analysis reveals the individual amino acids selectively shielded from modification in the protein–protein complex. The current report focuses on probing interactions between full-length HIV-1 integrase and its principal cellular partner lens epithelium-derived growth factor. This method has a generic application and is particularly attractive for studying large protein–protein interactions that are less amenable for crystallographic or NMR analysis.     

Hybridization by cosonication of pigeon erythrocyte membrane with exogenous lipid vesicles

Summary Concentrated mixtures of lipid vesicles and pigeon erythrocyte membrane were cosonicated in order to produce functional hybrid vesicles. From the properties of the resulting material, we conclude that hybrids were very probably formed. These properties were as follows: (i) The presence of membrane increased the sonic fragmentability of lipid vesicles. Sonic fragmentability was assessed by measuring sonication-induced release of previously trapped [¹⁴C]-choline and trapping of external [³H]-choline. (ii) Space enclosed by lipid was served by the membrane-like properties of³⁶Cl^– permeability and ATP-dependent⁴⁵Ca⁺⁺ uptake activity. (iii)³⁶Cl-permeability was more readily and fully induced into the more easily fragmented lipid vesicles. Further sonication caused loss of the induced³⁶Cl^–-permeability. This loss was less rapid with the less easily fragmented lipid vesicles; i.e., less easily fragmented lipids protected³⁶Cl^–-permeability better. (iv) Glycine uptake activity was partially protected from sonic damage by the presence of lipid vesicles. (v) On centrifugation in bovine serum albumin density gradients, cosonicated material showed lipid properties (enclosed choline and³²P_i space and [³H]-cholesterol) and membrane properties (³⁶Cl^–-permeability and ATP-dependent⁴⁵Ca²⁺ uptake) coinciding at a density intermediate between those reached by separately sonicated membrane and lipid vesicles. (vi) Electron micrographs showed the disappearance of pure membrane-like structures and the appearance of large amounts of new vesicles whose appearance is consistent with a hybrid structure.     

Liposomes induce chromosome aberrations in human cultured cells

The genotoxic effect of multilamellar lipid vesicles (MLV) was analysed on cultured heteroploid and diploid human cells. Dose-dependent reduction of cell survival and mitotic rate as well as induction of chromosome aberrations were observed. Chromatid and chromosome breaks and chromatid exchanges were found in 24-h culture after liposome treatment, whereas chromosome rearrangements were prevalent at 48 h. Neutral (PC/Chol) and positive (PC/SA) MLV showed a greater damage than negative (PS/PC; PS) MLV. Fibroblasts were the most sensitive cell type. In the case of PC/Chol MLV vesicles, control experiments with PC and Chol of controlled purity ruled out the possibility that the observed chromosome aberrations were caused by toxic oxidation products present in commercial preparations.     

Effects of polyphenol oxidase on lipolysis and proteolysis of red clover silage with and without a silage inoculant (Lactobacillus plantarum L54)

Polyphenol oxidase (PPO) has been shown to reduce proteolysis and lipolysis in red clover through deactivation of proteolytic and lipolytic enzymes and/or through formation of protein–phenol–lipid complexes. This experiment investigated the time course of both lipolysis and proteolysis in two red clover lines with different PPO activities with and without addition of a silage inoculant to help understand the action of PPO in the silo, and its potential effects on protein and glycerol-based lipid conservation, and to determine effects of a more rapid pH reduction with inoculation on PPO activity. Four silages were prepared from high or low PPO precision chopped red clover in 60 test-tube-silos, each containing 120 g fresh weight: (a) high PPO red clover without inoculation (H−), (b) low PPO red clover without inoculation (L−), (c) high PPO red clover with inoculation (H+), and (d) low PPO red clover with inoculation (L+). Each treatment had three replicates for each time point of 1, 2, 4, 8 and 90 days. The inoculant used was Lactobacillus plantarum strain L54 applied at a rate of 10⁶ CFU/g fresh weight. Silage pH was reduced (P < 0.001) by inoculation with no effect of PPO. Inoculation had no effect on either lipolysis or free amino acid release, although more (P < 0.01) soluble protein and less (P < 0.01) ammonia-N was in inoculated silages. H silages had a lower level of both proteolysis (release of free amino acids, P < 0.05) and lipolysis (loss of membrane lipid, P < 0.01) than L red clover silages. Results indicate that PPO reduced proteolysis and lipolysis in the silo and that inoculation had no adverse effects on PPO activity.     

Engineering trypsin for inhibitor resistance

The development of effective protease therapeutics requires that the proteases be more resistant to naturally occurring inhibitors while maintaining catalytic activity. A key step in developing inhibitor resistance is the identification of key residues in protease-inhibitor interaction. Given that majority of the protease therapeutics currently in use are trypsin-fold, trypsin itself serves as an ideal model for studying protease-inhibitor interaction. To test the importance of several trypsin-inhibitor interactions on the prime-side binding interface, we created four trypsin single variants Y39A, Y39F, K60A, and K60V and report biochemical sensitivity against bovine pancreatic trypsin inhibitor (BPTI) and M84R ecotin. All variants retained catalytic activity against small, commercially available peptide substrates [k_cat/K_M = (1.2 ± 0.3) × 10⁷ M⁻¹ s⁻¹. Compared with wild-type, the K60A and K60V variants showed increased sensitivity to BPTI but less sensitivity to ecotin. The Y39A variant was less sensitive to BPTI and ecotin while the Y39F variant was more sensitive to both. The relative binding free energies between BPTI complexes with WT, Y39F, and Y39A were calculated based on 3.5 µs combined explicit solvent molecular dynamics simulations. The BPTI:Y39F complex resulted in the lowest binding energy, while BPTI:Y39A resulted in the highest. Simulations of Y39F revealed increased conformational rearrangement of F39, which allowed formation of a new hydrogen bond between BPTI R17 and H40 of the variant. All together, these data suggest that positions 39 and 60 are key for inhibitor binding to trypsin, and likely more trypsin-fold proteases.     

Protein-RNA interactions: a structural analysis

A detailed computational analysis of 32 protein–RNA complexes is presented. A number of physical and chemical properties of the intermolecular interfaces are calculated and compared with those observed in protein–double-stranded DNA and protein–single-stranded DNA complexes. The interface properties of the protein–RNA complexes reveal the diverse nature of the binding sites. van der Waals contacts played a more prevalent role than hydrogen bond contacts, and preferential binding to guanine and uracil was observed. The positively charged residue, arginine, and the single aromatic residues, phenylalanine and tyrosine, all played key roles in the RNA binding sites. A comparison between protein–RNA and protein–DNA complexes showed that whilst base and backbone contacts (both hydrogen bonding and van der Waals) were observed with equal frequency in the protein–RNA complexes, backbone contacts were more dominant in the protein–DNA complexes. Although similar modes of secondary structure interactions have been observed in RNA and DNA binding proteins, the current analysis emphasises the differences that exist between the two types of nucleic acid binding protein at the atomic contact level.     

An ESR Study of the Cytotoxin II Interaction with Model Membranes

Cytotoxin II from the venom of the Central-Asian cobra Naja oxianaspin-labeled at Lys35 (SLCT II) was studied by ESR spectroscopy in aqueous solution and upon interaction with phospholipid vesicles from egg phosphatidylcholine or its mixture with dimyristoylphosphatidylglycerol (molar ratio 9 : 1). The distribution of SLCT II between the aqueous and lipid phases depended on the toxin and lipid concentrations and on the solution ionic strength. It was analyzed using the modified Gouy–Chapman theory that takes into account different charges of the cytotoxin in solution and in membrane. The analysis revealed two states of the cytotoxin–lipid complex. The first state corresponds to monomeric SLCT II hydrophobically interacting with the lipid membrane [a binding constant of (8 ± 3) × 10³M^–1] and carrying the charge of 4.4 ± 0.3. On the basis of these parameters and the spatial structure of cytotoxin II in dodecylphosphocholine micelles, we concluded that the cytotoxin is mainly incorporated into the region of polar groups of the lipid bilayer. The second state of SLCT II is realized at high cytotoxin concentrations in the membrane and corresponds to the formation of toxin–lipid complexes that disorganize the membrane bilayer structure.     

Microsphere-based co-immunoprecipitation in multiplex

Co-immunoprecipitation (co-IP) is a prominent technique for evaluating protein–protein interactions. Currently, large quantities of protein are required to perform co-IP followed by mass spectrometric or Western blot analyses of the interacting proteins. Here catenin–cadherin complexes were employed to establish a multiplexed microsphere-based co-immunoprecipitation (μco-IP) protocol that allows the analysis of different complexes of a given protein with various interacting proteins within a single experiment using a limited amount of sample material.     

GUVs Melt Like LUVs: The Large Heat Capacity of MLVs Is Not Due to Large Size or Small Curvature

The excess heat capacity functions (ΔC_p) associated with the main phase transition of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs) are very different. Two explanations are possible. First, the difference in vesicle size (curvature) results in different gel-fluid interactions in the membrane; those interactions have a large effect on the cooperativity of the phase transition. Second, there is communication between the bilayers in an MLV when they undergo the gel-fluid transition; this communication results in thermodynamic coupling of the phase transitions of the bilayers in the MLV and, consequently, in an apparent increase in the cooperativity of the transition. To test these hypotheses, differential scanning calorimetry was performed on giant unilamellar vesicles (GUVs) of pure dipalmitoylphosphatidylcholine. The ΔC_p curve of GUVs was found to resemble that of the much smaller LUVs. The transition in GUVs and LUVs is much broader (half-width ∼1.5°C) than in MLVs (∼0.1°C). This similarity in GUVs and LUVs indicates that their size has little effect on gel-fluid interactions in the phase transition. The result suggests that coupling between the transitions in the bilayers of an MLV is responsible for their apparent higher cooperativity in melting.     

Structure of DNA Complexes with Nonhistone Chromosomal Protein HMGB1 in the Presence of Manganese Ions

The complexes of DNA–HMGB1 protein–manganese ions have been studied using the circular dichroism (CD) technique. It was shown that the interactions of both the protein and metal ions with DNA in this three-component system differ from those in two-component complexes. The manganese ions did not affect the CD spectrum of the free HMGB1 protein. However, Mn²⁺ ions induced considerable changes in the CD spectrum of free DNA in the spectral range of 260–290 nm. The presence of Mn²⁺ ions prevented the formation of the ordered supramolecular structures typical of HMGB1–DNA complexes. The interaction of manganese ions with DNA had a marked influence on the local DNA structure, changing the properties of protein-binding sites and resulting in a marked decrease in cooperativity of HMGB1–DNA binding. Such changes in the mode of protein–DNA interactions occurred at concentrations as small as 0.01 mM Mn²⁺. Moreover, the changes in local DNA structure induced by the manganese ions promoted the appearance of new HMGB1 binding sites in the DNA double helix. At the same time, interactions with the HMGB1 protein induced alterations in the structure of the DNA double helix, which increased with an increase in the protein-to-DNA ratio. These alterations made the DNA–protein complex especially sensitive to manganese ions. Under these conditions the Mn²⁺ ions strongly affected the DNA structure, which was reflected in abrupt changes in the CD spectra of DNA in the complex in the range of 260–290 nm. Thus, structural changes in the DNA double helix in three-component DNA–HMGB1–Mn²⁺ complexes result from the combined and interdependent interactions of DNA with Mn²⁺ ions and HMGB1 molecules.     

Quaternary structure predictions and estimation of mutational effects on the free energy of dimerization of the OMPLA protein

This study represents an extension to the outer membrane phospholipase A protein (OMPLA) of the docking-based protocols previously developed for quaternary structure predictions of transmembrane oligomeric proteins and for estimating mutational effects on the thermodynamics of protein–protein and protein–DNA association.Predictions of the likely architecture of OMPLA homo-dimers were carried out on 31 different forms of the monomer, 30 of which were variants of the unbound state. In all the test cases but the ones characterized by combined deletions of the 98–110 and 145–153 segments (L2 and L3, respectively), native-like complexes could be predicted, independent of the bound or unbound state of the structural model, of side chain conformation and presence or absence of amino acid deletions at the putative inter-monomer interface.The protocol for estimating mutational effects on the thermodynamics of protein–protein association proved effective as well. In fact, it was possible to estimate correctly the effects of five mutants on the free energy of dimerization of the sulfonylated form of OMPLA.The integrity of L2 and either one of the L1, L3 and L4 loops turned out to be more important than sulfonylation for the achievement of the native dimeric architecture. On the other hand, sulfonylation seems to be essential for a favorable dimerization energetics.     

Prediction of yeast protein-protein interaction network: insights from the Gene Ontology and annotations

A map of protein–protein interactions provides valuable insight into the cellular function and machinery of a proteome. By measuring the similarity between two Gene Ontology (GO) terms with a relative specificity semantic relation, here, we proposed a new method of reconstructing a yeast protein–protein interaction map that is solely based on the GO annotations. The method was validated using high-quality interaction datasets for its effectiveness. Based on a Z-score analysis, a positive dataset and a negative dataset for protein–protein interactions were derived. Moreover, a gold standard positive (GSP) dataset with the highest level of confidence that covered 78% of the high-quality interaction dataset and a gold standard negative (GSN) dataset with the lowest level of confidence were derived. In addition, we assessed four high-throughput experimental interaction datasets using the positives and the negatives as well as GSPs and GSNs. Our predicted network reconstructed from GSPs consists of 40753 interactions among 2259 proteins, and forms 16 connected components. We mapped all of the MIPS complexes except for homodimers onto the predicted network. As a result, ~35% of complexes were identified interconnected. For seven complexes, we also identified some nonmember proteins that may be functionally related to the complexes concerned. This analysis is expected to provide a new approach for predicting the protein–protein interaction maps from other completely sequenced genomes with high-quality GO-based annotations.     

Incorporation of the Lipopolysaccharide and Polysaccharide from Aeromonas Salmonicida Into Liposomes

Abstract

Incorporation of the lipopolysaccharide (LPS) and polysaccharide (PS) from Aeromonas salmonicida into liposomes of varying lipid composition and lamellarity as a function of the LPS and PS concentration was investigated. Positively-charged multilamellar vesicles (MLV) composed of phosphatidylcholine (PC): cholesterol (CH): stearylamine (SA) (6:3:1, mole: mole: mole) incorporated the LPS more readily than negatively-charged liposomes composed of PC: CH: phosphatidylglycerol (PG) in the same molar ratios. Regardless of surface charge, more LPS was incorporated into MLV than into vesicles prepared by relatively mild sonication (SV) or large unilamellar vesicles prepared via extrusion through 200 nm pore size filters (LUVET₂₀₀). In contrast, SV and LUVET₂₀₀ incorporated more PS than did MLV. The total amount of liposomally-incorporated LPS or PS among the three vesicle types was proportional to the concentration of antigen in the hydrating solutions.     

Protective Role of the Polysaccharide-containing Capsular Components of Azospirillum brasilense

The involvement of the carbohydrate components of the Azospirillum brasilenseSp245 capsules in bacterial protection from the action of extreme factors was investigated. The survival of encapsulated and non-encapsulated azospirilla exposed to elevated (46–48°C) and below-freezing (–20 and –70°C) temperatures, extreme pH values (2 and 10), and to drying was studied. High-molecular-weight carbohydrate-containing complexes (lipopolysaccharide–protein complex and polysaccharide–lipid complex) were isolated from the capsular material of azospirilla. It was shown that the addition of these complexes to the suspension of decapsulated cells before exposing them to extreme factors enhanced their survival rates by 15 to 51%.     

Physicochemical Properties and Specific Structural Features of Protein–Lipid Composites of Increased Nutritive Value

Fractional and component compositions of protein–lipid composites with increased nutritive value (compared to the protein preparations from which they were produced) were studied based on solubility and electrophoretic behavior. Differences in the fractional compositions of proteins and the amounts of hydrogen, ionic, and hydrophobic bonds were found. It was demonstrated that the water-, salt-, and alkali-soluble fractions of proteins changed during the manufacturing of the composites with soybean and wheat bran flour; the water- and alkali-soluble fractions, with protein concentrate from bran. Heterogeneity of the compositions and specific conformational features of composite proteins resulting from disulfide bonds were found. It was demonstrated that, during the manufacturing of composites, proteins of soybean flour aggregated (with the involvement of disulfide bonds), whereas protein products from wheat bran disaggregated. Breaks of interchain (wheat) or intrachain (concentrate) disulphide bonds accompanied the disaggregation. Overall the properties and specific structural features of the protein–lipid composites studied depended on the nature of the protein (soybean or wheat), type of initial preparations (flour or concentrate), and method of their production (emulsifying or drying).     

Phospholipid-Dependence of Plant UDP-Glucose Sterol beta-d-Glucosyl Transferase : IV. Reconstitution into Small Unilamellar Vesicles

The phospholipid dependence of the UDP-glucose sterol glucosyl transferase (UDPG-SGTase) from maize coleoptiles was previously demonstrated using the partially purified and highly delipidated enzyme, in the presence of the detergent Triton X-100 (P Ullmann, P Bouvier-Navé, P Benveniste [1987] Plant Physiol 85: 51-55). We now report the reconstitution of the enzyme activity into unilamellar lipid vesicles. This was achieved by adding phospholipids, sterols and β-octylglucoside to the solubilized enzyme and passing the mixture through Sephadex G-50. The treatment led to almost complete removal of the detergents. The incorporation of UDPG-SGTase in the lipid vesicles was demonstrated by (a) coelution of the enzyme activity with the labeled lipid vesicles (average diameter: 260Å) on a Sephacryl S-1000 column and (b) flotation experiments on metrizamide density gradients. Release of dithiobis-(2-nitro-benzoic acid) (DTNB) from DTNB-preloaded vesicles was very slow, indicating good membrane integrity of the vesicles. Treatment of the intact vesicles with the nonpermeant reagent p-chloro-mercuribenzene sulfonate led to more than 95% inactivation of the total enzyme activity, i.e. the activity measured in the presence of Triton X-100 at permeabilizing concentration. This suggests an outward orientation for the active site of the enzyme. Finally, the enzyme was incorporated into vesicles of various phospholipid compositions and the kinetic parameters of the reactions were determined. Our results clearly show that the reconstituted UDPG-SGTase activity is stimulated to a large extent by negatively charged phospholipids.