Characterization and reassembly of a regular array in the cell wall of Clostridium difficile GAI 4131

The cell wall of Clostridium difficile GAI 4131 was revealed by electron microscopy to have an outer layer composed of a nearly square array and contained the two major proteins with molecular weights of 38 kDa and 42 kDa. The properties and reassembly of the two major proteins into the regular array were investigated. When the isolated cell walls were treated with hydrophobic bond-disrupting agents or a chelating agent specific for Ca2+, the two major proteins were effectively removed and the regularly arranged outer layer disappeared. The amino acid composition of the two major proteins differed from each other. The two major proteins also gave different peptide maps from each other upon proteolysis with Staphylococcus aureus V8 protease. The major proteins solubilized from the isolated cell walls with 8 M urea or 4 M guanidine hydrochloride could be reassembled into open-ended cylinders possessing the native regular pattern by dialysis against neutral buffer containing 5 mM CaCl2. The reassembled cylinders purified by centrifugation on a Percoll density gradient were composed of almost equal amounts of the 38 kDa and 42 kDa proteins and freed from the other proteins. These results suggest that the regular array in the outer cell wall layer is constructed from the two major cell wall proteins and requires Ca2+ for its assembly.     

Antibodies to 5 M urea soluble chromosomal proteins from HeLa cells

The tissue specificity of a chromosomal protein fraction, extractable from chromatin with 5 M urea at low ionic strength, has been examined in HeLa, A549 and HT 29 cells. Electrophoresis in polyacrylamide gels indicates that each cell type has a different content of 5 M urea soluble proteins which are distinguishable from the histones, from the tight DNA-binding proteins and from the high-mobility-group chromosomal proteins. Antibodies against 5 M urea soluble proteins extracted from HeLa cells were produced in mice. Although each of the mice tested prior to immunization contained a detectable amount of antibodies against both the 5 M urea soluble proteins and tight DNA-binding proteins, immunization elevated the level of the antibodies in the serum over 100-fold. The antibodies do not distinguish between the 5 M urea extracts obtained from different sources because most of the antibodies are directed against antigens shared by the cells studied. Immunofluorescence studies reveal that components which cross-react with 5 M urea soluble chromosomal proteins are also present in the cytoplasm. We conclude the following. (1) 5 M urea extracts from chromatin a group of proteins which differs among cells. (2) Mice contain detectable amounts of autoantibodies against these chromosomal proteins. (3) Immunization with the 5 M urea extractable fraction elicits antibodies against a restricted number of antigenic components which are shared among the cells studied. (4) 5 M urea extractable proteins are found both in the nucleus and cytoplasm; part of these may be cytoskeletal elements. Because the antisera do not react with histones, high-mobility-group proteins and tight DNA-binding proteins, they may be used for various functional studies on the 5 M urea extractable chromosomal protein fraction.     

Composition and DNA-binding properties of the nuclear matrix proteins from mammalian cell nuclei

A rapidly sedimenting DNA-protein complex was isolated from nuclear lysates in 2 M NaCl and characterized with regard to its polypeptide composition and the DNA-binding properties of the purified proteins. The complex consists of the nuclear matrix with attached DNA. Electrophoresis in SDS-polyacrylamide gels revealed two major and five minor polypeptide bands, mainly in the 60 to 75 kDa molecular weight region. The DNA-matrix complex dissociated into free DNA and proteins in the presence of 2 M NaCl and 5 M urea. The proteins could be purified by chromatography on hydroxyapatite and showed a strong tendency to reassociate at 0.15 M NaCl concentration in the absence of urea. DNA was bound to the reassociated proteins at 0.15 M NaCl concentration. Part of the DNA-protein complex was stable at 1 M NaCl concentration. The binding appeared to be random with regard to the DNA sequence.     

Proteins C23 and B23 are the major nucleolar silver staining proteins.

To examine the silver staining proteins of Novikoff hepatoma nucleoli, the nucleolar proteins were separated on two-dimensional polyacrylamide gels with an isoelectric focusing first dimension and an acid-urea gel second dimension. The nucleoli were sequentially extracted with (1) 0.6 M potassium acetate, pH 5.5 and (2) 2 M potassium acetate — 5 M urea — 10 mM Tris, pH 7.5. The silver staining method used for the detection of silver binding proteins in gels was similar to that used to stain the nucleolar granules on microscope slides. Two major silver staining proteins were found which were identified as (molecular weight × 10^?3/pI) proteins C23 (100/5.3) and B23 (37/5.1). These two proteins are the major acidic proteins in Novikoff hepatoma nucleoli.     

The procuticle of Drosophila: heterogeneity of urea-soluble proteins.

Proteins, soluble in 7 M urea, 4 M guanidine hydrochloride, or 2% sodium dodecyl sulfate, have been extracted from untanned larval cuticles of Drosophila melanogaster. A major protein fraction, apparent molecular weight 8000 - 10 000, is resolved into eight different components (five major, three minor) by gradient gel electrophoresis under nondenaturing conditions. Proteins extracted in 7 M urea have been resolved by diethylaminoethylcellulose chromatography into five fractions, three of which are greatly enriched for electrophoretically homogeneous proteins. The five fractions have different amino acid compositions. Electrophoretic variants involving four of the five major proteins have been obtained. Preliminary genetic analysis indicates that at least three of the five proteins are specified by separate structural genes.     

Calcium-binding proteins in the vorticellid spasmoneme

The proteins of the contractile spasmoneme from Vorticella convallaria, Carcheslium polypinum, and Zoothamnium geniculatum have been extracted in the detergent, sodium dodecyl sulfate (SDS), as well as urea and guanidine hydrochloride (GuCl). After SDS extraction, the molecular weight distribution of the proteins was examined by means of SDS-polyacrylamide gel electrophoresis. Significant amounts of material corresponding to the contractile proteins actin and tubulin are not present. The contractile organelles in the three species examined contain a group of closely related proteins of molecular weight near 20,000, which constitute a major part (40-60%) of the dry mass. The 20,000 mol wt proteins in Zoothamnium bind calcium with high affinity (pK congruent to 6) and are termed "spasmins." By means of urea polyacrylamide gel electrophorsis, it is demonstrated that in Carchesium and Zoothamnium certain spasmin components bind calcium even in the presence of 6 M urea. The binding of calcium in 6 M urea suggests a functional relationship between the spasmins and the calcium-binding proteins of striated muscle which behave similarly. The calcium binding in urea also indicates that the spasmins within a single spasmoneme have different calcium affinities, and this difference in calcium-binding properties may be an important factor in the physiological function of the organelle.     

Optimization of inclusion body solubilization and renaturation of recombinant human growth hormone from Escherichia coli

Recombinant human growth hormone (r-hGH) was expressed in Escherichia coli as inclusion bodies. In 10 h of fed-batch fermentation, 1.6 g/L of r-hGH was produced at a cell concentration of 25 g dry cell weight/L. Inclusion bodies from the cells were isolated and purified to homogeneity. Various buffers with and without reducing agents were used to solubilize r-hGH from the inclusion bodies and the extent of solubility was compared with that of 8 M urea as well as 6 M Gdn-HCl. Hydrophobic interactions as well as ionic interactions were found to be the dominant forces responsible for the formation of r-hGH inclusion bodies during its high-level expression in E. coli. Complete solubilization of r-hGH inclusion bodies was observed in 100 mM Tris buffer at pH 12.5 containing 2 M urea. Solubilization of r-hGH inclusion bodies in the presence of low concentrations of urea helped in retaining the existing native-like secondary structures of r-hGH, thus improving the yield of bioactive protein during refolding. Solubilized r-hGH in Tris buffer containing 2 M urea was found to be less susceptible to aggregation during buffer exchange and thus was refolded by simple dilution. The r-hGH was purified by use of DEAE-Sepharose ion-exchange chromatography and the pure monomeric r-hGH was finally obtained by using size-exclusion chromatography. The overall yield of the purified monomeric r-hGH was approximately 50% of the initial inclusion body proteins and was found to be biologically active in promoting growth of rat Nb2 lymphoma cell lines.     

The physical state of water in living cells and model systems. XII. The influence of the conformation of a protein on the solubility of Na+ (sulfate), sucrose, glycine and urea in the water in which the protein is also dissolved

In this report, we describe the result of an extensive investigation of the effects of the conformations of proteins on the solvency of the bulk-phase water in which the proteins are dissolved. The concentrations of the proteins used were usually between 20 to 40%; the temperature was 25 degrees +/- 1 degree C. To probe the solvency of the water, the apparent equilibrium distribution coefficients (or p-values) of 4 solutes were studied: Na+ (sulfate), glycine, sucrose, and urea. From 8 to 14 isolated proteins in three types of conformations were investigated: native; denatured by agents that unravel the secondary structure (e.g., alpha-helix, beta-pleated sheet) of the protein (i.e., 9 M urea, 3 M guanidine HCl); denatured by agents that only disrupt the tertiary structure but leave the secondary structure intact or even strengthened (i.e., 0.1 M sodium dodecylsulfate or SDS, 2 M n-propanol). The results are as follows: (1) as a rule, native proteins have no or weak effect on the solvency of the water for all 4 probes; (2) exposure to 0.1 M SDS and to 2 M n-propanol, as a rule, does not significantly decrease the p-value of all 4 probes; (3) exposure to 9 M urea and to 3 M guanidine HCl consistently lowers the p-values of sucrose, glycine and Na+ (sulfate) and equally consistently produces no effect on the p-value of urea. Sucrose, glycine, and Na+ are found in low concentrations in cell water while urea is not. These experiments were designed and carried out primarily to test two subsidiary theories of the AI hypotheses: the polarized multilayer (PM) theory of cell water; and the theory of size-dependent solute exclusion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biophysical and structural characterization of the small heat shock protein HspA from Thermosynechococcus vulcanus in 2 M urea

Small heat shock proteins (sHSPs) belong to the superfamily of molecular chaperones. They prevent aggregation of partially unfolded or misfolded client proteins, providing protection to organisms under stress conditions. Here, we report the biophysical and structural characterization of a small heat shock protein (HspA) from a thermophilic cyanobacterium Thermosynechococcus vulcanus in the presence of 2 M urea. HspA has been shown to be important for the protection of Photosystem II and the Phycobilisome antenna complex at elevated temperatures. Heterologously expressed HspA requires the presence of 1–2 M urea to maintain its solubility at concentrations required for most characterization methods. Spectroscopic studies reveal the presence of the β-sheet structure and intactness of the tertiary fold in HspA. In vitro assays show that the HspA maintains chaperone-like activity in protecting soluble proteins from thermal aggregation. Chromatography and electron microscopy show that the HspA exists as a mixture of oligomeric forms in the presence of 2 M urea. HspA was successfully crystallized only in the presence of 2 M urea. The crystal structure of HspA shows urea-induced loss of about 30% of the secondary structure without major alteration in the tertiary structure of the protein. The electron density maps reveal changes in the hydrogen bonding network which we attribute to the presence of urea. The crystal structure of HspA demonstrates a mixture of both direct interactions between urea and protein functionalities and interactions between urea and the surrounding solvent that indirectly affect the protein, which are in accordance with previously published studies.     

Soybean disease resistance protein RHG1-LRR domain expressed, purified and refolded from Escherichia coli inclusion bodies: preparation for a functional analysis

Introduction and expression of foreign genes in bacteria often results accumulation of the foreign protein(s) in inclusion bodies (IBs). The subsequent processes of refolding are slow, difficult and often fail to yield significant amounts of folded protein. RHG1 encoded by rhg1 was a soybean (Glycine max L. Merr.) transmembrane receptor-like kinase (EC 2.7.11.1) with an extracellular leucine-rich repeat domain. The LRR of RHG1 was believed to be involved in elicitor recognition and interaction with other plant proteins. The aim, here, was to express the LRR domain in Escherichia coli (RHG1-LRR) and produce refolded protein. Urea titration experiments showed that the IBs formed in E. coli by the extracellular domain of the RHG1 protein could be solubilized at different urea concentrations. The RHG1 proteins were eluted with 1.0-7.0M urea in 0.5M increments. Purified RHG1 protein obtained from the 1.5 and 7.0M elutions was analyzed for secondary structure through circular dichroism (CD) spectroscopy. Considerable secondary structure could be seen in the former, whereas the latter yielded CD curves characteristic of denatured proteins. Both elutions were subjected to refolding by slowly removing urea in the presence of arginine and reduced/oxidized glutathione. Detectable amounts of refolded protein could not be recovered from the 7.0M urea sample, whereas refolding from the 1.5M urea sample yielded 0.2mg/ml protein. The 7.0M treatment resulted in the formation of a homogenous denatured state with no apparent secondary structure. Refolding from this fully denatured state may confer kinetic and/or thermodynamic constraints on the refolding process, whereas the kinetic and/or thermodynamic barriers to attain the folded conformation appeared to be lesser, when refolding from a partially folded state.     

Larval cuticle proteins of Lucilia cuprina: Electrophoretic separation,quantification and developmental changes

Proteins from isolated cuticles of third instar larvae of the sheep blowfly, Lucilia cuprina, have been solubilized with water or 7 M urea or 2% SDS. While 7 M urea or 2% SDS extract significantly more protein than water, the same major proteins, in the same relative proportions, are extracted by all three solutions. More than 80% of the cuticular protein is extracted by 7 M urea or 2% SDS. Extracted proteins resolve into nine major bands when analysed by gradient polyacrylamide gel electrophoresis. These proteins are anionic, relatively low in molecular weight (13–28 kd) and are essentially free of carbohydrate. Only minor differences exist between the proteins of two morphologically distinct cuticular regions. Cuticle proteins, extracted from larvae at different developmental stages (first, second and third instars) display quantitatively and qualitatively unique electrophoretic profiles. A number of proteins are common to all stages however. The electrophoretic profiles of proteins extracted from larval cuticles at various times within an instar also differ although the differences are largely quantitative. This is particularly evident during the transition from the feeding to the wandering stages of the third instar; the weight of the cuticle relative to that of the larva increases and this is accompanied by marked changes in the electrophoretic profile of the cuticle proteins.     

Interrelation between the charge isoforms of mammalian ornithine decarboxylase

Ornithine decarboxylase (ODC) isolated from a variety of tissues has been separated, using DEAE ion-exchange chromatography, into multiple peaks of activity that appear to be related to control of this enzyme stability. Reports of these charge isoforms in current literature are generally unclear as to whether these represent a covalent posttranslational modification or merely an alteration in structural conformation or association. In this study we investigated the relationship of this form separation to the degree of enzyme polymerization, interaction with other proteins and buffer components, and the multiple isoelectric forms of this enzyme noted in denaturing concentrations of urea. High-performance chromatography techniques were used to demonstrate that two of the major enzyme forms, ODC I and II, are really monomers of the enzyme, while minor peaks of activity frequently observed to elute after ODC II contain various dimeric enzyme states. Pyridoxal 5'-phosphate (0.05 mM) added to isolated enzyme preparations composed of I and II monomers induced the formation of I and II dimers as well as a mixed I-II dimer. All three dimer forms were observed to be natural components of freshly isolated crude cell homogenates. The charge distinction between the monomer forms I and II was found to be maintained during ion-exchange chromatography in the presence of 8 M urea, and the enzyme isoforms demonstrated distinct bands on isoelectric focusing gels run in the presence of 9 M urea. Thus, although some of the multiple ornithine decarboxylase forms identified by ion-exchange chromatography of crude mammalian cell homogenates are related to enzyme conformation, the two major forms are distinctly charged protein states that can be visualized using two-dimensional gel electrophoresis of highly purified samples.     

Biochemical properties of phosphatidylcholine-binding proteins that share common antigenic determinants with Fc gamma 2b receptor

Biochemical and immunological properties of biosynthetically radiolabeled phosphatidylcholine-(PC-) binding proteins were investigated. The PC-binding proteins were extracted from the detergent lysate of biosynthetically radiolabeled P388D1 cells by affinity chromatography on PC-Sepharose and filtered through a Sephadex G-100 gel column in the presence of 6 M urea. Isoelectric focusing of the gel-filtered materials in the presence of 6 M urea revealed the presence of a major protein component of pIe of 5.8 and minor heterogeneous cellular proteins. The yield of the electrofocused PC-binding proteins based on protein determination by Lowry's method ranged from 0.7 to 4 mg per 10(9) cells. The purified PC-binding proteins appeared to be tightly associated with Triton X-100 and phospholipids in the weight ratio of 0.57 and 0.05 g/g of proteins, respectively. The majority of lipids that could be extracted from the PC-binding proteins by chloroform/methanol (2:1 v/v) are free fatty acids, whereas lipids extracted from Pronase-treated PC-binding proteins contained phosphatidylethanolamine. By amino acid analysis, the purified PC-binding proteins were found to consist of a minimum of 417 amino acid residues, suggesting a minimum molecular weight of about 38 000 for this protein. Results of radiolabeling experiments with [3H]glucosamine and amino acid analysis both showed the presence of a mole of glucosamine per a mole of the PC-binding proteins, suggesting their glycoprotein nature. About 40% of the purified PC-binding proteins coprecipitated with monoclonal anti-Fc gamma 2bR antibody (2.4G2) in detergent-containing buffer, whereas only 6% of the isolated IgG binding proteins reacted with this antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteins of Bacillus thuringiensis delta-endotoxin crystals]

Pure crystals (at least 99% purification) of sigma-endotoxin were isolated from Bac. thuringiensis var. galleriae. The complete dissolution of crystals might be achieved by the increase of pH up to 12 and higher or by a combined action of S = S-reducing and denaturing agents. Electrophoresis of the solubilized crystal proteins in 5% polyacrylamide gels containing 0,1% sodium dodecyl sulfate and 8 M urea reveals two major bands corresponding to molecular weights of 120000--140000 (65%) and 65000 (8-10%), and some minor components whose molecular weights varied from 65000 to 340000. Urea (3--8 M) causes to partial dissolution of the crystals; the component with molecular weight of 65000 is mainly found in the solution (component A). In dithioerythritol extracts at pH 9 the major component of the crystal is the protein with molecular weight 120000--140000 (component B). The crystals, alkali-soluble components and proteins isolated from crystals by selective extraction (3--8 M urea or 0.01 M dithioerythrytol, pH 9) were found toxic for the larvae of Galleria mellonella.     

Three-dimensional rearrangement of proteins in the tail of bacteriophage T4 on infection of its host

The contractile tail of bacteriophage T4 undergoes major structural transitions when the virus attaches to the host cell surface. The baseplate at the distal end of the tail changes from a hexagonal to a star shape. This causes the sheath around the tail tube to contract and the tail tube to protrude from the baseplate and pierce the outer cell membrane and the cell wall before reaching the inner cell membrane for subsequent viral DNA injection. Analogously, the T4 tail can be contracted by treatment with 3 M urea. The structure of the T4 contracted tail, including the head-tail joining region, has been determined by cryo-electron microscopy to 17 A resolution. This 1200 A-long, 20 MDa structure has been interpreted in terms of multiple copies of its approximately 20 component proteins. A comparison with the metastable hexagonal baseplate of the mature virus shows that the baseplate proteins move as rigid bodies relative to each other during the structural change.     

A comparison of surface proteins in embryonal carcinoma cells and their differentiated derivatives

Surface proteins from five cell lines (three embryonal carcinoma cell lines (F9, PCC4 and PCC3), teratocarcinoma-derived endodermal cells (PYS) and fibroblasts (line 3/A/1-D-3 differentiated from PCC3)) were compared by two dimensional polyacrylamide gel electrophoresis after selective iodination with ¹²⁵I in the presence of lactoperoxidase. The labeled proteins were solubilized either in Nonidet P40/urea/ampholyte/mercaptoethanol solution or in Nonidet P40 only. In total, about thirty major ¹²⁵I-labeled surface proteins were identified by their isoelectric point and molecular weight. 14 proteins are present in all five cell types, although their quantity or accessibility for labeling differs between differentiated and undifferentiated cells. Three proteins (200, 160 and 150 kilodaltons) are present in undifferentiated cells only. Two of them (160 and 150 kilodaltons) were solubilized by Nonidet P40/urea/ampholyte/mercaptoethanol, but not by Nonidet P40. One protein (50 kilodaltons) was found in nullipotent F9 cells only. About 14–15 proteins (including fibronectin) were released by Nonidet P40/urea/ampholyte/mercaptoethanol but not by Nonidet P40. They are presumably bound to submembrane or cytoskeleton structures by non-covalent bonds.     

Effects of low urea concentrations on protein-water interactions

Solvent properties of aqueous media (dipolarity/polarizability, hydrogen bond donor acidity, and hydrogen bond acceptor basicity) were measured in the coexisting phases of Dextran–PEG aqueous two-phase systems (ATPSs) containing .5 and 2.0 M urea. The differences between the electrostatic and hydrophobic properties of the phases in the ATPSs were quantified by analysis of partitioning of the homologous series of sodium salts of dinitrophenylated amino acids with aliphatic alkyl side chains. Furthermore, partitioning of eleven different proteins in the ATPSs was studied. The analysis of protein partition behavior in a set of ATPSs with protective osmolytes (sorbitol, sucrose, trehalose, and TMAO) at the concentration of .5 M, in osmolyte-free ATPS, and in ATPSs with .5 or 2.0 M urea in terms of the solvent properties of the phases was performed. The results show unambiguously that even at the urea concentration of .5 M, this denaturant affects partitioning of all proteins (except concanavalin A) through direct urea–protein interactions and via its effect on the solvent properties of the media. The direct urea–protein interactions seem to prevail over the urea effects on the solvent properties of water at the concentration of .5 M urea and appear to be completely dominant at 2.0 M urea concentration.     

Import into mitochondria of precursors containing hydrophobic passenger proteins: pretreatment of precursors with urea inhibits import

We have studied the import into isolated yeast mitochondria of three hydrophobic passenger proteins attached to the N-terminal cleavable presequence of mitochondrial ATPase subunit 9 from Neurospora crassa. One natural precursor (pN9) contained N. crassa subunit 9; two chimaeric precursors, N9L/Y8-1 and N9L/Y9-2, respectively contained yeast mitochondrial ATPase subunits 8 and 9. In the absence of urea, pN9 and N9L/Y8-1 are imported efficiently but N9L/Y9-2 is not imported. After pretreatment of precursors in 4 M urea, binding of pN9 to mitochondria is marginally affected while its import is substantially inhibited; the binding to mitochondria of chimaeric proteins, N9L/Y8-1 and N9L/Y9-2, is greatly enhanced but no import is observed. This behaviour of import precursors containing hydrophobic passenger proteins is contrasted with that of a hydrophilic chimaeric precursor pCOXIV-DHFR, whose binding and import are enhanced by pretreatment with a high concentration of urea (8 M). The import of N9L/Y8-1 is very sensitive to the presence of low concentrations of urea in the import reaction mixture, and is abolished above 0.5 M urea although precursor binding to mitochondria is increased. By contrast, neither the import nor binding of pCOXIV-DHFR is affected directly by urea up to 0.8 M. These deleterious effects of urea on import of the chimaeric precursors N9L/Y8-1 and N9L/Y9-2 are interpreted in terms of a non-productive binding of these precursors to mitochondria, brought about by exposure of their hydrophobic domains resulting from urea unfolding. The generalization that membrane translocation of mitochondrial import precursors is enhanced by their prior unfolding in urea thus does not apply in the case of these precursors containing hydrophobic passenger proteins.     

Surface properties of membrane systems. Transport of staphylococcal delta-toxin from aqueous to membrane phase

Hemolytic delta-toxin from Staphylococcus aureus was soluble in either water, methanol or chloroform/methanol (2 : 1, v/v). The toxin spread readily from distilled water into films with pressures (pi) of 10 dynes/cm on water and 30 dynes/cm on 6 M urea; from chloroform/methanol it produced 40 dynes/cm pressure on distilled water. The toxin adsorbed barely from water (pi = 1 dyne/ cm) but it did rapidly from 6 M urea (pi = 35 dynes/cm). The protein films had unusually high surface potentials, which increased with the film pressure and decreased with increasing both pH and urea concentration in the aqueous phase. The fluorescence of 1-aniline 8-naphthalene sulfonate with delta-toxin was much greater than that with RNAase and dipalmitoyl phosphatidylcholine itself, indicating probably a marked lipid-binding character of the toxin. By circular dichroism the alpha-helix content of delta-toxin was 42% in water, 45% in methanol, 24% in 6 M urea. Infrared spectroscopy showed predominant alpha-helix in both 2H2O and deuterated chloroform/methanol as well as in films spread from either solvent on 2H2O. In spreading from 6 M [2H]urea, in which the major infrared absorption was that of [2H]urea with peaks at 1600 and 1480 cm(-1), the delta-toxin film showed prevalently non-alpha-helix structures with major peak intensities at 1633 cm(-1) > 1680 cm(-1), indicating the appearance of new beta-aggregated and beta-antiparallel pleated sheet structures in the film. The data prove that (1) high pressure protein films can consist of alpha-helix as well as non-alpha-helix structures and, differently from another cytolytic protein, melittin, delta-toxin does not resume the alpha-helix conformation in going into the film phase from the extended chain in 6 M urea; (2) conformational changes are important in the transport of proteins from aqueous to lipid or membrane phase; (3) delta-toxin is by far more versatile in structural dynamics and more surface active than alpha-toxin.     

Exploring the hidden human urinary proteome via ligand library beads

The human urinary proteome has been reassessed and re-evaluated via a novel concentration/equalization technique, exploiting beads coated with hexameric peptide ligand libraries. These beads act by capturing the whole protein spectra contained in the sample, by drastically reducing the level of the most abundant species, while strongly concentrating the more dilute and rare ones. In a control urine sample, 134 unique proteins could be identified. The first bead eluate (in thiourea, urea, and CHAPS) permitted the identification of 317 gene products, whereas the second eluate (in 9 M urea, pH 3.8) allowed the identification of another 95 unique proteins. By eliminating redundancies, a total of 383 unique gene products could be identified in human urines. This represents a major increment as compared to data reported in recent literature. By comparing our data with those reported to the present, an additional 251 proteins could be added to the list, thus bringing the total unique gene products so far identified in human urines to ca. 800 species.