The structure and protein binding of amyloid-specific dye reagents

The self-assembling tendency and protein complexation capability of dyes related to Congo red and also some dyes of different structure were compared to explain the mechanism of Congo red binding and the reason for its specific affinity for beta-structure. Complexation with proteins was measured directly and expressed as the number of dye molecules bound to heat-aggregated IgG and to two light chains with different structural stability. Binding of dyes to rabbit antibodies was measured indirectly as the enhancement effect of the dye on immune complex formation. Self-assembling was tested using dynamic light scattering to measure the size of the supramolecular assemblies. In general the results show that the supramolecular form of a dye is the main factor determining its complexation capability. Dyes that in their compact supramolecular organization are ribbon-shaped may adhere to polypeptides of beta-conformation due to the architectural compatibility in this unique structural form. The optimal fit in complexation seems to depend on two contradictory factors involving, on the one hand, the compactness of the non-covalently stabilized supramolecular ligand, and the dynamic character producing its plasticity on the other. As a result, the highest protein binding capability is shown by dyes with a moderate self-assembling tendency, while those arranging into either very rigid or very unstable supramolecular entities are less able to bind.     

Supramolecular ligands: Monomer structure and protein ligation capability

The aim of this work was to define the chemical structure of compounds self-assembling in water solutions, which appear to interact with proteins as single ligands with their supramolecular nature preserved. For this purpose the ligation to proteins of his azo dyes, represented by Congo red and its derivatives with designed structural alterations, were tested. The three parameters which characterize the reactivity of supramolecular material were determined in the same conditions for all studied dyes. These were: A) stability of the assembly products; B) binding to heat-denatured protein (human IgG); and C) binding to native protein (rabbit antibodies in the immune complex) measured by the enhancement of hemagglutination. The structural differences between the Congo red derivatives concerned the symmetry of the molecule and the structure of its non-polar component, which occupies the central part of the dye molecule and is thought to be crucial for self-assembly. Other dyes were also studied for the same purpose: Evans blue and Trypan blue, bis-ANS and ANS, as well as a group of compounds with a structural design unlike that of bis azo dyes. Compounds with rigid elongated symmetric molecules with a large non-polar middle fragment are expected to form a ribbon-like supramolecular organization in assembling. They appeared to have ligation properties related to their self-assembling tendency. The compounds with different structures, not corresponding to his azo dyes, did not reveal ligation capability, at least in respect to native protein. The conditions of binding to denatured proteins seem less restrictive than the conditions of binding to native molecules. The molten hydrophobic protein interior becomes a new binding area allowing for complexation of even non-assembled molecules.     

Supramolecularity creates nonstandard protein ligands

Congo red and a group of structurally related dyes long used to stain amyloid proteins are known to associate in water solutions. The self-association of some dyes belonging to this group appears particularly strong. In water solutions their molecules are arranged in ribbon-like micellar forms with liquid crystalline properties. These compounds have recently been found to form complexes with some native proteins in a non-standard way. Gaps formed by the local distribution of beta-sheets in proteins probably represent the receptor sites for these dye ligands. They may result from higher structural instability in unfolding conditions, but also may appear as long range cooperative fluctuations generated by ligand binding. Immunoglobulins G were chosen as model binding proteins to check the mechanism of binding of these dyes. The sites of structural changes generated by antigen binding in antibodies, believed to act as a signal propagated to distant parts of the molecule, were assumed to be suitable sites for the complexation of liquid-crystalline dyes. This assumption was confirmed by proving that antibodies engaged in immune complexation really do bind these dyes; as expected, this binding affects their function by significantly enhancing antigen binding and simultaneously inhibiting C1q attachment. Binding of these supramolecular dyes by some other native proteins including serpins and their natural complexes was also shown. The strict dependence of the ligation properties on strong self-assembling and the particular arrangement of dye molecules indicate that supramolecularity is the feature that creates non-standard protein ligands, with potential uses in medicine and experimental science.     

Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence

Statistical analysis of 12 unstable and 32 stable proteins revealed that there are certain dipeptides, the occurrence of which is significantly different in the unstable proteins compared with those in the stable ones. Based on the impact of these dipeptides on the unstable proteins over the stable ones, a weight value of instability is assigned to each of the dipeptides. For a given protein the summation of these weight values normalized to the length of its sequence helps to distinguish between unstable and stable proteins. Results suggest that the in vivo instability of proteins is possibly determined by the order of certain amino acids in its sequence. An attempt is made to correlate metabolic stability of proteins with features of their primary sequence where weight values of instability for a protein of known sequence could thus be used as an index for predicting its stability characteristics.     

Profiling mitochondrial proteins in radiation-induced genome-unstable cell lines with persistent oxidative stress by mass spectrometry

Previous work by Morgan and coworkers on radiation-induced genome instability in Chinese hamster ovary (CHO) cell lines showed that unstable LS-12 cells had persistently elevated levels of reactive oxygen species (ROS) that were likely due to dysfunctional mitochondria. To further investigate the correlation between radiation-induced genome instability and dysfunctional mitochondria, we performed quantitative high-throughput mass spectrometry on samples enriched in mitochondrial proteins from three chromosomally unstable CHO cell lines and their stable unirradiated GM10115 parental cell line. Out of several hundred identified proteins, sufficient data were collected on 74 mitochondrial proteins to test for statistically significant differences in their abundance between unstable and stable cell lines. The LS-12 cell line, which exhibited the highest level of ROS among the three unstable cell lines, was characterized by eight significantly down-regulated mitochondrial proteins, all associated with the TCA (tricarboxylic acid). Elevated levels of ROS relative to the unirradiated parental control were also statistically significant for the CS-9 cell line. The protein profile of CS-9 revealed five significantly up-regulated mitochondrial proteins, three of which are involved in oxidative phosphorylation. Elevation of ROS in the unstable 115 cell line was nearly as large as that seen in CS-9 cells but was not statistically significant. The mitochondrial protein profile of 115 cells showed significant down-regulation of acetyl-CoA-acetyltransferase, which was also down-regulated in LS-12, and two other proteins with abundances that were significantly different from control levels but were not directly related to either the TCA or oxidative phosphorylation. These results provide further evidence that elevated ROS and mitochondrial dysfunction are associated with radiation-induced genome instability; however, additional work is required to establish a firm mechanistic relationship between these end points.     

Molecular biological differences between strains of Mycoplasma gallisepticum]

Differences in virulence of two Mycoplasma gallisepticum strains, S6 and A5969, are confirmed in experiments with chickens. Macromolecular discrepancies detected between these two strains are concerning the genomic size, electrophoretic spectra of DNA and proteins. Cross immunoblotting data with polyclonal and monoclonal antibodies reveal major immunogens of protein nature in both the strains. Homologous proteins with different electrophoretic mobility are detected in other four M. gallisepticum strains. A possible participation of these proteins of M. gallisepticum in adhesion to the host cells is discussed.     

On the possibility of applying noncovalent dyes for protein labeling in isoelectric focusing

Noncovalent fluorescent dyes are widely used for protein quantification and postcolumn detection in electrophoretic separations and recently some attempts to separate the precolumn labeled proteins using isoelectric focusing (IEF) have been made. In the present study, the possibility of applying the technique of protein labeling with noncovalent dyes for IEF is investigated. We found that fluorescent signal emitted by NanoOrange dye increases essentially in presence of carrier ampholyte (CA) components, which makes problematic a reliable protein detection in CA environment. Since in an isoelectric focusing mode the CA species are present in much greater concentration than the concentrations of fractionated proteins, the method of protein labeling with NanoOrange is not suitable for precolumn labeling and cannot be used for CA-IEF, at least without more detailed study of the dye-protein interaction mechanism.     

Phenylalanine hydroxylase-like antibody in human chorionic villi]

An antigen similar by electrophoretic mobility to liver phenylalanine hydroxylase (PH) and cross-reacting with monoclonal antibody PH8 against liver PH was detected in extracts of soluble proteins in 6 from 23 samples of chorionic villi. An antigen with electrophoretic mobility corresponding to 40-41 kDa was detected in extracts of membrane proteins from these 23 samples by immunoblotting with monoclonal antibody PH8. Its molecular weight was similar to that of major chymotryptic peptide of human liver PH. The content of the antigen varied with samples and was less than 20 ng/mg of the extracted protein. Two-dimensional gel electrophoresis revealed only 1 spot of the antigen. The antigen did not react with monoclonal antibodies PH7 and PH9 epitopes of which were located in N-terminal fragment of liver PH. These data suggest that the antigen of membrane fraction could be a PH protein without N-terminal domain.     

Production and detection of coliphage T4 endonuclease V polyclonal and monoclonal antibodies using staphylococcal protein-A hybrid proteins

To facilitate the production of antibodies against endonuclease V, a pyrimidine dimer-specific DNA glycosylase produced in bacteriophage T4-infected Escherichia coli, we constructed plasmids containing protein-A-endonuclease V fusion genes under control of the E. coli tac promoter. Induction with isopropyl-beta-D-thiogalactopyranoside produced large amounts of fusion proteins, which could easily be purified on human IgG agarose columns. The affinity-purified fusion proteins were injected into rabbits and mice to produce polyclonal and monoclonal antibodies, and also used for the screening of the monoclonal antibodies. These antibodies recognized endonuclease V on immunoblots, and also inhibited the DNA-glycosylase activity in vitro. Epitope mapping of monoclonal antibodies showed that they all (6/6) recognized determinants in the C-half of endonuclease V. A convenient way to detect primary antibodies on nitrocellulose was also developed using a crude protein extract containing protein-A-beta-galactosidase fusion protein and subsequent detection with a mixture of dyes.     

Monoclonal antibodies as probes for determining the microheterogeneity of the link proteins of cartilage proteoglycan

Monoclonal antibodies were raised against Swarm rat chondrosarcoma link protein 2. Two of the resultant hybridomas (9/30/6-A-1 and 9/30/8-A-4) were used in structural analyses of the link proteins. The 9/30/6-A-1 monoclonal antibody recognized an epitope which was only present on rat chondrosarcoma link protein 2. This epitope was absent in rat chondrosarcoma link protein 3 obtained after trypsin or clostripain treatment of rat chondrosarcoma proteoglycan aggregate, indicating that proteolytic digestion either removed or modified the epitope. Contrasting this, the 9/30/8-A-4 monoclonal antibody recognized an epitope present in link protein(s) 1, 2, or 3 isolated from cartilage of several animal species (rat, bovine, human, and chicken). Rat chondrosarcoma link protein 2 was digested with Staphylococcus aureus V8 protease, and the resulting peptides were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to immunolocation analyses. The 9/30/6-A-1 and 9/30/8-A-4 monoclonal antibodies recognized epitopes in two different halves of the link protein molecule. The 9/30/8-A-4 monoclonal antibody was used to identify proteolytic cleavage peptides common to the individual link proteins (1, 2, or 3) purified from cartilage proteoglycans of several animal species. Digestion of rat chondrosarcoma link protein 2 with endoglycosidase H or alpha-mannosidase increased its electrophoretic mobility to that of link protein 3 and removed or altered the determinant recognized by the 9/30/6-A-1 monoclonal antibody, indicating that a high-mannose oligosaccharide chain was part of the antigenic determinant. The 9/30/8-A-4 monoclonal recognition of epitope was unaffected by endo- or exoglycosidase treatment. Endo- and exoglycosidase treatment of bovine nasal cartilage link proteins also altered their electrophoretic mobility, indicating that high-mannose oligosaccharide structures on the various link proteins (1, 2, or 3) accounted for the microheterogeneity observed in sodium dodecyl sulfate-polyacrylamide gels.     

Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.

A discontinuous electrophoretic system for the isolation of membrane proteins from acrylamide gels has been developed using equipment for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Coomassie dyes were introduced to induce a charge shift on the proteins and aminocaproic acid served to improve solubilization of membrane proteins. Solubilized mitochondria or extracts of heart muscle tissue, lymphoblasts, yeast, and bacteria were applied to the gels. From cells containing mitochondria, all the multiprotein complexes of the oxidative phosphorylation system were separated within one gel. The complexes were resolved into the individual polypeptides by second-dimension Tricine-SDS-PAGE or extracted without SDS for functional studies. The recovery of all respiratory chain complexes was almost quantitative. The percentage recovery of functional activity depended on the respective protein complex studied and was zero for some complexes, but almost quantitative for others. The system is especially useful for small scale purposes, e.g., separation of radioactively labeled membrane proteins, N-terminal protein sequencing, preparation of proteins for immunization, and diagnostic studies of inborn neuromuscular diseases.     

Variations of Candida albicans electrophoretic karyotypes.

We previously described 14 rare spontaneous morphological mutants of Candida albicans that were associated with chromosomal aberrations (E. P. Rustchenko-Bulgac, F. Sherman, and J. B. Hicks, J. Bacteriol. 172:1276-1283, 1990). Improved conditions for separation of chromosomes, as well as hybridization probes, were used to investigate the variation of karyotypes of clinical isolates and additional morphological mutants. All 23 newly analyzed morphological mutants, representing frequently occurring and highly unstable colonial forms, had a variety of altered karyotypes. All chromosomal changes were similar to those previously observed in mutants m1 to m14. In this study, I particularly noted that the most frequent changes involved the long chromosome VIII, which carries ribosomal DNA cistrons. Two rates of instability were uncovered by analyzing the progenies from two highly unstable mutants. An unstable mutant proved to be able to continuously produce a large number of altered karyotypes that could result in a wide variety of different phenotypes. Furthermore, all four independent clinical isolates, FC18, C9, 3153A, and WO-1, common laboratory strains, revealed different electrophoretic karyotypes and distinct colonial morphologies on a synthetic medium, similar to spontaneous mutants. The differences of electrophoretic karyotypes observed among clinical isolates resembled the changes found among different kinds of spontaneous morphological mutants. These findings contribute to the understanding of natural karyotypic variability and are in agreement with the hypothesis that chromosomal alterations observed spontaneously under laboratory conditions provide this amictic species with genetic variability in nature.     

Principle of the supramolecular stacking of the cellular deoxyribonucleoprotein complex

Using cytofluorimetry with acridine orange staining and a modified thermal denaturation technique of cellular DNP, it has been shown that chromatin melting profiles of normal human nuclei (from lymphocytes and granulocytes) have distinct regularities. It is believed that these regularities reflect specific supramolecular chromatin organization. Parallel comparative analysis performed using electrophoretic fractionation and isoelectric focussing of nuclear proteins has revealed that: 1) peculiarities of chromatin melting profiles are independent of the quantity and molecular weights of chromatin proteins; 2) the lack of principal differences in chromatin melting profiles and the data on isoelectric points of nuclear proteins of granulocytes and lymphocytes from the same patient indicate that specific supramolecular organization of DNP-complex depends on the chromatin protein charge.     

Production of a monoclonal antibody directed against rat liver glutathione-insulin transhydrogenase

A hybridoma cell line secreting monoclonal antibody specific for glutathione-insulin transhydrogenase has been produced by fusing mouse myeloma cells with spleen cells from mice immunized to purified rat liver glutathione-insulin transhydrogenase. The secreted antibody isotypes were found to be: Ig gamma 1 heavy chains and kappa light chains. This monoclonal antibody has been used to screen glutathione-insulin transhydrogenase in various rat tissue extracts (liver, fat, heart, testis, spleen, lung and kidney) following separation on NaDodSO4/urea polyacrylamide disc-gel electrophoresis and electrophoretic transfer to nitrocellulose. Screening with the monoclonal antibody showed the presence of one immunoreactive protein band equal in molecular weight to that of purified rat liver GIT (Mr 53,000) in extracts of all tissues studied and a second immunoreactive protein band of lower molecular weight (Mr 49,000) in spleen and lung tissue extracts. Separation of these two proteins by HPLC using a TSK-DEAE column demonstrated that both proteins exhibit insulin degrading activity. These data indicate that GIT may occur in multiple forms in some tissues.     

Systematic comparison of surface coatings for protein microarrays

To process large numbers of samples in parallel is one potential of protein microarrays for research and diagnostics. However, the application of protein arrays is currently hampered by the lack of comprehensive technological knowledge about the suitability of 2-D and 3-D slide surface coatings. We have performed a systematic study to analyze how both surface types perform in combination with different fluorescent dyes to generate significant and reproducible data. In total, we analyzed more than 100 slides containing 1152 spots each. Slides were probed against different monoclonal antibodies (mAbs) and recombinant fusion proteins. We found two surface coatings to be most suitable for protein and antibody (Ab) immobilization. These were further subjected to quantitative analyses by evaluating intraslide and slide-to-slide reproducibilities, and the linear range of target detection. In summary, we demonstrate that only suitable combinations of surface and fluorescent dyes allow the generation of highly reproducible data.     

Rabies mRNA translation in Xenopus laevis oocytes.

Two rabies virus-specific mRNA species were identified by analysis of their encoded proteins after translation of the partially purified species in Xenopus laevis oocytes. One of these coded for the virion surface glycoprotein (G protein), and the other coded for the major structural protein of the virion nucleocapsid (N protein). The G-mRNA sedimented in a sucrose density gradient at about 18S, and the N-mRNA had a sedimentation coefficient of approximately 16S. Their respective translation products were identified in a radioimmunoassay with specific monoclonal antibody probes that recognized only G or N proteins. Immunoprecipitates formed between the radiolabeled viral antigens synthesized in programmed oocytes and their respective monoclonal antibodies were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The glycoprotein antigen translated from G-mRNA in oocytes migrated in the gel ahead of the virion G protein with a migration rate that was similar to that of nonglycosylated intracellular glycoproteins from virus-infected cells. The results suggested that the branched-chain carbohydrate of G protein was not required for recognition by the particular monoclonal antibody used. The nucleocapsid antigen translated from N-mRNA in oocytes migrated to the same position in the gel as marker virion N protein. Both the electrophoretic mobility of virus-specific antigens in sodium dodecyl sulfate-polyacrylamide gel and the antibody concentration dependence for immunoprecipitations were criteria for identifying the individual viral proteins encoded by the two rabies mRNA's.     

Detection of relationships among microsporidan isolates by electrophoretic analysis: hydrophilic extracts

Hydrophilic spore proteins were extracted from Nosema sp. and Nosema trichoplusiae. These proteins were subjected to electrophoretic analysis. The resulting electrophoretic spectra were found to be unstable when (1) two genera of hosts were used for spore propagation, (2) hosts were reared at a variety of temperatures, (3) protein was extracted from spores stored for different periods of time, or (4) spore incubation period was varied. Comparison of the major bands obtained from spore protein of the isolates indicated no overlap in relative migration values. Although variation in spectra was observed, the use of major band patterns indicate electrophoretic analysis of hydrophilic spore protein can provide characters useful in the separation and identification of microsporidan isolates. Nosema sp. and Nosema trichoplusiae are not considered to be closely related phylogenetically.     

Spectroscopic evidence for amyloid-like interfacial self-assembly of hydrophobin Sc3

Amphipathic fungal proteins called hydrophobins are able to self-assemble into insoluble supramolecular structures at hydrophobic/hydrophilic interfaces, but the molecular mechanism and underlying protein conformation changes are not known. Secondary-structure prediction indicated that hydrophobin Sc3 is an all-beta protein. Many amyloidogenic proteins self-assemble into insoluble amyloid fibrils while undergoing a change to an all-beta conformation. In this study we show that two dyes, thioflavin T, and Congo red, which are widely used for specific detection of stacked beta sheets, interact with Sc3 assemblies in the same way as with the amyloid beta-sheet fibrils. We conclude that Sc3, and probably other hydrophobins too, self-assemble at interfaces in the same manner as amyloidogenic proteins, i.e., through beta-sheet stacking.     

Catching and separating protein ligands by functional affinity electrophoresis

A new kind of affinity electrophoresis called functional affinity electrophoresis (FAEP) is a technique used to separate and/or capture proteins according to their functions in a native polyacrylamide gel. Protein A:immunoglobulin G, avidin:biotin, antibody:antigen, and concanavalin A:glycoprotein interactions are used to demonstrate this technique. Protein A, avidin, monoclonal anti-bovine serum albumin (BSA) antibody, and concanavalin A are embedded in distinct regions of a 7.5% native polyacrylamide gel. Some of each of the embedded proteins get covalently and/or noncovalently incorporated into the gel matrix network. Under electrophoresis conditions, these proteins do not show significant electrophoretic mobility or they migrate in a direction opposite to the protein analytes, as in avidin. We clearly observe that polyclonal anti-human myoglobin antibody, biotinylated insulin, BSA, and ovalbumin (glycoprotein) are captured and separated in distinct regions of a FAEP gel by protein A, avidin, monoclonal anti-BSA antibody, and concanavalin A, respectively.     

Multiple gene-like sequences related to the rabbit hepatic progesterone 21-hydroxylase cytochrome P-450 1

Rabbits exhibit phenotypic differences, 21H and 21L, in the rate of hepatic progesterone 21-hydroxylation that reflect 10-fold higher microsomal concentrations of cytochrome P-450 1 in 21H rabbits. A cDNA library in pBR322 was prepared from liver mRNA isolated from a 21H rabbit. A clone, p1-8, producing a hybrid protein resulting from the insertion of the cDNA into the beta-lactamase gene of the plasmid expressed 5 distinct epitopes that were recognized by a panel of monoclonal antibodies developed toward P-450 1. RNAs selected from total hepatic mRNA by filter hybridization with p1-8 yield at least two electrophoretically distinct proteins when translated in vitro and immunoprecipitated with the 3C3 monoclonal antibody. Only one of the two proteins is recognized by the 1F11 monoclonal antibody, which is highly specific for P-450 1, and the immunoprecipitated protein exhibits the electrophoretic mobility of P-450 1. The other protein remains unidentified. Northern blot analysis indicates that the 3' noncoding portion of p1-8 hybridizes to higher steady state concentrations of polyadenylated RNA in the 21H as compared to 21L rabbits. This correspondence in expression with that of P-450 1 in the 21H and 21L phenotypes further suggests that p1-8 encodes P-450 1 or a closely related protein. The cDNA is 1871 base pairs in length and encodes a protein of 487 amino acids. Southern blot analysis indicates that several independent, gene-like sequences hybridize with the 3' noncoding region of p1-8 under conditions of high stringency. These results indicate that P-450 1 is a member of an extensive multigene family.