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PubMed1995

Biosynthesis of melanin from dopamine. An investigation of early oligomerization products.

Matrix-assisted laser desorption/ionization (MALDI) and fast-atom bombardment (FAB) mass spectrometry experiments were applied to the study of the early stages of the oligomerization reaction of dopamine with mushroom tyrosinase. Ultrafiltration was employed to remove the enzyme at various reaction times, to prevent possible attachment of the protein to the highly reactive intermediates. Two sets of five samples each, obtained at different reaction times, in one case immediately lyophilized and in the other left to react under an oxygen stream for 24 h before lyophilization, were compared. FAB showed the presence of various species and of these, that at m/z 305 increased in abundance with reaction time in immediately lyophilized set of samples only. Accurate mass measurements and tandem mass spectrometric experiments indicated the structure of a dopamine protonated dimer for this ion. MALDI measurements showed that all samples were composed of clusters of oligomers differing in degree of oligomerization. Oligomerization increases with reaction time, resulting in the formation of species at 2643-2911 Da. These clusters in turn were formed of species with a different degree of oxidation, detected in both sets of samples.

Dopamine↗
PubMed2004

Oligomeric carbon and siloxane series observed by matrix-assisted laser desorption/ionisation and laser desorption/ionisation mass spectrometry during the analysis of soot formed in fuel-rich flames.

Oligomeric carbon and siloxane series have been observed by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS), during the analysis of the dichloromethane (DCM)-soluble fractions of condensable material recovered from fuel-rich flames. Laser desorption (LD) spectra showed a pattern of oligomeric dimethyl-siloxane structures with a spacing of 74 u. The siloxane series appears to have originated as contamination of samples by silicone oil used to lubricate connections of polymer tubing. This was confirmed by extracting silicone tubing and silicone grease with DCM followed by MALDI-MS analysis. A series of peaks with a mass spacing of 24 u was also observed, superimposed on the continuum of unresolved organic ions. This oligomeric series appears to correspond to polycyclic aromatics separated by (mainly) ethylene bridges. Thus LD-MS appears to have revealed a series of soot precursors, intermediate between polycyclic aromatics and particulate soot, which was not detected by MALDI-MS. More detailed work is necessary to define these species with precision.

Carbon↗
PubMed2005

Analyzing glycerol-mediated protein oligomerization by electrospray ionization mass spectrometry.

Glycerol is widely used as protein stabilizer, in both local and commercial preparations, so it has become necessary to develop methods for mass spectrometric analysis of protein preparations in the presence of glycerol. However, this stabilizing agent may cause signal suppression when present in high concentrations, and is also known to induce protein supercharging even at low concentrations. This work reports the use of electrospray ionization (ESI) mass spectrometry to characterize glycerol-mediated protein oligomerization. This phenomenon seems to involve the formation of strong non-covalent interactions between protein and glycerol involving close contact between the monomers, leading to formation of protein oligomers adducted with glycerol molecules under the characteristic analytical conditions of the ESI interface. At high orders of oligomerization a lower number of glycerol molecules is required to maintain the high oligomeric states than for the dimers and trimers, and it is possible that for the higher oligomers the monomers become so close to one another that non-covalent bonds between the side chains of the amino acid residues in the proteins may be established.

Animals↗
PubMed2001

Regulation of chicken gizzard ecto-ATPase activity by modulators that affect its oligomerization status.

The major ectonucleoside triphosphate phosphohydrolase in the chicken gizzard smooth muscle membranes is an ecto-ATPase, an integral membrane glycoprotein belonging to the E-ATPase (or E-NTPDase) family. The gizzard ecto-ATPase is distinguished by its unusual kinetic properties, temperature dependence, and response to a variety of modulators. Compounds that promote oligomerization of the enzyme protein, i.e., concanavalin A, chemical cross-linking agent, and eosin iodoacetamide, increase its activity. Compounds that inhibit some ion-motive ATPases, e.g., sulfhydryl reagents, xanthene derivatives, NBD-halides, and suramin, also inhibit the gizzard ecto-ATPase, but not another E-ATPase, the chicken liver ecto-ATP-diphosphohydrolase, which contains the same conserved regions as the ecto-ATPase. Furthermore, inhibition of the gizzard ecto-ATPase by these compounds as well as detergents is not prevented by preincubation of the membranes with the substrate, ATP, indicating that their interaction with the enzyme occurs at a locus other than the catalytic site. On the other hand, the inhibitory effect of these compounds, except suramin, is abolished or reduced if the membranes are preincubated with concanavalin A. It is concluded that these structurally unrelated modulators exert their effect by interfering with the oligomerization of the ecto-ATPase protein. Our findings suggest that, under physiological conditions, the gizzard smooth muscle ecto-ATPase may exhibit a range of activities determined by membrane events that affect the status of oligomerization of the enzyme.

4-Chloro-7-nitrobenzofurazan↗
PubMed2001

Recombinant phycobiliproteins. Recombinant C-phycocyanins equipped with affinity tags, oligomerization, and biospecific recognition domains.

A family of specific cloning vectors was constructed to express in the cyanobacterium Anabaena sp. PCC7120 recombinant C-phycocyanin subunits with one or more different tags, including the 6xHis tag, oligomerization domains, and the streptavidin-binding Strep2 tag. Such tagged alpha or beta subunits of Anabaena sp. PCC7120 C-phycocyanin formed stoichiometric complexes in vivo with appropriate wild-type subunits to give constructs with the appropriate oligomerization state and normal posttranslational modifications and with spectroscopic properties very similar to those of unmodified phycocyanin. All of these constructs were incorporated in vivo into the rod substructures of the light-harvesting complex, the phycobilisome. The C-terminal 114-residue portion of the Anabaena sp. PCC7120 biotin carboxyl carrier protein (BCCP114) was cloned and overexpressed and was biotinylated up to 20% in Escherichia coli and 40% in wild-type Anabaena sp. His-tagged phycocyanin beta--BCCP114 constructs expressed in Anabaena sp. were >30% biotinylated. In such recombinant phycocyanins equipped with stable trimerization domains, >75% of the fusion protein was specifically bound to streptavidin- or avidin-coated beads. Thus, the methods described here achieve in vivo production of stable oligomeric phycobiliprotein constructs equipped with affinity purification tags and biospecific recognition domains usable as fluorescent labels without further chemical manipulation.

Amino Acid Sequence↗
PubMed1997

Oligomerization of expanded-polyglutamine domain fluorescent fusion proteins in cultured mammalian cells.

Six inherited neurologic diseases, including Huntington's disease, result from the expansion of a CAG domain of the disease genes to produce a domain of more than 40 glutamines in the expressed protein. The mechanism by which expansion of this polyglutamine domain causes disease is unknown. Recent studies demonstrated oligomerization of polyglutamine-domain proteins in mammalian neurons. To study oligomerization of polyglutamine proteins and to identify heterologous protein interactions, varying length polyglutamine-green fluorescent protein fusion proteins were expressed in cultured COS-7 cells. The 19- and 35-glutamine fusion proteins (non-pathologic length) distributed diffusely throughout the cytoplasm. In contrast, 56- and 80-glutamine fusion proteins (pathologic length) formed fibrillar arrays resembling those previously observed in neurons in Huntington's disease and in a transgenic mouse model. These aggregates were intranuclear and intracytoplasmic. Intracytoplasmic aggregates were surrounded by collapsed intermediate filaments. The intermediate filament protein vimentin co-immunoisolated with expanded polyglutamine fusion proteins. This cellular model will expedite investigations into oligomerization of polyglutamine proteins and their interactions with other proteins.

Animals↗
PubMed1997

High-density lipoprotein from hypercholesterolemic animals has peroxidized lipids and oligomeric apolipoprotein A-I: its putative role in atherogenesis.

Oxidized lipoproteins have been involved in the pathogenesis of atherosclerosis and atherosclerotic lesions contain oxidized low density lipoprotein. Conversely, the presence of oxidized high density lipoprotein (HDL) in vivo has not been clearly established. Oxidation of HDL in vitro models produces an increase in peroxidized lipids and the appearance of apolipoprotein A-I (apo A-I) oligomers. We investigated the oxidative status of HDL in an in vivo model: the hypercholesterolemic chicken. The HDLs from control and hyperlipemic animals were analyzed for the content of lipid peroxides employing spectroscopic and fluorescence techniques, for the level of apo A-I oligomerization, and for susceptibility to in vitro oxidation. HDL from hypercholesterolemic chickens was more peroxidized (as detected by fluorescence), had higher amount of oligomeric apo A-I, and was oxidized to a greater extent by uv irradiation than that of control animals. We speculate that apo A-I oligomerization could be a key step in the atheroma formation.

Animals↗
PubMed1997

Oligomerization is an intrinsic property of calsequestrin in normal and transformed skeletal muscle.

In skeletal muscle fibers, the high-capacity medium-affinity Ca(2+)-binding protein calsequestrin functions as the major Ca(2+)-reservoir of the sarcoplasmic reticulum. To determine the oligomeric status of calsequestrin, immunoblotting of microsomal proteins following chemical crosslinking was performed. Diagonal non-reducing/reducing two-dimensional gel electrophoresis was employed to unequivocally differentiate between cross-linked species of 63 kDa calsequestrin and calsequestrin-like proteins of higher relative molecular mass. Since chronic low-frequency stimulation has a profound effect on the expression of many muscle-specific protein isoforms, we investigated normal and conditioned muscle fibers. Calsequestrin was found to exist in a wide range of high-molecular-mass clusters in normal and chronically stimulated skeletal muscle fibers. Hence, oligomerization is an intrinsic property of this important Ca(2+)-binding protein and does not appear to be influenced by the fast-to-slow transformation process. Although fiber-type specific differences exist in the physiology of the skeletal muscle Ca(2+)-regulatory system, oligomerization of calsequestrin seems to be essential for proper functioning.

Animals↗
PubMed1999

A genetic screen to identify sequences that mediate protein oligomerization in Escherichia coli.

Many proteins assemble as oligomeric complexes and in several cases a distinct domain mediates the interaction between the subunits. The identification of new oligomerization modules is relevant to comprehend both the architecture and the evolution of protein sequences and also for protein engineering applications. Using the bacteriophage lambda repressor dimerization assay, we searched Escherichia coli genomic libraries for sequences able to mediate protein oligomerization in vivo. We identified short peptides that can substitute very effectively the dimerizing domain of the repressor. Most of these peptides belong to open reading frames that are normally not expressed in the bacterial cell.

Amino Acid Sequence↗
PubMed1998

In vitro and in vivo studies on transthyretin oligomerization.

The oligomerization of transthyretin has been studied in vitro and in vivo. The results showed that wild-type transthyretin synthesized in vitro in the absence of microsomes did not form dimers and tetramers, but in the presence of microsomes the mature transthyretin which had been translocated into the microsomal lumen formed dimers and a small amount of tetramers which could be detected only by using a cross-linking reagent. Efficiency of tetramer formation depends upon the source of microsomes; in fact the amount of tetramer formed in liver microsomes was much higher than that in pancreas microsomes. Transthyretin synthesized in HepG2 cells appeared after SDS-PAGE analysis in mostly tetrameric form, while that synthesized in transfected COS-1 cells appeared mainly as dimers. Brefeldin A treatment and pulse-chase experiments in HepG2 cells showed that transthyretin tetramer was formed in the endoplasmic reticulum. These results strongly indicate that transthyretin tetramer is formed most efficiently in the endoplasmic reticulum lumen of hepatocytes. Transthyretin without the signal peptide [S(-)] and transthyretin with a mutation that prevents processing of the signal peptide Msc failed to form dimers and tetramers in vitro. In the transfected COS-1 cells, however, S(-) transthyretin did form dimers while Msc transthyretin failed to oligomerize. These results show that the cleavage of the signal peptide and some cellular factors are required for transthyretin oligomerization.

Animals↗
PubMed1995

Constitutive forms of the enhancer-binding protein NtrC: evidence that essential oligomerization determinants lie in the central activation domain.

Nitrogen regulatory protein C (NtrC) is a bacterial enhancer-binding protein that activates transcription by the sigma 54-holoenzyme. To activate transcription, NtrC must hydrolyze ATP, a reaction that depends upon its being phosphorylated and forming an appropriate oligomer. In this paper we characterize "constitutive" mutant forms of the NtrC protein from Salmonella typhimurium; unlike wild-type NtrC, these forms are able to hydrolyze ATP and activate transcription in vitro without being phosphorylated. The amino acids altered in NtrCconstitutive proteins are located in both the N-terminal regulatory domain and the central domain, which is directly responsible for transcriptional activation. The residues that are altered are not conserved among activators of the sigma 54-holoenzyme, and are not identical even among NtrC proteins from members of different subgroups of the proteobacteria (purple bacteria). NtrCconstitutive proteins are phosphorylated normally; phosphorylation increases their ability to hydrolyze ATP and activate transcription. Moreover, the oligomerization of these proteins that occurs when they bind to an enhancer also increases the ATPase activity of both unmodified and phosphorylated forms. Removal of the N-terminal regulatory domain from two NtrCconstitutive proteins with amino acid substitutions in the central domain (NtrCS160F and NtrCV2881) leaves them active, indicating that essential oligomerization determinants lie outside the regulatory domain. This conclusion is confirmed by the observation that the ATPase activity of delta N-NtrCS160F is greatly stimulated when it binds to an enhancer, and by the ability of this protein to activate transcription synergistically with a form of NtrC incapable of DNA-binding. Together with previous results indicating that oligomerization determinants do not lie in the C-terminal DNA-binding domain of NtrC; these results provide evidence that they lie in the central domain.

Alleles↗
PubMed1996

Structure of the oligomerization and L-arginine binding domain of the arginine repressor of Escherichia coli.

The structure of the oligomerization and L-arginine binding domain of the Escherichia coli arginine repressor (ArgR) has been determined using X-ray diffraction methods at 2.2 A resolution with bound arginine and at 2.8 A in the unliganded form. The oligomeric core is a 3-fold rotationally symmetric hexamer formed from six identical subunits corresponding to the 77 C-terminal residues (80 to 156) of ArgR. Each subunit has an alpha/beta fold containing a four-stranded antiparallel beta-sheet and two antiparallel alpha-helices. The hexamer is formed from two trimers, each with tightly packed hydrophobic cores. In the absence of arginine, the trimers stack back-to-back through a dyad-symmetric, sparsely packed hydrophobic interface. Six molecules of arginine bind at the trimer-trimer interface, each making ten hydrogen bonds to the protein including a direct ion pair that crosslinks the two protein trimers. Solution experiments with wild-type ArgR and oligomerization domain indicate that the hexameric form is greatly stabilized upon arginine binding. The crystal structures and solution experiments together suggest possible mechanisms of how arginine activates ArgR to bind to its DNA targets and provides a stereochemical basis for interpreting the results of mutagenesis and biochemical experiments with ArgR.

Amino Acid Sequence↗
PubMed2001

Structural characterization of the N-terminal oligomerization domain of the bacterial chromatin-structuring protein, H-NS.

The H-NS protein plays a key role in condensing DNA and modulating gene expression in bacterial nucleoids. The mechanism by which this is achieved is dependent, at least in part, on the oligomerization of the protein. H-NS consists of two distinct domains; the N-terminal domain responsible for protein oligomerization, and the C-terminal DNA binding domain, which are separated by a flexible linker region. We present a multidimensional NMR study of the amino-terminal 64 residues of H-NS (denoted H-NS1-64) from Salmonella typhimurium, which constitute the oligomerization domain. This domain exists as a homotrimer, which is predicted to be self-associated through a coiled-coil configuration. NMR spectra show an equivalent magnetic environment for each monomer indicating that the polypeptide chains are arranged in parallel with complete 3-fold symmetry. Despite the limited resonance dispersion, an almost complete backbone assignment for 1H(N), 1H(alpha), 15N, 13CO and 13C(alpha) NMR resonances was obtained using a suite of triple resonance experiments applied to uniformly 15N-, 13C/15N- and 2H/13C/15N-labelled H-NS1-64 samples. The secondary structure of H-NS1-64 has been identified on the basis of the analysis of 1H(alpha), 13C(alpha), 13Cbeta and 13CO chemical shifts, NH/solvent exchange rates, intra-chain H(N)-H(N) and medium-range nuclear Overhauser enhancements (NOEs). Within the context of the homotrimer, each H-NS1-64 protomer consists of three alpha-helices spanning residues 2-8, 12-20 and 22-53, respectively. A topological model is presented for the symmetric H-NS1-64 trimer based upon the combined analysis of the helical elements and the pattern of backbone amide group 15N nuclear relaxation rates within the context of axially asymmetric diffusion tensor. In this model, the longest of the three helices (helix 3, residues 22-53) forms a coiled-coil interface with the other chains in the homotrimer. The two shorter N-terminal helices fold back onto the outer surface of the coiled-coil core and potentially act to stabilise this configuration.

Bacterial Proteins↗
PubMed2002

Oligomeric state of membrane transport proteins analyzed with blue native electrophoresis and analytical ultracentrifugation.

Blue native electrophoresis is used widely for the analysis of non-dissociated protein complexes with respect to composition, oligomeric state and molecular mass. However, the effects of detergent or dye binding on the mass and stability of the integral membrane proteins have not been studied. By comparison with analytical ultracentrifugation, we have evaluated whether the oligomeric state of membrane transport proteins is reflected reliably with blue native electrophoresis. For the analysis we have used two well-characterized transporters, that is, the major facilitator superfamily protein LacS and the phosphotransferase system EII(Mtl). For another member of the major facilitator superfamily, the xyloside transporter XylP from Lactobacillus pentosus, the complete analysis of the quaternary structure determined by analytical ultracentrifugation and freeze-fracture electron microscopy is presented. Our experiments show that during blue native electrophoresis the detergent bound to the proteins is replaced by the amphipathic Coomassie brilliant blue (CBB) dye. The mass of the bound CBB dye was quantified. Provided this additional mass of bound CBB dye is accounted for and care is taken in the choice and concentration of the detergent used, the mass of LacS, XylP and EII(Mtl) and four other membrane (transport) proteins could be deduced within 10 % error. Our data underscore the fact that the oligomeric state of many membrane transport proteins is dimeric.

Bacterial Proteins↗
PubMed2000

Effects of oligomerization on the epitopes of the human immunodeficiency virus type 1 envelope glycoproteins.

To understand the differential expression of epitopes on monomeric and oligomeric forms of the envelope glycoproteins, nine human monoclonal antibodies (mAbs) were derived from the cells of human immunodeficiency virus-infected subjects by selection with soluble oligomeric gp140 (o.140). These nine mAbs and 12 human mAbs selected with V3 peptides, viral lysates, and rgp120, specific for the V2, V3, C5, CD4-binding domain (CD4bd), and gp41, were tested in a binding assay to compare the exposure of these regions on monomeric gp120 or gp41 and on o.140. None of the 21 mAbs were oligomer specific. However, mAbs to V3 and CD4bd were "oligomer sensitive," whereas mAbs to V2 and the distal epitope of C5 tended to be "monomer sensitive" (i.e., to react better with the oligomer or monomer, respectively). The majority of anti-gp41 mAbs reacted similarly with monomer and oligomer. Although the uncleaved o.140 used in this study differs from the cleaved gp120/41 oligomer found on the native virus particle, these results suggest that new epitopes are not introduced by oligomerization of viral envelope proteins, that such oligomer-specific epitopes, if they exist, are not highly immunogenic, and/or that they are not efficiently selected using soluble o.140.

Antibodies, Monoclonal↗
PubMed1992

Mo-MuLV nucleotide sequence exhibits three levels of oligomeric repetitions, suggesting a stepwise molecular evolution.

An exhaustive computer-assisted analysis of the Moloney murine leukemia virus nucleotide sequence shows numerous deviations in the oligomeric distribution, suggesting three overlapping levels of a stepwise duplicative evolution. (1) The sequence fits the universal rule of TG/CT excess which has been proposed as the construction principle of all sequences, and maintains some degree of symmetry between the two complementary strands. (2) Oligomeric repeating units share a core consensus regularly scattered throughout the sequence. This consensus is not merely predictable from the doublet frequencies and codon usage, but could correspond to an intermediary stage in a so-called periodic-to-chaotic transition. (3) Probable stepwise local duplications could be accounted for by slippagelike mechanisms. Comparison with the human spumaretrovirus (HSRV) shows similar segments in the overrepresented oligomers of the two sequences. The intermediary stage of transition oligomeric repeating units is not so clearly suggested in HSRV, perhaps because of numerous stepwise local duplications. In any case, a common evolutionary origin for the two viruses is not ruled out.

Base Sequence↗
PubMed1995

Characterization of Vibrio cholerae El Tor cytolysin as an oligomerizing pore-forming toxin.

V. cholerae El Tor cytolysin is a secreted, water-soluble protein of M(r) 60,000 that may be relevant to the pathogenesis of acute diarrhea. In this communication, we demonstrate that the toxin binds to and oligomerizes in target membranes to form SDS-stable aggregates of M(r) 200,000-250,000 that generate small transmembrane pores. Pores formed in erythrocytes were approximately 0.7 nm in size, as demonstrated by osmotic protection experiments. Binding was shown to occur in a temperature-independent manner preceding the temperature-dependent oligomerization step. Pores were also shown to be formed in L929 and HEp-2 cells, human fibroblasts and keratinocytes, albeit with highly varying efficacy. At neutral pH and in the presence of serum, human fibroblasts were able to repair a limited number of lesions. The collective data identify V. cholerae El Tor cytolysin as an oligomerizing toxin that damages cells by creating small transmembrane pores.

Adenosine Triphosphate↗
PubMed1979

Different specific activities of the monomeric and oligomeric forms of plasmid DNA in transformation of B. subtilis and E. coli.

(1) The low residual transforming activity in preparations of monomeric, supercoiled, circular (CCC) forms of the plasmids pC194 and pHV14 could be attributed to the presence in such isolates of a small number of contaminating multimeric molecules. (2) E. coli derived preparations of pHV14, as in vitro recombinant plasmid capable of replication in both E. coli and B. subtilis, contain oligomeric forms of plasmid DNA in addition to the prevalent monomeric CCC form. The specific transforming activity of pHV14 DNA for E. coli is independent of the degree of oligomerization, whereas in transformation of B. subtilis the specific activity of the purified monomeric CCC molecules is at least four orders of magnitude less than that of the unfractionated preparation. (3) Oligomerization of linearized pHV14 DNA by T4 ligase results in a substantial increase of specific transforming activity when assayed with B. subtilis and causes a decrease when used to transform E. coli.

Bacillus subtilis↗