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At least 667 records · Page 37Linked to original sources
PubMed1993

Determination of the number and relative molecular mass of subunits in an oligomeric protein by two-dimensional electrophoresis. Application to the subunit structure analysis of rat liver amidophosphoribosyltransferase.

To determine simultaneously the relative molecular mass (M(r)) of a native oligomeric protein, and the number and M(r) of its subunits, a method using two-dimensional electrophoresis was developed. To determine the M(r) of a native oligomeric protein, pore gradient gel electrophoresis was performed for the first dimension. Native proteins were dissociated into their subunits by sodium dodecyl sulphate (SDS) in a gel slice, then applied to SDS polyacrylamide gel electrophoresis for the second dimension to determine the M(r) of subunits. The advantage, accuracy, limitations and application of the method are discussed.

Amidophosphoribosyltransferase↗
PubMed1994

Controlling oligomerization of pharmaceutical proteins.

The degree of oligomerization (or in some cases aggregation) often determines the physiological half-life and uptake rate of a protein preparation. High-resolution crystal structures of insulin and other pharmacologically interesting proteins have aided in the design of mutants with altered quaternary structure and physiological uptake rates. Analysis of the contacts between natural oligomers and protein complexes can indicate sequences that may enhance protein oligomerization. These sequences can be altered to produce monomeric protein.

Amino Acid Sequence↗
PubMed1985

Oligomerization of simian virus 40 tumor antigen may be involved in viral DNA replication.

Biological implications of the oligomerization of simian virus 40 (SV40) large T antigen for viral DNA replication were studied by using two temperature-sensitive SV40 A-gene mutants, tsA 58 and tsA 1499. Both mutants are defective at elevated temperature for viral DNA replication whereas tsA 58 is like most other tsA mutants additionally heat sensitive for cell transformation. We found that in contrast to tsA 58 encoded T antigen, tsA 1499 T antigen is thermostable in the ability to bind specifically to the origin of replication of SV40 DNA. Detailed structural analysis of tsA 1499 T antigen by sucrose density gradient centrifugation revealed that it is strictly temperature sensitive for the formation of homologous oligomers but, as we reported previously (M. Montenarh, M. Kohler, and R. Henning, 1984, J. Virol, 49, 658-664), not for the association with the cellular phosphoprotein p53. These observations are compatible with the idea that, in addition to the specific origin-binding ability as well as other functional features, the oligomerization of T antigen may be essential for viral DNA replication.

Animals↗
PubMed1990

Folding and oligomerization properties of a soluble and secreted form of the paramyxovirus hemagglutinin-neuraminidase glycoprotein.

The paramyxovirus SV5 hemagglutinin-neuraminidase (HN) glycoprotein (a type II integral membrane protein) was converted into a soluble and secreted form (HN-F) by replacing the HN signal/anchor domain with a hydrophobic domain that can act as a cleavable signal sequence. Approximately 40% of the HN-F synthesized was secreted from cells (t1/2 approximately 2.5-3 hr). The extracellular HN-F molecules were identified as disulfide-linked dimers and the majority of the population of molecules were resistant to endoglycosidase H digestion. Examination of the oligomeric form of the secreted HN-F, by sucrose density gradient sedimentation, indicated that under conditions where HN was a tetramer, HN-F was found to be a dimer, and no extracellular HN-F monomeric species could be detected. Secreted HN-F was fully reactive with conformation-specific monoclonal antibodies and was enzymatically active as shown by HN-F having neuraminidase activity. Examination of the intracellular HN-F species indicated that HN-F monomers were slowly converted to the disulfide-linked form and that under the sucrose density gradient sedimentation conditions used the HN-F monomers aggregated. Some of the HN-F monomers were degraded intracellularly. These data are discussed in relationship to the seemingly different folding and oligomerization requirements for the intracellular transport of soluble and membrane bound forms of a glycoprotein. The soluble and biologically active form of HN may be suitable for further structural and enzymatic studies.

Antibodies, Monoclonal↗
PubMed1991

The N-terminal heptad repeat region of reovirus cell attachment protein sigma 1 is responsible for sigma 1 oligomer stability and possesses intrinsic oligomerization function.

The oligomerization domain of the reovirus cell attachment protein (sigma 1) was probed using the type 3 reovirus sigma 1 synthesized in vitro. Trypsin cleaved the sigma 1 protein (49K molecular weight) approximately in the middle and yielded a 26K N-terminal fragment and a 23K C-terminal fragment. Under conditions which allowed for the identification of intact sigma 1 in the oligomeric form (approximately 200K) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the N-terminal 26K fragment was found to exist as stable trimers (80K) and, to a less extent, as dimers (54K), whereas the C-terminal fragment remained in the monomeric form. A polypeptide (161 amino acids) containing the N-terminal heptad repeat region synthesized in vitro was capable of forming stable dimers and trimers. Using various criteria, we demonstrated that the stability of the intact sigma 1 oligomer is conferred mainly by the N-terminal heptad repeat region. Our results are summarized in a model in which individual heptad repeats are held together in a three-stranded alpha-helical coiled-coil structure via both hydrophobic and electrostatic interactions.

Amino Acid Sequence↗
PubMed1992

Characterization of an oligomerization domain and RNA-binding properties on rotavirus nonstructural protein NS34.

Intermolecular interactions between polypeptide chains often play essential roles in such biological phenomena as replication, transcription, translation, transport, ligand binding, and assembly. We have initiated studies of the functions of the rotavirus SA114F gene 7 product by sequence analysis and expression in insect cells. This nonstructural protein, NS34, is a slightly acidic protein, and its secondary structure is predicted to be 78% alpha-helix, with several heptad repeats of hydrophobic amino acids being present in its carboxy half. NS34 was found in oligomers when analyzed in insect cells, in SA11-infected MA104 cells, and in cell-free translation reactions. Investigation of the multiple electrophoretically distinct forms of NS34 showed they were all composed of homooligomers. Deletion mutants constructed and tested for oligomerization showed that the carboxy terminus of the protein, containing the predicted heptad repeats, was responsible for oligomerization. A basic region present in NS34 of group A rotaviruses, found to be 40% conserved in NS34 of group C rotavirus, is a candidate for a functional domain of this protein. NS34, which was found to be associated with the cytoskeleton fraction of cells, also interacts with viral RNA. These results make it likely that NS34 plays a central role in the replication and assembly of genomic RNA structures.

Amino Acid Sequence↗
PubMed1980

Defined oligomeric SV40 DNA: a sensitive probe of general recombination in somatic cells.

We have constructed well defined oligomeric molecules of simian virus 40 (SV40) DNA as probes for investigating mechanisms by which cultured somatic cells recombine DNA. Restriction enzyme fragments from different temperature-sensitive mutants were joined in a head-to-tail orientation to create partial dimers 1.84 genome lengths in size. These molecules are too large to fit into a viral capsid. Therefore an assay that depends on production of progeny virus after infection with oligomeric DNA is a selective measure of precise conversion of oligomers to monomers. By constructing oligomers from appropriate combinations of temperature-sensitive DNAs, we have been able to study the conversion process in several defined regions of the SV40 genome. Our results indicate that conversion of oligomers to monomers occurs uniformly throughout the genome and is not dependent on normal viral DNA replication. These data indicate that conversion occurs primarily by general, homology-dependent recombination. At least one secondary mechanism that generates a low level of wild-type progeny was also detected. Studies with heteroduplex molecules indicate that repair of mismatched bases may be the secondary mechanism.

Animals↗
PubMed1986

Oligomerization is essential for transport of vesicular stomatitis viral glycoprotein to the cell surface.

Using ts045, a temperature sensitive strain of Vesicular stomatitis virus, we show that oligomerization of G protein is a prerequisite for its transport from RER to the Golgi apparatus and for its subsequent maturation. While wild-type G forms an oligomer in the RER, ts045 G synthesized at the nonpermissive temperature does not. When the permissive temperature is reinstated, ts045 G forms an oligomer and moves to the Golgi. The state of oligomerization was determined by chemical cross-linking and by the ability of a microinjected monoclonal antibody specific for the carboxy-terminal five amino acids of the cytoplasmic tail of G to cause patching of G in intracellular membranes. We conclude that formation of an oligomer of G protein, probably a trimer, is necessary for G protein maturation.

Biological Transport↗
PubMed1993

Acetylcholine receptor assembly: subunit folding and oligomerization occur sequentially.

The temperature sensitivity of nicotinic acetylcholine receptors (AChRs) from T. californica was used to identify steps in AChR subunit folding and oligomerization. Assembly intermediates were isolated by lowering to an assembly-permissive temperature. The earliest identifiable assembly intermediates, alpha beta gamma trimers, form minutes after subunit synthesis. alpha beta gamma delta tetramers are formed slowly by the addition of delta subunits to trimers, and finally a second alpha subunit is added to form alpha 2 beta gamma delta pentamers. Between these oligomerization steps, subunits fold as monitored by alpha-bungarotoxin-binding site formation, appearance of antigenic epitopes, changes in apparent molecular weight, and changes in detergent solubility. Subunit folding requires specific combinations of subunits and correlates in time with subunit additions, suggesting that these subunit folding events contribute to subunit recognition site formation during assembly.

Animals↗
PubMed1989

Secondary structure of detergent-solubilized phospholamban, a phosphorylatable, oligomeric protein of cardiac sarcoplasmic reticulum.

The structure of phospholamban, a 30-kDa oligomeric protein integral to cardiac sarcoplasmic reticulum, was probed using ultraviolet absorbance and circular dichroism spectroscopy. Purified phospholamban was examined in three detergents: octyl glucoside, n-dodecyloctaethylene glycol monoether (C12E8) and sodium dodecyl sulfate (SDS). Ultraviolet absorption spectra of phospholamban reflected its aromatic amino acid content: absorption peaks at 275-277 nm and 253, 259, 265 and 268 nm were attributed to phospholamban's one tyrosine and two phenylalanines, respectively. Phospholamban phosphorylated at serine 16 by the catalytic subunit of cAMP-dependent protein kinase exhibited no absorbance changes when examined in C12E8 or SDS. Circular dichroism spectroscopy at 250-190 nm demonstrated that phospholamban possesses a very high content of alpha-helix in all three detergents and is unusually resistant to denaturation. Dissociation of phospholamban subunits by boiling in SDS increased the helical content, suggesting that the highly ordered structure is not dependent upon oligomeric interactions. The purified COOH-terminal tryptic fragment of phospholamban, containing residues 26-52 and comprising the hydrophobic, putative membrane-spanning domain, also exhibited a circular dichroism spectrum characteristic of alpha-helix. Circular dichroism spectra of phosphorylated and dephosphorylated phospholamban were very similar, indicating that phosphorylation does not alter phospholamban secondary structure significantly. The results are consistent with a two-domain model of phospholamban in which each domain contains a helix and phosphorylation may act to rotate one domain relative to the other.

Adenosine Triphosphatases↗
PubMed1995

Actions of picrotoxinin analogues on an expressed, homo-oligomeric GABA receptor of Drosophila melanogaster.

The actions of picrotoxinin and four of its analogues were tested on a Drosophila melanogaster homo-oligomeric GABA (gamma-aminobutyric acid) receptor formed when RDL (resistance to dieldrin) subunits were expressed in Xenopus oocytes. In agreement with previously reported studies on native insect GABA receptors and native expressed vertebrate GABA receptors, acetylation of the bridgehead hydroxyl group (picrotoxinin acetate) greatly reduced the activity of the molecule, but surprisingly, substitution with flourine at the same position also reduced the activity. Conversion of the terminal isopropenyl group to an acetyl (alpha-picrotoxinone) or hydration of the double bond (picrotin) also reduced activity, in agreement with findings for native insect and mammalian receptors. The present results suggest that interactions of convulsants with homo-oligomeric and multimeric GABA receptors are qualitatively similar. Thus, the RDL homo-oligomer exhibits a pharmacological profile for picrotoxinin analogues resembling that of native GABA receptors.

Acetylation↗
PubMed1992

Oligomerization-mediated activation of plasmid-borne genes in Escherichia coli.

A plasmid carrying a weakly expressed neomycin phosphotransferase (neo) gene from the transposable element Tn5 was found to confer elevated levels of antibiotic resistance on its host cell when it existed in a non-monomeric state. This activation of the neo gene appeared to be a generalized effect which can be exerted on any plasmid-encoded gene, since specific sequences were not required for enhanced neo expression, and the activity of a plasmid-borne chloramphenicol acetyltransferase gene could be similarly induced by oligomerization. The potential role that multiple origins of replication present in such oligomeric plasmids play in these observed increases in gene expression is discussed.

DNA Transposable Elements↗
PubMed1990

Two methods for rapid assembly and oligomerization of synthetic genes: construction of human calcitonin-encoding sequences.

Two fast, simple and reliable methods for construction of synthetic genes are described. The first represents a one-vial method for assembly and cloning of monomeric genes, and the second allows a simultaneous assembly, oligomerization and cloning of synthetic genes. Both methods consist of ligation of the synthetic oligodeoxyribonucleotides (oligos) building up the gene directly into the cloning vector, followed by screening of the recombinant clones for the presence of specific gene constructs. The average length of the oligomeric genes thus constructed is directly proportional to the molar ratio between the vector DNA and the synthetic oligos used. Both methods are illustrated by the construction of a series of human calcitonin-encoding genes.

Amino Acid Sequence↗
PubMed1992

Structure/function relationships in the Escherichia coli mannitol permease: identification of regions important for membrane insertion, substrate binding and oligomerization.

The Escherichia coli mannitol permease (EIIMtl) of the phosphoenolpyruvate-dependent phosphotransferase system is a 68-kDa membrane protein that carries out the concomitant transport and phosphorylation of D-mannitol. Previous studies indicated that there are ca. 6 membrane-spanning helices within the N-terminal half of the protein, while the hydrophilic C-terminal half was shown to be exposed in the cytoplasm. In the present study, an analysis of C-terminally truncated EIIMtl mutants showed that proteins from which only the cytoplasmic domain has been deleted were present in the membrane at > or = 50% the amount of the intact protein. However, deletion proteins smaller than ca. 34 kDa were present in the membrane at only about 20% the amount of the intact protein. We also constructed a plasmid that encodes the first 43 amino acid residues of ELLMtl fused to residues 378 to 637 (the C-terminal domain). The corresponding protein was associated with the cytoplasmic membrane. These results show that the first 43 amino acid residues of the N terminus are sufficient for membrane localization, although the region comprising the last 2 membrane-spanning helices appears to be important for maximum stability and/or efficient membrane insertion of the complete N-terminal domain. Further studies of these deletion proteins showed that binding of mannitol to the permease occurs even if the entire cytoplasmic domain is absent, but is abolished if the last putative membrane-spanning region is removed. Finally, regions of the protein within the membrane-bound domain were identified that influence the oligomerization state of the protein. These results further define domains of this multifunctional transport protein that are important for membrane insertion, stability, substrate binding and oligomerization.

Autoradiography↗
PubMed1992

Interrelationships between protein phosphorylation and oligomerization in transport and chemotaxis via the Escherichia coli mannitol phosphotransferase system.

The membrane-bound enzymes II of the bacterial carbohydrate phosphotransferase system (PTS) are multifunctional: they are required for the transport, phosphorylation and chemotactic sensing of their substrates. An oligomer (minimally a dimer) of at least one of these PTS permeases, the Escherichia coli mannitol permease, appears to be necessary for this protein to optimally carry out these functions. Much indirect evidence is consistent with this hypothesis, and recent experiments show that transport and phosphorylation of, and chemotaxis to, mannitol in E. coli involves an intersubunit phosphotransfer reaction, which can only occur in a protein oligomer. Membrane topological studies of the mannitol permease also argue in favour of an oligomeric structure in the membrane which may be necessary to form the hydrophilic channel through which mannitol must traverse the phospholipid bilayer. The possibility that the oligomerization state of the mannitol permease is a target for regulation of its activity in vivo is proposed, but has not yet been explored experimentally.

Biological Transport, Active↗
PubMed1994

Structural and functional basis for hormone binding and receptor oligomerization.

Most single-pass transmembrane receptors undergo a change in oligomeric state upon hormone binding. Recent mutational, biophysical and structural studies of the human growth hormone and tumor necrosis factor receptor complexes have revealed much about the mechanisms and molecular bases for binding and oligomerization. Principles learned from these examples and others should apply to many other hormone-receptor complexes.

Animals↗
PubMed1992

Folding and assembly of oligomeric proteins in Escherichia coli.

High levels of expression of oligomeric proteins in heterologous systems are frequently associated with misfolding and accumulation of the polypeptides in inclusion bodies. This reflects aspects of the folding and assembly pathways of oligomeric proteins, which generally proceed from either folding intermediates or native-like metastable species that are not in their final conformation. Methods for optimizing the yield of correctly assembled oligomers are discussed.

Amino Acid Sequence↗
PubMed1994

Selective deletions in the 90 kDa heat shock protein (hsp90) impede hetero-oligomeric complex formation with the glucocorticosteroid receptor (GR) or hormone binding by GR.

We have developed an in vivo system using coexpression of human glucocorticosteroid receptor (hGR) and chick hsp90 alpha (chsp90) in recombinant virus-infected Sf9 cells to study the formation of hetero-oligomeric complexes. We detected, in the cytosol, hGR complexes containing chsp90 as shown by the displacement of the [3H]triamcinolone acetonide bound hGR "8S" peak on glycerol/sucrose gradients by specific antibodies directed against chsp90 (BF4 and D7 alpha). We took advantage of this system and of the immunoadsorption of hGR containing complexes with anti-hGR antibody to analyze the effect of deletions introduced into the hsp90 molecule on the formation of complexes with the hGR. Deletion of the hydrophilic region "A", between amino-acids 221 and 290, abolished the formation of hGR/chsp90 complexes. Deletion of the hydrophilic region "B" (between amino-acids 530 and 581) or deletion of a leucine repeat region "Z" in the middle of the molecule (amino-acids 392 to 419) still allowed formation of hetero-oligomeric complexes detected by immunoadsorption but the hGR complexes formed with mutated chsp90s were devoid of steroid binding properties. These results are consistent with (1) the possible involvement of the "A" region in the interaction of hsp90 with steroid receptors and (2) a role of B and Z regions in the hsp90 structure for maintaining the steroid binding property of the hGR.

Animals↗