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PubMed2005

Oligomerization endows enormous stability to soybean agglutinin: a comparison of the stability of monomer and tetramer of soybean agglutinin.

Soybean agglutinin is a tetrameric legume lectin, each of whose subunits are glycosylated. This protein shows a very high degree of stability when compared to the other proteins of the same family. In a previous work, it was shown that the unusual stability of the protein is due to a high degree of subunit interactions. In this study we present the thermodynamic parameters for the stability of soybean agglutinin monomer. The monomeric species is found at pH 2 and below which it is most populated at pH 1.9, as evident from size-exclusion chromatographic and dynamic light scattering studies. The analyses of circular dichroism and fluorescence spectroscopy suggest that the monomer is well folded, and that it has certain characteristic features when compared to its tetrameric counterpart. The conformational stabilities of the tetramer and the monomer at the temperature of their maximum stabilities (310 K) are 59.2 kcal/mol and 9.8 kcal/mol, respectively, indicating that oligomerization contributes significantly to the stability of the native molecule. Also, the T(g) difference for the two forms of the protein is approximately 40 K, whereas the difference in DeltaC(p) is only 1.6 kcal/mol/K. This suggests that the major hydrophobic core is present in the monomer itself, and that oligomerization involves mainly ionic interactions.

Biophysical Phenomena↗
PubMed2006

Oligomerization and aggregation of bovine pancreatic ribonuclease A: characteristic events observed by FTIR spectroscopy.

Nonnative protein aggregation, which is a common feature in biotechnology, is also a clinical feature in more than 20 serious degenerative diseases. We studied the specific events of bovine pancreatic ribonuclease A thermal aggregation by a combination of second derivative infrared analysis and two-dimensional infrared correlation spectroscopy. By comparing the events that occur in reversible and irreversible thermal unfolding processes, certain events that were related to protein aggregation were characterized. Particularly, a band that appeared at high temperatures was assigned to the cross beta-structures in oligomers. The effect of pH, NaCl, and ethanol on ribonuclease A oligomerization as well as further aggregation induced by heat were studied and dissimilar effects of these additives were found. Basic pH and NaCl could accelerate the thermal aggregation but did not affect the formation of oligomers, whereas ethanol could increase both the aggregation rate and the population of oligomers. Our results suggested that the aggregation of RNase A might be initiated by hydrophobic interactions, controlled by oligomerization and mediated by electrostatic interactions. Moreover, the strategy of using second derivative and two-dimensional infrared analysis might provide a potential powerful tool to study the events that are directly related to the initiation of protein aggregation.

Animals↗
PubMed2006

Curvature and hydrophobic forces drive oligomerization and modulate activity of rhodopsin in membranes.

G protein-coupled receptors (GPCRs) are essential components of cellular signaling pathways. They are the targets of many current pharmaceuticals and are postulated to dimerize or oligomerize in cellular membranes in conjunction with their functional mechanisms. We demonstrate using fluorescence resonance energy transfer how association of rhodopsin occurs by long-range lipid-protein interactions due to geometrical forces, yielding greater receptor crowding. Constitutive association of rhodopsin is promoted by a reduction in membrane thickness (hydrophobic mismatch), but also by an increase in protein/lipid molar ratio, showing the importance of interactions extending well beyond a single annulus of boundary lipids. The fluorescence data correlate with the pK(a) for the MI-to-MII transition of rhodopsin, where deprotonation of the retinylidene Schiff base occurs in conjunction with helical movements leading to activation of the photoreceptor. A more dispersed membrane environment optimizes formation of the MII conformation that results in visual function. A flexible surface model explains both the dispersal and activation of rhodopsin in terms of bilayer curvature deformation (strain) and hydrophobic solvation energy. The bilayer stress is related to the lateral pressure profile in terms of the spontaneous curvature and associated bending rigidity. Transduction of the strain energy (frustration) of the bilayer drives protein oligomerization and conformational changes in a coupled manner. Our findings illuminate the physical principles of membrane protein association due to chemically nonspecific interactions in fluid lipid bilayers. Moreover, they yield a conceptual framework for understanding how the tightly regulated lipid compositions of cellular membranes influence their protein-mediated functions.

Animals↗
PubMed1995

Purification and oligomeric state of the major lens fiber cell membrane proteins.

Purification of the lens fiber cell membrane proteins MP20 and MP26, and the partial co-purification of the lens connexin-related proteins MP70 and connexin 46 has been achieved using anion- and cation-exchange chromatography of lens fiber cell membrane proteins solubilized in n-octyl-beta-D-glucopyranoside (octyl glucoside). The apparent molecular weights of the solubilized protein-detergent complexes were significantly greater than that expected for the monomeric proteins. The purified proteins retained their ability to be phosphorylated by cAMP-dependent protein kinase, and to bind calmodulin in a calcium and magnesium dependent manner. The heterobifunctional covalent chemical crosslinking agent N-5-azido-2-nitro-benzoyloxysuccinimide (ANB-NOS), and the thiol oxidant cupric phenanthroline were used to identify the oligomeric states of these proteins. Crosslinking of either the purified proteins or native lens membranes generated a ladder of crosslinked MP20 or MP26 homo-oligomers. The largest detectable crosslinked homo-oligomer of MP20 was at least a hexamer, while for MP26 the largest crosslinked homo-oligomer was at least a tetramer. The possible oligomeric states of MP70 and connexin 46 could not be determined with the crosslinking reagents used in this study. The procedure described here for the purification of detergent-solubilized major lens proteins should provide a valuable approach in future studies aimed at clarifying the roles of these different lens membrane proteins.

Affinity Labels↗
PubMed1977

Selective transport of an oligomeric IgA into canine saliva.

Evidence is presented which shows that an oligomeric IgA myeloma protein possessing J-chain but lacking secretory component, is selectively transferred from serum into canine saliva. The data also demonstrate that 125I-label remains with the IgA during the transfer process. These data interpreted in the light of findings in humans support the concept that the oligomeric form of IgA, devoid of secretory component, is required to achieve selective transport.

Animals↗
PubMed2001

The Bcr N-terminal oligomerization domain contributes to the full oncogenicity of P190 Bcr/Abl in transgenic mice.

The Bcr/Abl P190 oncoprotein is responsible for the development of Philadelphia-chromosome positive acute lymphoblastic leukemia (ALL). The Bcr moiety in Bcr/Abl activates the Abl tyrosine kinase, an ingredient essential for the transforming capability of Bcr/Abl. Residues 1-63 of Bcr form an N-terminal oligomerization domain and are key to Abl activation in vitro. Mice transgenic for P190 BCR/ABL reproducibly develop an aggressive B-lineage lymphoblastic leukemia/lymphoma. Here we test the hypothesis that residues 1-63 of Bcr have a major in vivo contribution to the oncogenicity of Bcr/Abl P190 by the generation of mice transgenic for an N-terminal deleted form of P190. We find that although the transgene is expressed in the bone marrow of mice at an early age, the incidence of leukemogenesis is greatly diminished as compared to mice transgenic for non-mutated P190 Bcr/Abl. Sporadic hematological malignancies which did develop showed decreased levels of phosphotyrosine as compared to those of wild-type P190 transgenics, although Ras was activated. These results demonstrate that the Bcr oligomerization domain contributes to the oncogenicity of Bcr/Abl in vivo.

Animals↗
PubMed2005

53BP1 oligomerization is independent of its methylation by PRMT1.

p53 binding protein 1 (53BP1) participates in the repair of DNA double stranded breaks (DSBs) where it is recruited to or near sites of DNA damage. Although little is known about the biochemical functions of 53BP1, the protein possesses several motifs that are likely important for its role as a DNA damage response element. This includes two BRCA1 C-terminal repeats, tandem Tudor domains, and a variety of phosphorylation sites. Here we show that a glycine-arginine rich (GAR) stretch of 53BP1 lying upstream of the Tudor motifs is methylated. We demonstrate that arginine residues within this region are important for asymmetric methylation by the PRMT1 methyltransferase. We further show that sequences upstream of the Tudor domains that do not include the GAR stretch are sufficient for 53BP1 oligomerization in vivo. Thus, although Tudor domains bind methylated proteins, 53BP1 homo-oligomerization occurs independently of Tudor function. Lastly, we find that deficiencies in 53BP1 generate a "hyper-rec" phenotype. Collectively, these data provide new insight into 53BP1, an important component in maintaining genomic stability.

Amino Acid Sequence↗
PubMed2006

Oligomerization of p53 precedes its association with dynein and nuclear accumulation.

Previous studies have identified several proteins that associate with microtubules and the dynein motor complex including p53, the glucocorticoid and the vitamin D receptors, and the APC (adenomatous polyposis coli) protein; but neither the residues important for this interaction nor the physical state of the proteins involved have been clarified. We observed in SN12C cells harboring a mutant p53 truncated at amino acid 336, impaired nuclear localization and impaired association with dynein. This finding was confirmed and extended by examining a series of truncated p53 proteins that identified residues 336 to 348 as crucial for association with dynein and nuclear transport. Point mutations identified the importance of residues involved in p53 oligomerization in this process, establishing a p53 oligomer as the cargo for dynein transport. The association of cytosolic p53 oligomers with dynein occurs independent of microtubules indicating that following this association, the p53/dynein complex then associates with microtubules and is transported to the peri-nuclear region. These studies suggest that mutations or modifications that affect p53 oligomerization not only interfere with DNA binding but also with its intracellular distribution. They also highlight the importance of an intact microtubule network in the trafficking of crucial cellular proteins.

Active Transport, Cell Nucleus↗
PubMed2000

Active site interactions in oligomeric structures of inorganic pyrophosphatases.

Recent progress in studies of the mode of action of cytoplasmic inorganic pyrophosphatases is mainly due to the analysis of a dozen and a half structures of the apoenzyme, its complexes, and mutants. However, despite considerable research on the mechanism of action of these enzymes, many important problems remain unclear. Among them is the problem of active site interactions in oligomeric structures and their role in catalysis; this review focuses on this problem. The abundant experimental data requires generalization and comprehensive analysis. A characteristic feature of the spatial structure of inorganic pyrophosphatases is a flexible system of noncovalent interactions between protein groups penetrating the whole molecule of the oligomeric enzyme. Binding of metal ions, sulfate (an analog of the product of the enzymatic reaction), and affinity phosphorus-containing inhibitors at the active site or site-directed mutagenesis induce rearrangements in the set of hydrogen and ionic interactions, which change active site properties and in some instances, cause molecule asymmetry. In the trimeric form of Escherichia coli pyrophosphatase obtained by dissociation of a hexamer, active sites also interact with each other, which is manifested by negative cooperativity upon substrate binding. The association of trimers into the hexamer leads to perfect organization of active sites and to their coordinated functioning, probably due to the restoration of communication channels between the trimers.

Binding Sites↗
PubMed2000

Oligomeric state of the muscle glycogen phosphorylase b in the system of reversed micelles of aerosol OT in octane.

The oligomeric state and formation of supramolecular structures of glycogen phosphorylase b from rabbit skeletal muscle was studied in the system of aerosol OT (AOT) reversed micelles in octane. The sedimentation experiments have shown that the enzyme oligomeric state depends on the degree of micelle hydration. The enzyme monomer, dimer, trimer, tetramer, hexamer, and octamer were observed, depending on the degree of hydration.

Animals↗
PubMed2001

A novel action of alzheimer's amyloid beta-protein (Abeta): oligomeric Abeta promotes lipid release.

Interactions between amyloid beta-protein (Abeta) and lipids have been suggested to play important roles in the pathogenesis of Alzheimer's disease. However, the molecular mechanism underlying these interactions has not been fully understood. We examined the effect of Abeta on lipid metabolism in cultured neurons and astrocytes and found that oligomeric Abeta, but not monomeric or fibrillar Abeta, promoted lipid release from both types of cells in a dose- and time-dependent manner. The main components of lipids released after the addition of Abeta were cholesterol, phospholipids, and monosialoganglioside (GM1). Density-gradient and electron microscopic analyses of the conditioned media demonstrated that these Abeta and lipids formed particles and were recovered from the fractions at densities of approximately 1.08-1.18 g/ml, which were similar to those of high-density lipoprotein (HDL) generated by apolipoproteins. The lipid release mediated by Abeta was abolished by concomitant treatment with Congo red and the PKC inhibitor, H7, whereas it was not inhibited with N-acetyl-l-cysteine. These Abeta-lipid particles were not internalized into neurons, whereas HDL-like particles produced by apolipoprotein E were internalized. Our findings indicate that oligomeric Abeta promotes lipid release from neuronal membrane, which may lead to the disruption of neuronal lipid homeostasis and the loss of neuronal function.

Alzheimer Disease↗
PubMed2002

Expression, purification, and characterization of the oligomeric states of rubredoxin oxidoreductase from Methanobacterium thermoautotrophicum.

The rubredoxin oxidoreductase from Methanobacterium thermoautotrophicum was successfully overexpressed in Escherichia coli with 6x His-Tag and purified by using Ni-NTA technology. It was characterized by SDS and native polyacrylamide gel electrophoresis (PAGE), as well as size-exclusion chromatography. The protein was pure, judged by SDS-PAGE, but three or more oligomeric species were observed by native PAGE and size-exclusion chromatography. The smallest rubredoxin oxidoreductase species is the dimer. The multiple species are stable and remain in their respective oligomeric states, judged by the chromatographic and electrophoretic results. A model is proposed in order to explain the structural basis for these results.

Amino Acid Sequence↗
PubMed1992

Mouse mammary tumor virus gene expression is suppressed by oligomeric ellagitannins, novel inhibitors of poly(ADP-ribose) glycohydrolase.

Oligomeric ellagitannins (nobotanins B, E, and K) were found to be potent inhibitors of poly(ADP-ribose) glycohydrolase purified from mouse mammary tumor 34I cells. Kinetic analysis revealed that the inhibition of nobotanin B (dimer) was competitive with respect to the substrate poly(ADP-ribose), whereas nobotanin E (trimer) and nobotanin K (tetramer) exhibited mixed-type inhibition. These results suggest that the dimeric structure of ellagitannin may have a functional domain that competes with poly(ADP-ribose) on the poly(ADP-ribose) glycohydrolase molecule. To determine the inhibitory effects of oligomeric ellagitannins on poly(ADP-ribose) glycohydrolase in vivo, we examined their effects on de-poly(ADP-ribosyl)ation of some chromosomal proteins in intact 34I cells that was induced by glucocorticoid treatment. Nobotanin B caused concentration-dependent inhibition of glucocorticoid-induced de-poly(ADP-ribosyl)ation of HMG 14 and 17 and histone H1 in intact 34I cells. Interestingly, this inhibition was associated with suppression of the glucocorticoid-sensitive mouse mammary tumor virus (MMTV) mRNA synthesis. In contrast, nobotanin E and K had little inhibitory effect on either de-poly(ADP-ribosyl)ation of these proteins or induction of MMTV transcription after glucocorticoid treatment. Nobotanin B but not E and K was taken into 34I cells. These results may suggest that the suppression of glucocorticoid-sensitive MMTV transcription results from in vivo inhibition of poly(ADP-ribose) glycohydrolase by nobotanin B. These results also indicate the importance of de-poly(ADP-ribosyl)ation of HMG 14 and 17 and histone H1 in regulation of transcription of the glucocorticoid-sensitive MMTV gene.

Animals↗
PubMed1992

[Dependence of epidermal growth factor activation on its affinity and oligomerization].

Epidermal growth factor receptor (EGF-R) oligomerization has been followed on A-431 cells using covalent labeling by 125I-EGF and EGF-dependent autophosphorylation of receptor-kinase. High molecular weight complexes corresponding to monomeric, dimeric, and trimeric forms of EGF-R are detected. The process of oligomerization occurs effectively at 37 degrees C while at 4 degrees C no oligomer formation is detected. PMA or ATP treatment reduces the number of high-affinity EGF-binding sites but has no influence on dimer formation. Dimerisation of the EGF-R in the absence of the ligand has been established on formalin-fixed A-431 cells.

Adenosine Triphosphate↗
PubMed1992

Assembly of the oligomeric membrane pore formed by Staphylococcal alpha-hemolysin examined by truncation mutagenesis.

The alpha-hemolysin (alpha HL) from Staphylococcus aureus causes the lysis of susceptible cells such as rabbit erythrocytes (rRBCs). Lysis is associated with the formation of a hexameric pore in the plasma membrane. Here we show that truncation mutants of alpha HL missing 2 to 22 N-terminal amino acids form oligomers on the surfaces of rRBCs but fail to lyse the cells. By contrast, mutants missing 3 or 5 amino acids at the C terminus are very inefficient at oligomerization but do lyse rRBCs, albeit extremely slowly. The C-terminal truncation mutants, retarded as monomers on the cell surface, undergo a conformational change in which the protease-sensitive loop located near the midpoint of the polypeptide chain becomes occluded. Judged by this criterion, polypeptides truncated at the N terminus, frozen as nonlytic oligomers, are in a similar conformation. A second proteolytic site near the N terminus of the polypeptide becomes inaccessible in the lytic pore formed by the wild-type polypeptide, supporting the idea that a second conformational change occurs upon pore formation. These findings suggest a pathway for assembly of the lytic pore in which alpha HL first binds to target cells as a monomer, which is converted to a nonlytic oligomeric intermediate before formation of the pore. In keeping with this model, an N-terminal truncation mutant blocks the slow lysis induced by a C-terminal truncation mutant, presumably by diverting the weakly lytic subunits into inactive oligomers.

Amino Acid Sequence↗
PubMed1992

Glucose transporter oligomeric structure determines transporter function. Reversible redox-dependent interconversions of tetrameric and dimeric GLUT1.

This study investigates the relationship between human erythrocyte glucose transport protein (GLUT1) oligomeric structure and glucose transporter function. Oligomeric structure was analyzed by hydrodynamic studies of cholate-solubilized GLUT1, by chemical cross-linking studies of membrane-resident GLUT1 and by using conformation-specific antibodies. Transporter function (substrate binding) was analyzed by equilibrium cytochalasin B and D-glucose binding measurements. Erythrocyte-resident glucose transporter is a GLUT1 homotetramer, binds 1 mol of cytochalasin B/2 mol of GLUT1, and presents at least two binding sites to D-glucose. Native structure and function appear to be stabilized by intramolecular disulfide bonds and are preserved during GLUT1 purification by the omission of reductant. Native structure is independent of in vitro and in vivo membrane GLUT1 density but is transformed to dimeric GLUT1 by alkaline reduction. Dimeric GLUT1 binds 1 mol of cytochalasin B/mol of GLUT1, presents a single population of binding sites to D-glucose, and is obtained upon GLUT1 purification in the presence of reductant. Native structure and function are restored by treatment of dimeric GLUT1 with glutathione-disulfide (K0.5 glutathione disulfide = 29 microM). We propose that native structure is established prior to transporter translocation to the plasma membrane and that intrasubunit disulfide bonds promote cooperative subunit interactions that stabilize transporter structure and function.

Binding Sites↗
PubMed2004

[From diversity of molecular forms to functional specialization of oligomeric proteins, nicotinic acetylcholine receptor, acetylcholinesterase and Na+, K+-ATPase].

The review is devoted to the issue of diversity of molecular forms of oligomeric proteins using as examples members of the three protein classes: nicotinic acetylcholine receptor, acetylcholinesterase, and Na,K-ATPase. The data are presented on the molecular structure of proteins, subunit compositions, and isoforms of subunits, as well as on some features of gene expression. Particular emphasis has been made on the functional specialization of different molecular forms of one and the same oligomeric protein. The three above proteins, which serve seemingly quite different cellular processes, demonstrate many common principles of molecular mechanisms of physiological function.

Acetylcholinesterase↗
PubMedDate not supplied

[Thermal denaturation of oligomeric proteins: structural and functional modifications, the sequence of steps].

On the basis of literature and own experimental data changes in the structural and functional properties of some oligomeric proteins of the blood system (hemoglobin, lactate dehydrogenase, superoxide dismutase, catalase) exposed to the influence of temperature in a broad range were analyzed. The many-phase character of the temperature modification of protein molecules with different values of functional and kinetic parameters for each of revealed stages was discovered. At a critical temperature and at higher values, the dissociation of oligomeric proteins into separate subunits was shown to occur along with the typical "loosening" of the protein globule. It was shown that low-molecular components (subunits) can subsequently associate with one another and with oligometic proteins, which leads to irreversible denaturation and to the unusual physicochemical behavior of protein molecules. Schemes of processes underlying the temperature modifications of the proteins studied were elaborated.

Animals↗